Publications

Purpose A Phase II study to screen for anti-melanoma activity of the heat shock protein 90 (HSP90) inhibitor, 17-AAG (17-allylamino-17-demethoxygeldanamycin) was performed. The primary endpoint was the rate of disease stabilisation in patients with progressive, metastatic melanoma treated with 17-AAG. Secondary endpoints were to determine: the toxicity of 17-AAG, the duration of response(s), median survival and further study the pharmacokinetics and pharmacodynamics of 17-AAG. Patients and Methods Patients with metastatic melanoma (progressive disease documented </=6 months of entering study) were treated with weekly, intravenous 17-AAG. A Simon one sample two stage minimax design was used. A stable disease rate of >/=25% at 6 months was considered compatible with 17-AAG having activity. Results Fourteen patients (8 male: 6 female) were entered, eleven received 17-AAG (performance status 0 or 1). Median age was 60 (range 29-81) years. The majority (93%) received prior chemotherapy and had stage M1c disease (71%). Toxicity was rarely >/= Grade 2 in severity and commonly included fatigue, headache and gastrointestinal disturbances. One of eleven patients treated with 17-AAG had stable disease for 6 months and median survival for all patients was 173 days. The study was closed prematurely prior to completion of the first stage of recruitment and limited planned pharmacokinetic and pharmacodynamic analyses. Conclusion Some evidence of 17-AAG activity was observed although early study termination meant study endpoints were not reached. Stable disease rates can be incorporated into trials screening for anti-melanoma activity and further study of HSP90 inhibitors in melanoma should be considered.

Histone deacetylase (HDAC) inhibitors are currently approved for cutaneous T-cell lymphoma and are in mid-late stage trials for other cancers. The HDAC inhibitors LAQ824 and SAHA increase phosphocholine (PC) levels in human colon cancer cells and tumor xenografts as observed by magnetic resonance spectroscopy (MRS). In this study, we show that belinostat, an HDAC inhibitor with an alternative chemical scaffold, also caused a rise in cellular PC content that was detectable by (1)H and (31)P MRS in prostate and colon carcinoma cells. In addition, (1)H MRS showed an increase in branched chain amino acid and alanine concentrations. (13)C-choline labeling indicated that the rise in PC resulted from increased de novo synthesis and correlated with an induction of choline kinase α expression. Furthermore, metabolic labeling experiments with (13)C-glucose showed that differential glucose routing favored alanine formation at the expense of lactate production. Additional analysis revealed increases in the choline/water and phosphomonoester (including PC)/total phosphate ratios in vivo. Together, our findings provide mechanistic insights into the impact of HDAC inhibition on cancer cell metabolism and highlight PC as a candidate noninvasive imaging biomarker for monitoring the action of HDAC inhibitors.

Our biological understanding of the molecular basis of cancer has benefited from advances in basic research, accelerated recently by cancer genome sequencing and other high-throughput, genome-wide profiling technologies. Given the diverse heterogeneity among tumors, the traditional cytotoxic chemotherapy and one-size-fits-all approaches to cancer discovery and development are not appropriate for molecularly targeted agents. Selection of new drug targets is based on achieving cancer selectivity through exploiting specific dependencies and vulnerabilities predicted from tumor genetics. Discovery of highly target-selective agents is enhanced by integrating multiple modern technologies, particularly structure-based design. Efficient clinical evaluation requires smart, hypothesis-testing studies using validated pharmacodynamic and predictive biomarkers. We discuss and exemplify biomarker-driven clinical development and the concept of the Pharmacologic Audit Trail. We detail the exciting approaches offered by drugging the cancer genome, focusing on blocking oncogene addiction, drugging the oncogenic lipid kinome, addressing nononcogene addiction, exploiting synthetic lethality, and overcoming apoptotic resistance, leading to personalized molecular medicine.

The early clinical hypothesis for inhibiting HSP90 in cancers was based on the dependence of certain key client proteins in malignant cells--including a host of well-characterized oncoproteins--on the activity of HSP90 for their function and stability. The additional concept has been established that cancer cells have heightened dependence on the efficient maintenance of intracellular proteomic homeostasis, central components of which are HSP90 and other heat shock proteins. We evaluate the evidence that inhibiting HSP90 in cancer exploits both of these biological vulnerabilities very effectively, we review the current status of the discovery and development of HSP90 inhibitors and we identify routes to improve their clinical efficacy, based on emerging knowledge.

Heat shock protein (Hsp) 90 is an ATP-dependent molecular chaperone that is exploited by malignant cells to support activated oncoproteins, including many cancer-associated kinases and transcription factors, and it is essential for oncogenic transformation. Originally viewed with skepticism, Hsp90 inhibitors are now being actively pursued by the pharmaceutical industry, with 17 agents having entered clinical trials. Investigators established Hsp90's druggability using the natural products geldanamycin and radicicol, which mimic the unusual ATP structure adopted in the chaperone's N-terminal nucleotide-binding pocket and cause potent and selective blockade of ATP binding/hydrolysis, inhibit chaperone function, deplete oncogenic clients, and show antitumor activity. Preclinical data obtained with these natural products have heightened interest in Hsp90 as a drug target, and 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin) has shown clinical activity (as defined by Response Evaluation Criteria in Solid Tumors) in HER2+ breast cancer. Many optimized synthetic, small-molecule Hsp90 inhibitors from diverse chemotypes are now in clinical trials. Here, we review the discovery and development of Hsp90 inhibitors and assess their potential. There has been significant learning from studies of the basic biology of Hsp90, as well as translational drug development involving this chaperone, enhanced by the use of Hsp90 inhibitors as chemical probes. Success will likely lie in treating cancers that are addicted to particular driver oncogene products (e.g., HER2, ALK, EGFR, and BRAF) that are sensitive Hsp90 clients, as well as malignancies (especially multiple myeloma) in which buffering of proteotoxic stress is critical for survival. We discuss approaches for enhancing the effectiveness of Hsp90 inhibitors and highlight new chaperone and stress-response pathway targets, including HSF1 and Hsp70.

Acquired resistance to selective FLT3 inhibitors is an emerging clinical problem in the treatment of FLT3-ITD(+) acute myeloid leukaemia (AML). The paucity of valid pre-clinical models has restricted investigations to determine the mechanism of acquired therapeutic resistance, thereby limiting the development of effective treatments. We generated selective FLT3 inhibitor-resistant cells by treating the FLT3-ITD(+) human AML cell line MOLM-13 in vitro with the FLT3-selective inhibitor MLN518, and validated the resistant phenotype in vivo and in vitro. The resistant cells, MOLM-13-RES, harboured a new D835Y tyrosine kinase domain (TKD) mutation on the FLT3-ITD(+) allele. Acquired TKD mutations, including D835Y, have recently been identified in FLT3-ITD(+) patients relapsing after treatment with the novel FLT3 inhibitor, AC220. Consistent with this clinical pattern of resistance, MOLM-13-RES cells displayed high relative resistance to AC220 and Sorafenib. Furthermore, treatment of MOLM-13-RES cells with AC220 lead to loss of the FLT3 wild-type allele and the duplication of the FLT3-ITD-D835Y allele. Our FLT3-Aurora kinase inhibitor, CCT137690, successfully inhibited growth of FLT3-ITD-D835Y cells in vitro and in vivo, suggesting that dual FLT3-Aurora inhibition may overcome selective FLT3 inhibitor resistance, in part due to inhibition of Aurora kinase, and may benefit patients with FLT3-mutated AML.

The molecular chaperone HSP90 maintains the activity and stability of a diverse set of "client" proteins that play key roles in normal and disease biology. Around 20 HSP90 inhibitors that deplete the oncogenic clientele have entered clinical trials for cancer. However, the full extent of the HSP90-dependent proteome, which encompasses not only clients but also proteins modulated by downstream transcriptional responses, is still incompletely characterized and poorly understood. Earlier large-scale efforts to define the HSP90 proteome have been valuable but are incomplete because of limited technical sensitivity. Here we discuss previous large-scale surveys of proteome perturbations induced by HSP90 inhibitors in light of a significant new study using state-of-the-art SILAC technology combined with more sensitive high-resolution mass spectrometry (MS) that extends the catalog of proteomic changes in inhibitor-treated cancer cells. Among wide-ranging changes, major functional responses include downregulation of protein kinase activity and the DNA damage response alongside upregulation of the protein degradation machinery. Despite this improved proteomic coverage, there was surprisingly little overlap with previous studies. This may be due in part to technical issues but is likely also due to the variability of the HSP90 proteome with the inhibitor conditions used, the cancer cell type and the genetic status of client proteins. We suggest future proteomic studies to address these factors, to help distinguish client protein components from indirect transcriptional components and to address other key questions in fundamental and translational HSP90 research. Such studies should also reveal new biomarkers for patient selection and novel targets for therapeutic intervention.

The discovery and development of small molecule cancer drugs has been revolutionised over the last decade. Most notably, we have moved from a one-size-fits-all approach that emphasized cytotoxic chemotherapy to a personalised medicine strategy that focuses on the discovery and development of molecularly targeted drugs that exploit the particular genetic addictions, dependencies and vulnerabilities of cancer cells. These exploitable characteristics are increasingly being revealed by our expanding understanding of the abnormal biology and genetics of cancer cells, accelerated by cancer genome sequencing and other high-throughput genome-wide campaigns, including functional screens using RNA interference. In this review we provide an overview of contemporary approaches to the discovery of small molecule cancer drugs, highlighting successes, current challenges and future opportunities. We focus in particular on four key steps: Target validation and selection; chemical hit and lead generation; lead optimization to identify a clinical drug candidate; and finally hypothesis-driven, biomarker-led clinical trials. Although all of these steps are critical, we view target validation and selection and the conduct of biology-directed clinical trials as especially important areas upon which to focus to speed progress from gene to drug and to reduce the unacceptably high attrition rate during clinical development. Other challenges include expanding the envelope of druggability for less tractable targets, understanding and overcoming drug resistance, and designing intelligent and effective drug combinations. We discuss not only scientific and technical challenges, but also the assessment and mitigation of risks as well as organizational, cultural and funding problems for cancer drug discovery and development, together with solutions to overcome the 'Valley of Death' between basic research and approved medicines. We envisage a future in which addressing these challenges will enhance our rapid progress towards truly personalised medicine for cancer patients.

Two recent papers published in Nature demonstrate the power of systematic high-throughput pharmacologic profiling of very large, diverse, molecularly-characterized human cancer cell line panels to reveal linkages between genetic profile and targeted-drug sensitivity. Known oncogene addictions are confirmed while surprising complexities and biomarker relationships with clinical potential are revealed.

Evidence that the phosphoinositide 3-kinase (PI3K) pathway is deregulated in ovarian cancer is largely based on the analysis of surgical specimens sampled at diagnosis and may not reflect the biology of advanced ovarian cancer. We aimed to investigate PI3K signaling in cancer cells isolated from patients with advanced ovarian cancer. Ascites samples were analyzed from 88 patients, of whom 61 received further treatment. Cancer cells were immunomagnetically separated from ascites, and the signaling output of the PI3K pathway was studied by quantifying p-AKT, p-p70S6K, and p-GSK3β by ELISA. Relevant oncogenes, such as PIK3CA and AKT, were sequenced by PCR-amplified mass spectroscopy detection methods. In addition, PIK3CA and AKT2 amplifications and PTEN deletions were analyzed by FISH. p-p70S6K levels were significantly higher in cells from 37 of 61 patients who did not respond to subsequent chemotherapy (0.7184 vs. 0.3496; P = 0.0100), and this difference was greater in patients who had not received previous chemotherapy. PIK3CA and AKT mutations were present in 5% and 0% of samples, respectively. Amplification of PIK3CA and AKT2 and deletion of PTEN was seen in 10%, 10%, and 27% of samples, respectively. Mutations of PIK3CA and amplification of PIK3CA/AKT2 or deletion of PTEN did not correlate with levels of p-AKT, p-p70S6K, and p-GSK3β. In patients with advanced ovarian cancer, there is an association between levels of p-p70S6K and response to subsequent chemotherapy. There is no clear evidence that this is driven specifically by PIK3CA or AKT mutations or by amplifications or deletion of PTEN.

Aberrant expression of chromatin-modifying enzymes (CMEs) is associated with a range of human diseases, including cancer. CMEs are now an important target area in drug discovery. Although the role that histone and protein (lysine) methyltransferases (PMTs) play in the regulation of transcription and cell growth is increasingly recognized, few small-molecule inhibitors of this class of enzyme have been reported. Here we describe an assay suitable for primary compound screening for the identification of PMT inhibitors. The assay followed the methylation of histones in the presence of the PMT SET7/9 and the radioactive cofactor S-adenosyl-methionine using scintillating microplates (FlashPlate) and was used to screen approximately 65 000 compounds (% coefficient of variation = 10%; Z' = 0.6). The hits identified from a library of more than 63 000 diverse small molecules included a series of rhodanine compounds with micromolar activity. A screen of the National Cancer Institute Diversity Set (2000 compounds) identified an orsein derivative that inhibited SET7/9 (~20 µM) and showed modest growth inhibition associated with the expected cellular phenotype of reduced histone methylation in a human tumor cell line. The assay represents a useful tool for the identification of inhibitors of PMT activity.

Deregulated phosphatidylinositol 3-kinase pathway signaling through AGC kinases including AKT, p70S6 kinase, PKA, SGK and Rho kinase is a key driver of multiple cancers. The simultaneous inhibition of multiple AGC kinases may increase antitumor activity and minimize clinical resistance compared with a single pathway component.

Over the past decade, whole genome sequencing and other 'omics' technologies have defined pathogenic driver mutations to which tumor cells are addicted. Such addictions, synthetic lethalities and other tumor vulnerabilities have yielded novel targets for a new generation of cancer drugs to treat discrete, genetically defined patient subgroups. This personalized cancer medicine strategy could eventually replace the conventional one-size-fits-all cytotoxic chemotherapy approach. However, the extraordinary intratumor genetic heterogeneity in cancers revealed by deep sequencing explains why de novo and acquired resistance arise with molecularly targeted drugs and cytotoxic chemotherapy, limiting their utility. One solution to the enduring challenge of polygenic cancer drug resistance is rational combinatorial targeted therapy.

Heat shock protein 90 (HSP90) is a molecular chaperone responsible for the conformational maintenance of a number of client proteins that play key roles in cell cycle arrest, DNA damage repair and apoptosis following radiation. HSP90 inhibitors exhibit antitumor activity by modulating the stabilisation and activation of HSP90 client proteins. We sought to evaluate NVP-AUY922, the most potent HSP90 inhibitor yet reported, in preclinical radiosensitization studies.

Acquired resistance to selective FLT3 inhibitors, is an emerging clinical problem in the treatment of FLT3-ITD+ acute myeloid leukaemia (AML). The paucity of valid pre-clinical models has limited investigations to determine the mechanism of acquired therapeutic resistance, thereby limiting the development of effective treatments. We generated selective FLT3 inhibitor-resistant cells by treating the FLT3-ITD+ human AML cell line MOLM-13 in vitro with the FLT3-selective inhibitor MLN518, and validated the resistant phenotype in vivo and in vitro. The resistant cells, MOLM-13-RES, harboured a new D835Y tyrosine kinase domain (TKD) mutation on the FLT3-ITD+ allele. Acquired TKD mutations, including D835Y, have recently been identified in FLT3-ITD+ patients relapsing after treatment with the novel FLT3 inhibitor, AC220. Consistent with this clinical pattern of resistance, MOLM-13-RES cells displayed high relative resistance to AC220 and Sorafenib. Furthermore, treatment of MOLM-13-RES cells with AC220 lead to loss of the FLT3 wild type allele and duplication of the FLT3-ITD-D835Y allele. Our FLT3-Aurora kinase inhibitor, CCT137690, successfully inhibited growth of FLT3-ITD-D835Y cells in vitro and in vivo, suggesting that dual FLT3-Aurora inhibition may overcome selective FLT3 inhibitor resistance, in part due to inhibition of Aurora kinase, and may benefit patients with FLT3-mutated AML.

A potential therapeutic strategy for targeting cancer that has gained much interest is the inhibition of the ATP binding and ATPase activity of the molecular chaperone Hsp90. We have determined the structure of the human Hsp90α N-terminal domain in complex with a series of 5-aryl-4-(5-substituted-2-4-dihydroxyphenyl)-1,2,3-thiadiazoles. The structures provide the molecular details for the activity of these inhibitors. One of these inhibitors, ICPD 34, causes a structural change that affects a mobile loop, which adopts a conformation similar to that seen in complexes with ADP, rather than the conformation generally seen with the pyrazole/isoxazole-resorcinol class of inhibitors. Competitive binding to the Hsp90 N-terminal domain was observed in a biochemical assay, and these compounds showed antiproliferative activity and induced apoptosis in the HCT116 human colon cancer cell line. These inhibitors also caused induction of the heat shock response with the upregulation of Hsp72 and Hsp27 protein expression and the depletion of Hsp90 clients, CRAF, ERBB2 and CDK4, thus confirming that antiproliferative activity was through the inhibition of Hsp90. The presence of increased levels of the cleavage product of PARP indicated apoptosis in response to Hsp90 inhibitors. This work provides a framework for the further optimization of thiadiazole inhibitors of Hsp90. Importantly, we demonstrate that the thiadiazole inhibitors display a more limited core set of interactions relative to the clinical trial candidate NVP-AUY922, and consequently may be less susceptible to resistance derived through mutations in Hsp90.

Optimization of the imidazo[4,5-b]pyridine-based series of Aurora kinase inhibitors led to the identification of 6-chloro-7-(4-(4-chlorobenzyl)piperazin-1-yl)-2-(1,3-dimethyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine (27e), a potent inhibitor of Aurora kinases (Aurora-A K(d) = 7.5 nM, Aurora-B K(d) = 48 nM), FLT3 kinase (K(d) = 6.2 nM), and FLT3 mutants including FLT3-ITD (K(d) = 38 nM) and FLT3(D835Y) (K(d) = 14 nM). FLT3-ITD causes constitutive FLT3 kinase activation and is detected in 20-35% of adults and 15% of children with acute myeloid leukemia (AML), conferring a poor prognosis in both age groups. In an in vivo setting, 27e strongly inhibited the growth of a FLT3-ITD-positive AML human tumor xenograft (MV4-11) following oral administration, with in vivo biomarker modulation and plasma free drug exposures consistent with dual FLT3 and Aurora kinase inhibition. Compound 27e, an orally bioavailable dual FLT3 and Aurora kinase inhibitor, was selected as a preclinical development candidate for the treatment of human malignancies, in particular AML, in adults and children.

Two classes of trisubstituted pyrimidines related to PI-103 1 have been prepared and their inhibitory activities against phosphatidylinositol 3-kinase (PI3K) p110α were determined. From those with direct 6-aryl substitution compound 11a was the most potent inhibitor with an IC₅₀ value of 62 nM, and showed similar activity against other class 1a PI3K isoforms tested, p110β and p110γ. When a linking chain was introduced, as in the second exemplified class, compound 15f inhibited p110α with IC₅₀ 142 nM, and showed greater selectivity towards p110α. Compounds of both classes showed promising inhibition of cellular proliferation in IGROV-1 ovarian cancer cells. Among compounds designed to replace the 3-phenolic motif with structural isosteres, analogues incorporating a 4-indazolyl group possessed enzyme and cellular activities comparable to the parent phenols.

AKT is frequently deregulated in cancer, making it an attractive anticancer drug target. CCT128930 is a novel ATP-competitive AKT inhibitor discovered using fragment- and structure-based approaches. It is a potent, advanced lead pyrrolopyrimidine compound exhibiting selectivity for AKT over PKA, achieved by targeting a single amino acid difference. CCT128930 exhibited marked antiproliferative activity and inhibited the phosphorylation of a range of AKT substrates in multiple tumor cell lines in vitro, consistent with AKT inhibition. CCT128930 caused a G(1) arrest in PTEN-null U87MG human glioblastoma cells, consistent with AKT pathway blockade. Pharmacokinetic studies established that potentially active concentrations of CCT128930 could be achieved in human tumor xenografts. Furthermore, CCT128930 also blocked the phosphorylation of several downstream AKT biomarkers in U87MG tumor xenografts, indicating AKT inhibition in vivo. Antitumor activity was observed with CCT128930 in U87MG and HER2-positive, PIK3CA-mutant BT474 human breast cancer xenografts, consistent with its pharmacokinetic and pharmacodynamic properties. A quantitative immunofluorescence assay to measure the phosphorylation and total protein expression of the AKT substrate PRAS40 in hair follicles is presented. Significant decreases in pThr246 PRAS40 occurred in CCT128930-treated mouse whisker follicles in vivo and human hair follicles treated ex vivo, with minimal changes in total PRAS40. In conclusion, CCT128930 is a novel, selective, and potent AKT inhibitor that blocks AKT activity in vitro and in vivo and induces marked antitumor responses. We have also developed a novel biomarker assay for the inhibition of AKT in human hair follicles, which is currently being used in clinical trials.

A phase I study to define toxicity and recommend a phase II dose of the HSP90 inhibitor alvespimycin (17-DMAG; 17-dimethylaminoethylamino-17-demethoxygeldanamycin). Secondary endpoints included evaluation of pharmacokinetic profile, tumor response, and definition of a biologically effective dose (BED).

Purpose: Preoperative chemotherapy has demonstrated a survival benefit for patients with potentially resectable esophageal cancer; however, currently it is not possible to predict the benefit of this treatment for an individual patient. This prospective study was designed to correlate gene expression profiles with clinical outcome in this setting. Patients and Methods: Eligible patients were deemed to have resectable disease after staging by computed tomography, endoscopic ultrasound, and leperoscopy as indicated and following discussion at the multidisciplinary team meeting. All patients received neoadjuvant platinum and fluoropyrimidine-based chemotherapy; and clinical data were entered prospectively onto a study-specific database. Total RNA was isolated from pretreatment tumor biopsies obtained at baseline endoscopy and analyzed using a cDNA array consisting of 22,000 cDNA clones. Results: Of the patients with adequate follow-up accrued between 2002 and 2005, 35 satisfied the quality control measures for the microarray profiling. Median follow-up was 938 days. Supervised hierarchical clustering of normalized data revealed 165 significantly differentially expressed genes based on overall survival (OS; P < .01) with 2 distinct clusters: a poor outcome group: N = 17 (1 year OS 46.2%) and a good outcome group: N = 18 (1 year OS 100%). Genes identified included those previously associated with esophageal cancer and, interestingly, a group of genes encoding proteins involved in the regulation of the TOLL receptor-signaling pathway. Conclusion: This initial study has highlighted groups of tumors with distinct gene expression profiles based on survival and warrants further validation in a larger cohort. This approach may further our understanding of individual tumor biology and thus facilitate the development of tailored treatment.

Despite the promise of the new generation of molecularly targeted drugs, intrinsic and acquired resistance is proving to be as problematic as with cytotoxic drugs. Two recent papers have identified novel ways by which non-small cell lung cancers can exhibit resistance to EGFR inhibitors and suggest new therapeutic workarounds.

Metastatic renal cell carcinoma (RCC) is a molecularly heterogeneous disease that is intrinsically resistant to chemotherapy and radiotherapy. Although therapies targeted to the molecules vascular endothelial growth factor and mammalian target of rapamycin have shown clinical effectiveness, their effects are variable and short-lived, underscoring the need for improved treatment strategies for RCC. Here, we used quantitative phosphoproteomics and immunohistochemical profiling of 346 RCC specimens and determined that Src kinase signaling is elevated in RCC cells that retain wild-type von Hippel-Lindau (VHL) protein expression. RCC cell lines and xenografts with wild-type VHL exhibited sensitivity to the Src inhibitor dasatinib, in contrast to cell lines that lacked the VHL protein, which were resistant. Forced expression of hypoxia-inducible factor (HIF) in RCC cells with wild-type VHL diminished Src signaling output by repressing transcription of the Src activator protein tyrosine phosphatase 1B (PTP1B), conferring resistance to dasatinib. Our results suggest that a HIF-regulated VHL-PTP1B-Src signaling pathway determines the sensitivity of RCC to Src inhibitors and that stratification of RCC patients with antibody-based profiling may identify patients likely to respond to Src inhibitors in RCC clinical trials.

The phosphoinositide 3-kinases (PI3Ks) constitute an important family of lipid kinase enzymes that control a range of cellular processes through their regulation of a network of signal transduction pathways, and have emerged as important therapeutic targets in the context of cancer, inflammation and cardiovascular diseases. Since the mid-late 1990s, considerable progress has been made in the discovery and development of small molecule ATP-competitive PI3K inhibitors, a number of which have entered early phase human trials over recent years from which key clinical results are now being disclosed. This review summarizes progress made to date, primarily on the discovery and characterization of class I and dual class I/IV subtype inhibitors, together with advances that have been made in translational and clinical research, notably in cancer.

Rational drug discovery and development requires biomarkers to inform on target modulation and treatment efficacy. Many aspects of metabolism are altered in cancer, compared to normal tissues, and are often regulated by oncogene activation. Non-invasive imaging of spatio-temporal effects of molecularly targeted anticancer agents on tumor metabolism has considerable potential in the development and use of personalized molecular medicine approaches to cancer treatment. Here we describe how non-invasive monitoring of metabolism, using primarily magnetic resonance spectroscopy (MRS), can be used to follow treatment with novel molecularly targeted anticancer agents. We discuss how the regulation of metabolic pathways by oncogenic signaling can affect MRS-detectable metabolic signals together with the physiological factors that can influence the measured changes. Finally, the translation of these metabolic measurements from pre-clinical models to patients will be discussed.

The cyclin-dependent kinase (CDK) inhibitor seliciclib (1, CYC202) is in phase II clinical development for the treatment of cancer. Here we describe the synthesis of novel purines with greater solubility, lower metabolic clearance, and enhanced potency versus CDKs. These compounds exhibit novel selectivity profiles versus CDK isoforms. Compound αSβR-21 inhibits CDK2/cyclin E with IC(50)=30 nM, CDK7-cyclin H with IC(50)=1.3 μM, and CDK9-cyclinT with IC(50)=0.11 μM; it (CCT68127) inhibits growth of HCT116 colon cancer cells in vitro with GI(50)=0.7 μM; and shows antitumour activity when dosed p.o. at 50mg/kg to mice bearing HCT116 solid human tumour xenografts.

Lead optimization studies using 7 as the starting point led to a new class of imidazo[4,5-b]pyridine-based inhibitors of Aurora kinases that possessed the 1-benzylpiperazinyl motif at the 7-position, and displayed favorable in vitro properties. Cocrystallization of Aurora-A with 40c (CCT137444) provided a clear understanding into the interactions of this novel class of inhibitors with the Aurora kinases. Subsequent physicochemical property refinement by the incorporation of solubilizing groups led to the identification of 3-((4-(6-bromo-2-(4-(4-methylpiperazin-1-yl)phenyl)-3H-imidazo[4,5-b]pyridin-7-yl)piperazin-1-yl)methyl)-5-methylisoxazole (51, CCT137690) which is a potent inhibitor of Aurora kinases (Aurora-A IC(50) = 0.015 +/- 0.003 muM, Aurora-B IC(50) = 0.025 muM, Aurora-C IC(50) = 0.019 muM). Compound 51 is highly orally bioavailable, and in in vivo efficacy studies it inhibited the growth of SW620 colon carcinoma xenografts following oral administration with no observed toxicities as defined by body weight loss.

The molecular chaperone heat shock protein 90 (Hsp90) is required for the correct folding and stability of a number of client proteins that are important for the growth and maintenance of cancer cells. Heat shock protein 72 (Hsp72), a co-chaperone of Hsp90, is also emerging as an attractive cancer drug target. Both proteins bind and hydrolyze adenosine triphosphate (ATP), and ATPase activity is essential for their function. Inhibition of Hsp90 ATPase activity leads to the degradation of client proteins, resulting in cell growth inhibition and apoptosis. Several small-molecule inhibitors of the ATPase activity of Hsp90 have been described and are currently being evaluated clinically for the treatment of cancer. A number of methods for the measurement of ATPase activity have been previously used, but not all of these are ideally suited to screening cascades in drug discovery projects. The authors have evaluated the use of commercial reagents (Transcreener ADP) for the measurement of ATPase activity of both yeast and human Hsp90 (ATP K(m) approximately 500 microM) and human Hsp72 (ATP K(m) ~1 microM). The low ATPase activity of human Hsp90 and its stimulation by the co-chaperone Aha1 was measured with ease using reduced incubation times, generating robust data (Z' = 0.75). The potency of several small-molecule inhibitors of both Hsp90 and Hsp72 was determined using the Transcreener reagents and compared well to that determined using other assay formats.

The phosphatidylinositide 3-kinase (PI3K) pathway is very commonly activated in a wide range of human cancers and is a major driving force in oncogenesis. One of the class I lipid kinase members of the PI3K family, p110alpha, is probably the most commonly mutated kinase in the human genome. Alongside genetic, molecular biological, and biochemical studies, chemical inhibitors have been extremely helpful tools in understanding the role of PI3K enzymes in signal transduction and downstream physiological and pathological processes, and also in validating PI3Ks as therapeutic targets. Although they have been valuable in the past, the early and still frequently employed inhibitors, wortmannin and LY294002, have significant limitations as chemical tools. Here, we discuss the case history of the discovery and properties of an increasingly used chemical probe, the pan-class I PI3K and mammalian target of rapamycin (mTOR) inhibitor PI-103 (a pyridofuropyrimidine), and its very recent evolution into the thienopyrimidine drug GDC-0941, which exhibits excellent oral anticancer activity in preclinical models and is now undergoing phase I clinical trials in cancer patients. We also illustrate the impact of structural biology on the design of PI3K inhibitors and on the interpretation of their effects. The challenges and outlook for drugging the PI3 kinome are discussed in the more general context of the role of structural biology and chemical biology in innovative drug discovery.

The HSF1-mediated stress response pathway is steadily gaining momentum as a critical source of targets for cancer therapy. Key mediators of this pathway include molecular chaperones such as heat shock protein (HSP) 90. There has been considerable progress in targeting HSP90 and the preclinical efficacy and signs of early clinical activity of HSP90 inhibitors have provided proof-of-concept for targeting this group of proteins. The HSP70 family of molecular chaperones are also key mediators of the HSF-1-stress response pathway and have multiple additional roles in protein folding, trafficking and degradation, as well as regulating apoptosis. Genetic and biochemical studies have supported the discovery of HSP70 inhibitors which have the potential for use as single agents or in combination to enhance the effects of classical chemotherapeutic or molecularly targeted agents including HSP90 inhibitors. Here we provide a perspective on the progress made so far in designing agents which target the HSP70 family.

The dismal prognosis of glioblastoma (GB) indicates the urgent need for new therapies for these tumors. Heat shock protein 90 (HSP90) inhibitors induce the proteasome-mediated degradation of many oncogenic client proteins involved in all of the hallmark characteristics of cancer. Here, we explored the mechanistic potential of the potent synthetic diarylisoxazole amide resorcinol HSP90 inhibitor, NVP-AUY922, in adult and pediatric GB. In vitro antiproliferative potency (nanomolar range) was seen in both adult and pediatric human GB cell lines with different molecular pathologies. A cytostatic effect was observed in all GB lines; more apoptosis was observed at lower concentrations in the SF188 pediatric GB line and at 144 hours in the slower growing KNS42 pediatric GB line, as compared with the adult GB lines U87MG and SF268. In vitro combination studies with inhibitors of phosphoinositide 3-kinase/mammalian target of rapamycin (PI-103) or mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (PD-0325901) supported the hypothesis that sustained inhibition of ERK up to 72 hours and at least temporary inhibition of AKT were necessary to induce apoptosis in GB lines. In athymic mice bearing established s.c U87MG GB xenografts, NVP-AUY922 (50 mg/kg i.p x 3 days) caused the inhibition of ERK1/2 and AKT phosphorylation and induced apoptosis, whereas 17-AAG used at maximum tolerated dose was less effective. NVP-AUY922 antitumor activity with objective tumor regression resulted from antiproliferative, proapoptotic, and antiangiogenic effects, the latter shown by decreased microvessel density and HIF1alpha levels. Our results have established a mechanistic proof of concept for the potential of novel synthetic HSP90 inhibitors in adult and pediatric GB, alone or in combination with phosphoinositide 3-kinase/mammalian target of rapamycin and mitogen-activated protein/ERK kinase inhibitors.

Vps34 is the primordial member of the PI3 kinase family involved in vesicular trafficking, nutrient signaling, and autophagy. A report in Science unveils the Vps34 structure, providing new insights into the catalytic mechanism, explaining why Vsp34 is so difficult to inhibit, and facilitating design of chemical tools and potential drugs.

Animal experiments remain essential to understand the fundamental mechanisms underpinning malignancy and to discover improved methods to prevent, diagnose and treat cancer. Excellent standards of animal care are fully consistent with the conduct of high quality cancer research. Here we provide updated guidelines on the welfare and use of animals in cancer research. All experiments should incorporate the 3Rs: replacement, reduction and refinement. Focusing on animal welfare, we present recommendations on all aspects of cancer research, including: study design, statistics and pilot studies; choice of tumour models (e.g., genetically engineered, orthotopic and metastatic); therapy (including drugs and radiation); imaging (covering techniques, anaesthesia and restraint); humane endpoints (including tumour burden and site); and publication of best practice.

The phosphoinositide 3-kinase (PI3K) pathway is a major target for cancer drug development. PI-103 is an isoform-selective class I PI3K and mammalian target of rapamycin inhibitor. The aims of this work were as follows: first, to use magnetic resonance spectroscopy (MRS) to identify and develop a robust pharmacodynamic (PD) biomarker for target inhibition and potentially tumor response following PI3K inhibition; second, to evaluate mechanisms underlying the MRS-detected changes. Treatment of human PTEN null PC3 prostate and PIK3CA mutant HCT116 colon carcinoma cells with PI-103 resulted in a concentration- and time-dependent decrease in phosphocholine (PC) and total choline (tCho) levels (P < 0.05) detected by phosphorus ((31)P)- and proton ((1)H)-MRS. In contrast, the cytotoxic microtubule inhibitor docetaxel increased glycerophosphocholine and tCho levels in PC3 cells. PI-103-induced MRS changes were associated with alterations in the protein expression levels of regulatory enzymes involved in lipid metabolism, including choline kinase alpha (ChoK(alpha)), fatty acid synthase (FAS), and phosphorylated ATP-citrate lyase (pACL). However, a strong correlation (r(2) = 0.9, P = 0.009) was found only between PC concentrations and ChoK(alpha) expression but not with FAS or pACL. This study identified inhibition of ChoK(alpha) as a major cause of the observed change in PC levels following PI-103 treatment. We also showed the capacity of (1)H-MRS, a clinically well-established technique with higher sensitivity and wider applicability compared with (31)P-MRS, to assess response to PI-103. Our results show that monitoring the effects of PI3K inhibitors by MRS may provide a noninvasive PD biomarker for PI3K inhibition and potentially of tumor response during early-stage clinical trials with PI3K inhibitors.

Chemical probes for interrogating biological processes are of considerable current interest. Cell permeable small molecule tools have a major role in facilitating the functional annotation of the human genome, understanding both physiological and pathological processes, and validating new molecular targets. To be valuable, chemical tools must satisfy necessary criteria and recent publications have suggested objective guidelines for what makes a useful chemical probe. Although recognizing that such guidelines may be valuable, we caution against overly restrictive rules that may stifle innovation in favor of a "fit-for-purpose" approach. Reviewing the literature and providing examples from the cancer field, we recommend a series of "fitness factors" to be considered when assessing chemical probes. We hope this will encourage innovative chemical biology research while minimizing the generation of poor quality and misleading biological data, thus increasing understanding of the particular biological area, to the benefit of basic research and drug discovery.

Anticancer drug development remains slow, costly and inefficient. One way of addressing this might be the use of predictive biomarkers to select patients for Phase I/II trials. Such biomarkers, which predict response to molecular-targeted agents, have the potential to enrich these trials with patients more likely to benefit. Doing so could maximize the efficiency of anticancer drug development by facilitating earlier clinical qualification of predictive biomarkers and generating valuable information on cancer biology. In this review, we suggest a new model of early clinical trial design, which incorporates patient selection through predictive molecular biomarkers for selected targeted agents.

A series of benzo-macrolactones has been prepared by chemical synthesis, and evaluated as inhibitors of heat shock protein 90 (Hsp90), an emerging attractive target for novel cancer therapeutic agents. A new synthesis of these resorcylic acid macrolactone analogues of the natural product radicicol is described in which the key steps are the acylation and ring opening of a homophthalic anhydride to give an isocoumarin, followed by a ring-closing metathesis to form the macrocycle. The methodology has been extended to a novel series of macrolactones incorporating a 1,2,3-triazole ring.

The development of novel molecularly targeted cancer therapeutics remains slow and expensive with many late-stage failures. There is an urgent need to accelerate this process by improving early clinical anticancer drug evaluation through modern and rational trial designs that incorporate predictive, pharmacokinetic, pharmacodynamic, pharmacogenomic and intermediate end-point biomarkers. In this article, we discuss current approaches and propose strategies that will potentially maximize benefit to patients and expedite the regulatory approvals of new anticancer drugs.

The serine/threonine kinase AKT plays a pivotal role in signal transduction events involved in malignant transformation and chemoresistance and is an attractive target for the development of cancer therapeutics. Fragment-based lead discovery, combined with structure-based drug design, has recently identified AT7867 as a novel and potent inhibitor of both AKT and the downstream kinase p70 S6 kinase (p70S6K) and also of protein kinase A. This ATP-competitive small molecule potently inhibits both AKT and p70S6K activity at the cellular level, as measured by inhibition of GSK3 beta and S6 ribosomal protein phosphorylation, and also causes growth inhibition in a range of human cancer cell lines as a single agent. Induction of apoptosis was detected by multiple methods in tumor cells following AT7867 treatment. Administration of AT7867 (90 mg/kg p.o. or 20 mg/kg i.p.) to athymic mice implanted with the PTEN-deficient U87MG human glioblastoma xenograft model caused inhibition of phosphorylation of downstream substrates of both AKT and p70S6K and induction of apoptosis, confirming the observations made in vitro. These doses of AT7867 also resulted in inhibition of human tumor growth in PTEN-deficient xenograft models. These data suggest that the novel strategy of AKT and p70S6K blockade may have therapeutic value and supports further evaluation of AT7867 as a single-agent anticancer strategy. Mol Cancer Ther; 9(5); 1100-10. (C) 2010 AACR.

The Wnt signaling pathway is frequently deregulated in cancer due to mutations in genes encoding APC, beta-catenin, and axin. To identify small-molecule inhibitors of Wnt signaling as potential therapeutics, a diverse chemical library was screened using a transcription factor reporter cell line in which the activity of the pathway was induced at the level of Disheveled protein. A series of deconvolution studies was used to focus on three compound series that selectively killed cancer cell lines with constitutive Wnt signaling. Activities of the compounds included the ability to induce degradation of beta-catenin that had been stabilized by a glycogen synthase kinase-3 (GSK-3) inhibitor. This screen illustrates a practical approach to identify small-molecule inhibitors of Wnt signaling that can seed the development of agents suitable to treat patients with Wnt-dependent tumors. Cancer Res; 70(14); 5963-73. (C) 2010 AACR.

A series of resorcylic acid macrolactones, analogues of the natural product radicicol has been prepared by chemical synthesis, and evaluated as inhibitors of heat shock protein 90 (Hsp90), an emerging attractive target for novel cancer therapeutic agents. The synthesis involves acylation of an ortho-toluic acid dianion, esterification, followed by a ring-closing metathesis to form the macrocycle. Subsequent manipulation of the protected hydroxymethyl side chain allows access to a range of new analogues following de-protection of the two phenolic groups. Co-crystallization of one of the new macrolactones with the N-terminal domain of yeast Hsp90 confirms that it binds in a similar way to the natural product radicicol and to our previous synthetic analogues, but that the introduction of the additional hydroxymethyl substituent appears to result in an unexpected change in conformation of the macrocyclic ring. As a result of this conformational change, the compounds bound less favorably to Hsp90.

Molecular chaperone heat shock protein 90 (Hsp90) inhibitors are promising targeted cancer therapeutic drugs, with the advantage that they deplete multiple oncogenic client proteins and modulate all the classical hallmarks of cancer. They are now in clinical trial and show potential for activity in melanoma and other malignancies. Here we explore the metabolic response to Hsp90 inhibition in human melanoma cells using magnetic resonance spectroscopy. We show that, concomitant with growth inhibition and re-differentiation, Hsp90 inhibition in human melanoma cells is associated with increased glycerophosphocholine content. This was seen with both the clinical geldanamycin-based Hsp90 drug 17-AAG and the structurally dissimilar Hsp90 inhibitor CCT018159. The effect was noted in both BRAF mutant SKMEL28 and BRAF wildtype CHL-1 melanoma cells. Elevated content of the -CH2+CH3 fatty acyl chains and cytoplasmic mobile lipid droplets was also observed in 17-AAG-treated SKMEL28 cells. Importantly, the phospholipase A2 inhibitor bromoenol lactone prevented the rise in glycerophosphocholine seen with 17-AAG, suggesting a role for phospholipase A2 activation in the Hsp90 inhibitor-induced metabolic response. Our findings provide a basis for using metabolic changes as non-invasive indicators of Hsp90 inhibition and potentially as biomarkers of anticancer activity with Hsp90 drugs in malignant melanoma and possibly in other cancers.

Sensitivity to temozolomide is restricted to a subset of glioblastoma patients, with the major determinant of resistance being a lack of promoter methylation of the gene encoding the repair protein DNA methyltransferase MGMT, although other mechanisms are thought to be active. There are, however, limited preclinical data in model systems derived from pediatric glioma patients. We screened a series of cell lines for temozolomide efficacy in vitro, and investigated the differential mechanisms of resistance involved. In the majority of cell lines, a lack of MGMT promoter methylation and subsequent protein overexpression were linked to temozolomide resistance. An exception was the pediatric glioblastoma line KNS42. Expression profiling data revealed a coordinated upregulation of HOX gene expression in resistant lines, especially KNS42, which was reversed by phosphoinositide 3-kinase pathway inhibition. High levels of HOXA9/HOXA10 gene expression were associated with a shorter survival in pediatric high-grade glioma patient samples. Combination treatment in vitro of pathway inhibition and temozolomide resulted in a highly synergistic interaction in KNS42 cells. The resistance gene signature further included contiguous genes within the 12q13-q14 amplicon, including the Akt enhancer PIKE, significantly overexpressed in the KNS42 line. These cells were also highly enriched for CD133 and other stem cell markers. We have thus shown an in vitro link between phosphoinositide 3-kinase-mediated HOXA9/HOXA10 expression, and a drug-resistant, progenitor cell phenotype in MGMT-independent pediatric glioblastoma. Cancer Res; 70 (22); 9243-52. (C) 2010 AACR.

Nek2 is a serine/threonine protein kinase that localizes to the centrosome and is implicated in mitotic regulation. Overexpression of Nek2 induces premature centrosome separation and nuclear defects indicative of mitotic errors, whereas depletion of Nek2 interferes with cell growth. As Nek2 expression is upregulated in a range of cancer cell lines and primary human tumors, inhibitors of Nek2 may have therapeutic value in cancer treatment. The authors used a radiometric proximity assay in a high-throughput screen to identify small-molecule inhibitors of Nek2 kinase activity. The assay was based on the measurement of the radiolabeled phosphorylated product of the kinase reaction brought into contact with the surface of wells of solid scintillant-coated microplates. Seventy nonaggregating hits were identified from approximately 73,000 compounds screened and included a number of toxoflavins and a series of viridin/wortmannin-like compounds. The viridin-like compounds were >70-fold selective for Nek2 over Nek6 and Nek7 and inhibited the growth of human tumor cell lines at concentrations consistent with their biochemical potencies. An automated mechanism-based microscopy assay in which centrosomes were visualized using pericentrin antibodies confirmed that 2 of the viridin inhibitors reduced centrosome separation in a human tumor cell line. The data presented show that pharmacological inhibition of Nek2 kinase results in the expected phenotype of disruption to centrosome function associated with growth inhibition and further supports Nek2 as a target for cancer drug discovery. (Journal of Biomolecular Screening 2010:918-927)

Although paediatric high grade gliomas resemble their adult counterparts in many ways, there appear to be distinct clinical and biological differences. One important factor hampering the development of new targeted therapies is the relative lack of cell lines derived from childhood glioma patients, as it is unclear whether the well-established adult lines commonly used are representative of the underlying molecular genetics of childhood tumours. We have carried out a detailed molecular and phenotypic characterisation of a series of paediatric high grade glioma cell lines in comparison to routinely used adult lines.

The epidermal growth factor receptor (EGFR) is amplified and overexpressed in adult glioblastoma, with response to targeted inhibition dependent on the underlying biology of the disease. EGFR has thus far been considered to play a less important role in pediatric glioma, although extensive data are lacking. We have sought to clarify the role of EGFR in pediatric high-grade glioma (HGG).

The cochaperone CDC37 promotes the association of HSP90 with the protein kinase subset of client proteins to maintain their stability and signalling functions. HSP90 inhibitors induce depletion of clients, which include several oncogenic kinases. We hypothesized that the targeting of CDC37 using siRNAs would compromise the maturation of these clients and increase the sensitivity of cancer cells to HSP90 inhibitors. Here, we show that silencing of CDC37 in human colon cancer cells diminished the association of kinase clients with HSP90 and reduced levels of the clients ERBB2, CRAF, CDK4 and CDK6, as well as phosphorylated AKT. CDC37 silencing promoted the proteasome-mediated degradation of kinase clients, suggesting a degradation pathway independent from HSP90 binding. Decreased cell signalling through kinase clients was also demonstrated by reduced phosphorylation of downstream substrates and colon cancer cell proliferation was subsequently reduced by the inhibition of the G1/S-phase transition. Furthermore, combining CDC37 silencing with the HSP90 inhibitor 17-AAG induced more extensive and sustained depletion of kinase clients and potentiated cell cycle arrest and apoptosis. These results support an essential role for CDC37 in concert with HSP90 in maintaining oncogenic protein kinase clients and endorse the therapeutic potential of targeting CDC37 in cancer.

High-throughput screening led to the identification of isothiazolones 1 and 2 as inhibitors of histone acetyltransferase (HAT) with IC50s of 3 microM and 5 microM, respectively. Analogues of these hit compounds with variations of the N-phenyl group, and with variety of substituents at C-4, C-5 of the thiazolone ring, were prepared and assayed for inhibition of the HAT enzyme PCAF. Potency is modestly favoured when the N-aryl group is electron deficient (4-pyridyl derivative 10 has IC(50)=1.5 microM); alkyl substitution at C-4 has little effect, whilst similar substitution at C-5 causes a significant drop in potency. The ring-fused compound 38 has activity (IC(50)=6.1 microM) to encourage further exploration of this bicyclic structure. The foregoing SAR is consistent with an inhibitory mechanism involving cleavage of the S-N bond of the isothiazolone ring by a catalytically important thiol residue.

Gliomas are primary brain tumors with poor prognosis that exhibit frequent abnormalities in phosphatidylinositol 3-kinase (PI3 kinase) signaling. We investigated the molecular mechanism of action of the isoform-selective class I PI3 kinase and mTOR inhibitor PI-103 in human glioma cells. The potent inhibitory effects of PI-103 on the PI3 kinase pathway were quantified. PI-103 and the mTOR inhibitor rapamycin both inhibited ribosomal protein S6 phosphorylation but there were clear differences in the response of upstream components of the PI3 kinase pathway, such as phosphorylation of Thr(308)-AKT, that were inhibited by PI-103 but not rapamycin. Gene expression profiling identified altered expression of genes encoding regulators of the cell cycle and cholesterol metabolism, and genes modulated by insulin or IGF1 signaling, rapamycin treatment or nutrient starvation. PI-103 decreased expression of positive regulators of G(1)/S phase progression and increased expression of the negative cell cycle regulator p27(kip1). A reversible PI-103-mediated G(1) cell cycle arrest occurred without significant apoptosis, consistent with the altered gene expression detected. PI-103 induced vacuolation and processing of LC-3i to LC-3ii, which are features of an autophagic response. In contrast to PI-103, LY294002 and PI-387 induced apoptosis, indicative of likely off-target effects. PI-103 interacted synergistically or additively with cytotoxic agents used in the treatment of glioma, namely vincristine, BCNU and temozolomide. Compared to individual treatments, the combination of PI-103 with temozolomide significantly improved the response of U87MG human glioma xenografts. Our results support the therapeutic potential for PI3 kinase inhibitors with a PI-103-like profile as therapeutic agents for the treatment of glioma.

Interest in HSP90 inhibitors has grown rapidly in the last decade. The heightened dependence of malignant cells on molecular chaperones to maintain multiple oncogenic signalling pathways gives HSP90 broad anticancer appeal. New HSP90-directed agents are continually emerging, several of which are under clinical evaluation. In parallel, dissection of the functional mechanism of the chaperone system has emphasised the importance of cochaperones that regulate HSP90. As we begin to fully elucidate the roles of these HSP90 accessory proteins, it is becoming increasingly clear that they too have potential as additional routes to disrupt chaperone activity. CDC37, a predominantly kinase client-associated cochaperone that promotes malignant transformation, has particular promise. Recently, we demonstrated that, similar to HSP90 inhibitors, siRNA-mediated CDC37 silencing caused the proteasomal degradation of kinase client proteins and inhibited the proliferation of cancer cells. Importantly, depleting CDC37 does not induce the unwanted, antiapoptotic heat shock response that is characteristic of pharmacologic HSP90 inhibition. Furthermore, CDC37 silencing sensitises cancer cells to HSP90 inhibitors by potentiating kinase client depletion and the induction of apoptosis, suggesting that simultaneously modulating HSP90 and CDC37 could be beneficial. Here we discuss the therapeutic possibilities of targeting CDC37 for cancer treatment in light of recent significant findings.

Dramatic responses to epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitors may be seen in non-small cell lung cancers (NSCLCs) with a sensitizing mutation of the EGFR TK domain. It is not known how to predict response in patients with squamous cell carcinoma of the head and neck (SCCHN), where EGFR TK mutations are less frequent and where response rates in unselected patients are disappointing. We have characterized the intrinsic sensitivity of a panel of 18 SCCHN cell lines to gefitinib, an EGFR TK inhibitor, and have investigated correlations between putative markers of response and intrinsic sensitivity. Induction of G1 arrest was only seen in cell lines with GI(50) < 1 microM. Expression of EGFR, by three techniques, correlated with sensitivity to gefitinib. ERB-B2 expression appeared to influence sensitivity to gefitinib but ERB-B3 expression did not. While EGFR tyrosine kinase mutations were not detected, EGFR gene amplification was confirmed by fluorescence in situ hybridization in the most sensitive cell line. The number of cytosine adenine dinucleotide repeats in intron 1 of the EGFR gene did not correlate with sensitivity. E-cadherin expression was detected in cell lines with a range of sensitivities, whereas amphiregulin was secreted predominantly by sensitive cell lines. MET expression was an independent predictor of sensitivity to gefitinib, although neither expression nor phosphorylation of insulin-like growth factor 1 receptor correlated with intrinsic resistance. Breast receptor kinase (BRK) was more highly expressed in the sensitive cell lines, but siRNA knockdown of neither BRK nor MET affected sensitivity. Our data suggest that overexpression of EGFR and multiple related cell surface receptors may be associated with sensitivity to gefitinib and that differences between our data and the literature highlight that biomarkers of response are tumour type- and cell line-dependent.

Heat shock protein 90 (HSP90) inhibitors, such as 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin), which is currently in phase II/phase III clinical trials, are promising new anticancer agents. Here, we explored acquired resistance to HSP90 inhibitors in glioblastoma (GB), a primary brain tumor with poor prognosis. GB cells were exposed continuously to increased 17-AAG concentrations. Four 17-AAG-resistant GB cell lines were generated. High-resistance levels with resistance indices (RI = resistant line IC(50)/parental line IC(50)) of 20 to 137 were obtained rapidly (2-8 weeks). After cessation of 17-AAG exposure, RI decreased and then stabilized. Cross-resistance was found with other ansamycin benzoquinones but not with the structurally unrelated HSP90 inhibitors, radicicol, the purine BIIB021, and the resorcinylic pyrazole/isoxazole amide compounds VER-49009, VER-50589, and NVP-AUY922. An inverse correlation between NAD(P)H/quinone oxidoreductase 1 (NQO1) expression/activity and 17-AAG IC(50) was observed in the resistant lines. The NQO1 inhibitor ES936 abrogated the differential effects of 17-AAG sensitivity between the parental and resistant lines. NQO1 mRNA levels and NQO1 DNA polymorphism analysis indicated different underlying mechanisms: reduced expression and selection of the inactive NQO1*2 polymorphism. Decreased NQO1 expression was also observed in a melanoma line with acquired resistance to 17-AAG. No resistance was generated with VER-50589 and NVP-AUY922. In conclusion, low NQO1 activity is a likely mechanism of acquired resistance to 17-AAG in GB, melanoma, and, possibly, other tumor types. Such resistance can be overcome with novel HSP90 inhibitors.

Phospholipase C-gamma (PLC-gamma) has been identified as a possible biological target for anticancer drug therapy but suitable inhibitors are lacking. Therefore, in order to identify active compounds (hits) virtual high throughput screening was performed. The crystal structure of the PLC-delta isoform was used as a model docking scaffold since no crystallographic data are available on its gamma counterpart. A pilot screen was performed using approximately 9.2x10(4) compounds, where the robustness of the methodology was tested. This was followed by the main screening effort where approximately 4.4x10(5) compounds were used. In both cases, plausible compounds were identified (virtual hits) and a selection of these was experimentally tested. The most potent compounds were in the single digit micro-molar range as determined from the biochemical (Flashplate) assay. This translated into approximately 15 microM in a functional assay in cells. About 30% of the virtual hits showed activity against PLC-gamma (IC(50)<50 microM).

Structure-based approaches now impact across the whole continuum of drug discovery, from new target selection through the identification of hits to the optimization of lead compounds. Optimal application of structure-based design involves close integration with other discovery technologies, including fragment-based and virtual screening. Here, we illustrate the use of structural information and of structure-based drug design approaches in the discovery of small-molecule inhibitors for cancer drug targets and provide an outlook on the exploitation of structural information in future cancer drug discovery. Examples include high profile protein kinase targets and structurally related PI3 kinases, histone deacetylases, poly(ADP-ribose)polymerase and the molecular chaperone HSP90. Structure-based design approaches have also been successfully applied to the protein-protein interaction targets p53-MDM2 and the Bcl-2 family.

A dose-escalation, phase I study evaluated the safety, pharmacokinetics, pharmacogenomics, and efficacy of ES-285, a novel agent isolated from a marine mollusc, in adult cancer patients. Patients received a 24-hour i.v. infusion of ES-285 once every 3 weeks until disease progression or unacceptable toxicity. The starting dose was 4 mg/m(2). Dose escalation in cohorts of at least three patients proceeded according to the worst toxicity observed in the previous cohort. Twenty-eight patients were treated with 72 courses of ES-285 across eight dose levels. No dose-limiting toxicities were seen between 4 and 128 mg/m(2). Two of four patients treated at 256 mg/m(2) had dose-limiting reversible grade 3 transaminitis; one patient at 256 mg/m(2) also had transient grade 3 central neurotoxicity. One of three patients subsequently treated at 200 mg/m(2) died following drug-related central neurotoxicity. Other toxicities included phlebitis, nausea, fatigue, and fever. Pharmacokinetic studies indicated dose proportionality with high volume of distribution (median V(ss) at 256 mg/m(2) was 2,389 liters; range, 1,615-4,051 liters) and long elimination half life (median t(1/2) at 256 mg/m(2) was 28 h; range, 21-32 h). The three patients with dose-limiting toxicity had the highest drug exposure. Pharmacogenomic studies of paired surrogate tissue samples identified changes in gene expression following treatment that correlated with increasing dose. Disease stabilization for 6 to 18 weeks was recorded in nine patients. Using this schedule, 128 mg/m(2) was considered safe and feasible. At this dose, pharmacologically relevant concentrations of the drug were safely achieved with pharmacogenomic studies indicating changes in the expression of genes of potential mechanistic relevance.

The phosphatidylinositide 3-kinase pathway is frequently deregulated in human cancers and inhibitors offer considerable therapeutic potential. We previously described the promising tricyclic pyridofuropyrimidine lead and chemical tool compound PI-103. We now report the properties of the pharmaceutically optimized bicyclic thienopyrimidine derivatives PI-540 and PI-620 and the resulting clinical development candidate GDC-0941. All four compounds inhibited phosphatidylinositide 3-kinase p110alpha with IC(50) < or = 10 nmol/L. Despite some differences in isoform selectivity, these agents exhibited similar in vitro antiproliferative properties to PI-103 in a panel of human cancer cell lines, with submicromolar potency in PTEN-negative U87MG human glioblastoma cells and comparable phosphatidylinositide 3-kinase pathway modulation. PI-540 and PI-620 exhibited improvements in solubility and metabolism with high tissue distribution in mice. Both compounds gave improved antitumor efficacy over PI-103, following i.p. dosing in U87MG glioblastoma tumor xenografts in athymic mice, with treated/control values of 34% (66% inhibition) and 27% (73% inhibition) for PI-540 (50 mg/kg b.i.d.) and PI-620 (25 mg/kg b.i.d.), respectively. GDC-0941 showed comparable in vitro antitumor activity to PI-103, PI-540, and PI-620 and exhibited 78% oral bioavailability in mice, with tumor exposure above 50% antiproliferative concentrations for >8 hours following 150 mg/kg p.o. and sustained phosphatidylinositide 3-kinase pathway inhibition. These properties led to excellent dose-dependent oral antitumor activity, with daily p.o. dosing at 150 mg/kg achieving 98% and 80% growth inhibition of U87MG glioblastoma and IGROV-1 ovarian cancer xenografts, respectively. Together, these data support the development of GDC-0941 as a potent, orally bioavailable inhibitor of phosphatidylinositide 3-kinase. GDC-0941 has recently entered phase I clinical trials.

SR4554 is a fluorine-containing 2-nitroimidazole, designed as a hypoxia marker detectable with 19F magnetic resonance spectroscopy (MRS). In an initial phase I study of SR4554, nausea/vomiting was found to be dose-limiting, and 1400 mg m(-2) was established as MTD. Preliminary MRS studies demonstrated some evidence of 19F retention in tumour. In this study we investigated higher doses of SR4554 and intratumoral localisation of the 19F MRS signal.

HSP70 family members are highly conserved proteins that function as molecular chaperones. Their principle role is to aid protein folding and promote the correct cellular localizations of their respective substrates. The function of HSP70 isoforms can be exhibited independently or with the HSP90 chaperone system in which HSP70 is important for substrate recruitment. In addition to their chaperone role, HSP70 isoforms promote cell survival by inhibiting apoptosis at multiple points within both the intrinsic and extrinsic cell death pathways. Consistent with this cytoprotective function, increased expression of HSP70 isoforms is commonly associated with the malignant phenotype. We recently reported that dual silencing of the major constitutive (HSC70) and inducible (HSP72) isoforms of HSP70 in cancer cells could phenocopy the effects of a pharmacologic HSP90 inhibitor to induce proteasome-dependent degradation of HSP90 client proteins CRAF, CDK4 and ERBB2. This was accompanied by a G(1) cell cycle arrest and extensive apoptosis which was not seen in non-tumorigenic human cell lines. Here we discuss the possible implications of our research for the development of HSP70 family modulators which offer not only the possibility of inhibiting HSP70 activity but also the simultaneous inhibition of HSP90, resulting in extensive tumor-specific apoptosis.

Compared with conventional chemotherapy, rationally designed molecularly targeted agents may be more likely to have antitumor activity in selected tumor subgroups driven by the oncogenic signals targeted by these compounds and a different side-effect profile. The use of biomarkers in the early clinical trials of these new anticancer agents has the potential to increase study participants' benefit from early clinical trials, accelerate the drug development process, maximize the ability to generate important biological information about human cancer, and decrease the risk of late and costly drug attrition.

Heat shock protein 90 (Hsp90) is a promising cancer drug target, as multiple oncogenic proteins are destabilized simultaneously when it loses its activity in tumor cells. Highly selective Hsp90 inhibitors, including the natural antibiotics geldanamycin (GdA) and radicicol (RAD), inactivate this essential molecular chaperone by occupying its nucleotide binding site. Often cancer drug therapy is compromised by the development of resistance, but a resistance to these Hsp90 inhibitors should not arise readily by mutation of those amino acids within Hsp90 that facilitate inhibitor binding, as these are required for the essential ATP binding/ATPase steps of the chaperone cycle and are tightly conserved. Despite this, the Hsp90 of a RAD-producing fungus is shown to possess an unusually low binding affinity for RAD but not GdA. Within its nucleotide binding site a normally conserved leucine is replaced by isoleucine, though the chaperone ATPase activity is not severely affected. Inserted into the Hsp90 of yeast, this conservative leucine to isoleucine substitution recreated this lowered affinity for RAD in vitro. It also generated a substantially enhanced resistance to RAD in vivo. Co-crystal structures reveal that the change to isoleucine is associated with a localized increase in the hydration of an Hsp90-bound RAD but not GdA. To the best of our knowledge, this is the first demonstration that it is possible for Hsp90 inhibitor resistance to arise by subtle alteration to the structure of Hsp90 itself.

Inhibitors of the Hsp90 molecular chaperone are showing considerable promise as potential molecular therapeutic agents for the treatment of cancer. Here we describe novel 2-aminothieno[2,3-d]pyrimidine ATP competitive Hsp90 inhibitors, which were designed by combining structural elements of distinct low affinity hits generated from fragment-based and in silico screening exercises in concert with structural information from X-ray protein crystallography. Examples from this series have high affinity (IC50 = 50-100 nM) for Hsp90 as measured in a fluorescence polarization (FP) competitive binding assay and are active in human cancer cell lines where they inhibit cell proliferation and exhibit a characteristic profile of depletion of oncogenic proteins and concomitant elevation of Hsp72. Several examples (34a, 34d and 34i) caused tumor growth regression at well tolerated doses when administered orally in a human BT474 human breast cancer xenograft model.

Early clinical trials represent a crucial bridge between preclinical drug discovery and the especially resource-intense randomized phase III trial-the definitive regulatory hurdle for drug approval. High attrition rates and rising costs, when coupled with the extraordinary opportunities opened up by cancer genomics and the promise of personalized medicine call for new approaches in the conduct and design of phase I/II trials. The key challenge lies in increasing the odds for successful and efficient transition of a compound through the drug development pipeline. The incorporation of scientifically and analytically validated biomarkers into rationally designed hypothesis-testing clinical trials offers a promising way forward to achieving this objective. In this article, we provide an overview of biomarkers in early clinical trials, including examples where they have been particularly successful, and the caveats and pitfalls associated with indiscriminate application. We describe the use of pharmacodynamic end points to demonstrate the proof of modulation of target, pathway, and biologic effect, as well as predictive biomarkers for patient selection and trial enrichment. Establishing the pharmacologic audit trail provides a means to assess and manage risk in a drug development program and thus increases the rationality of the decision-making process. Accurate preclinical models are important for pharmacokinetic-pharmacodynamic-efficacy modeling and biomarker validation. The degree of scientific and analytical validation should ensure that biomarkers are fit-for purpose, according to the stage of development and the impact on the trial; specifically they are either exploratory or used to make decisions within the trial. To be maximally useful at an early stage, these must be in place before the commencement of phase I trials. Validation and qualification of biomarkers then continues through clinical development. We highlight the impact of modern technology platforms, such as genomics, proteomics, circulating tumor cells, and minimally invasive functional and molecular imaging, with respect to their potential role in improving the success rate and speed of drug development and in interrogating the consequences of therapeutic intervention and providing a unique insight into human disease biology. With these technologies already having an impact in the clinic today, we predict that further future advances will come from the application of network analysis to clinical trials, leading to individualized systems-based medicine for cancer.

To study the interactions of the heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) and carboplatin in vitro and in vivo.

AHA1 (activator of HSP90 ATPase) is a cochaperone of the ATP-dependent molecular chaperone, HSP90, which is involved in the maturation, stabilization/degradation, and function of oncogenic proteins. HSP90 operates in a multimeric complex driven by the binding and hydrolysis of ATP. Treatment of cells with the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) results in the degradation of client proteins via the ubiquitin-proteasome pathway. As AHA1 increases the ATPase activity of HSP90, we hypothesized that modulation of AHA1 expression could influence the activity of client proteins and/or the cellular response to 17-AAG. We show that the basal expression of AHA1 is different across a panel of human cancer cell lines, and that treatment with 17-AAG resulted in sustained AHA1 up-regulation. Increasing the expression of AHA1 did not affect the sensitivity to 17-AAG, but did increase C-RAF activity and the levels of phosphorylated MEK1/2 and ERK1/2 without affecting total levels of these proteins or of client proteins C-RAF, ERBB2, or CDK4. Conversely, small interfering RNA-selective knockdown of >80% of AHA1 expression decreased C-RAF activity and reduced the levels of MEK1/2 and ERK1/2 phosphorylation. Moreover, the AHA1 knockdown resulted in a significant (P < 0.05) increase in sensitivity to 17-AAG, due in part to a 2- to 3-fold increase in apoptosis. These results show that the reduction of AHA1 levels could decrease the phosphorylation of key signal transduction proteins, and for the first time, separate the activation and stabilization functions of HSP90. Furthermore, AHA1 knockdown could sensitize cancer cells to 17-AAG. We conclude that modulation of AHA1 might be a potential therapeutic strategy to increase sensitivity to HSP90 inhibitors.

The molecular chaperone Hsp90 (90 kDa heat-shock protein) is a remarkably versatile protein involved in the stress response and in normal homoeostatic control mechanisms. It interacts with 'client proteins', including protein kinases, transcription factors and others, and either facilitates their stabilization and activation or directs them for proteasomal degradation. By this means, Hsp90 displays a multifaceted ability to influence signal transduction, chromatin remodelling and epigenetic regulation, development and morphological evolution. Hsp90 operates as a dimer in a conformational cycle driven by ATP binding and hydrolysis at the N-terminus. The cycle is also regulated by a group of co-chaperones and accessory proteins. Here we review the biology of the Hsp90 molecular chaperone, emphasizing recent progress in our understanding of structure-function relationships and the identification of new client proteins. In addition we describe the exciting progress that has been made in the development of Hsp90 inhibitors, which are now showing promise in the clinic for cancer treatment. We also identify the gaps in our current understanding and highlight important topics for future research.

Fragment-based screening identified 7-azaindole as a protein kinase B inhibitor scaffold. Fragment elaboration using iterative crystallography of inhibitor-PKA-PKB chimera complexes efficiently guided improvements in the potency and selectivity of the compounds, resulting in the identification of nanomolar 6-(piperidin-1-yl)purine, 4-(piperidin-1-yl)-7-azaindole, and 4-(piperidin-1-yl)pyrrolo[2,3- d]pyrimidine inhibitors of PKBbeta with antiproliferative activity and showing pathway inhibition in cells. A divergence in the binding mode was seen between 4-aminomethylpiperidine and 4-aminopiperidine containing molecules. Selectivity for PKB vs PKA was observed with 4-aminopiperidine derivatives, and the most PKB-selective inhibitor (30-fold) showed significantly different bound conformations between PKA and PKA-PKB chimera.

Oncogenic BRAF and NRAS mutations are frequent in malignant melanoma. BRAF that is activated by the common V600E and other mutations, as well as by upstream NRAS mutations, has been shown to require the molecular chaperone heat shock protein 90 (HSP90) for stabilization and is depleted by the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG)]. Here, we explore the possible relationship between tumor BRAF and NRAS mutations and clinical response to 17-AAG in six patients with metastatic malignant melanoma who received pharmacologically active doses of 17-AAG as part of a phase I clinical trial. One patient with disease stabilization for 49 months had a (G13D)NRAS mutation and (WT)BRAF. A second patient who had stable disease for 15 months had a (V600E)BRAF mutation and (WT)NRAS. These preliminary results suggest that BRAF and NRAS mutation status should be determined in prospective phase II studies of HSP90 inhibitors in melanoma.

We describe the biological properties of NVP-AUY922, a novel resorcinylic isoxazole amide heat shock protein 90 (HSP90) inhibitor. NVP-AUY922 potently inhibits HSP90 (K(d) = 1.7 nmol/L) and proliferation of human tumor cells with GI(50) values of approximately 2 to 40 nmol/L, inducing G(1)-G(2) arrest and apoptosis. Activity is independent of NQO1/DT-diaphorase, maintained in drug-resistant cells and under hypoxic conditions. The molecular signature of HSP90 inhibition, comprising induced HSP72 and depleted client proteins, was readily demonstrable. NVP-AUY922 was glucuronidated less than previously described isoxazoles, yielding higher drug levels in human cancer cells and xenografts. Daily dosing of NVP-AUY922 (50 mg/kg i.p. or i.v.) to athymic mice generated peak tumor levels at least 100-fold above cellular GI(50). This produced statistically significant growth inhibition and/or regressions in human tumor xenografts with diverse oncogenic profiles: BT474 breast tumor treated/control, 21%; A2780 ovarian, 11%; U87MG glioblastoma, 7%; PC3 prostate, 37%; and WM266.4 melanoma, 31%. Therapeutic effects were concordant with changes in pharmacodynamic markers, including induction of HSP72 and depletion of ERBB2, CRAF, cyclin-dependent kinase 4, phospho-AKT/total AKT, and hypoxia-inducible factor-1alpha, determined by Western blot, electrochemiluminescent immunoassay, or immunohistochemistry. NVP-AUY922 also significantly inhibited tumor cell chemotaxis/invasion in vitro, WM266.4 melanoma lung metastases, and lymphatic metastases from orthotopically implanted PC3LN3 prostate carcinoma. NVP-AUY922 inhibited proliferation, chemomigration, and tubular differentiation of human endothelial cells and antiangiogenic activity was reflected in reduced microvessel density in tumor xenografts. Collectively, the data show that NVP-AUY922 is a potent, novel inhibitor of HSP90, acting via several processes (cytostasis, apoptosis, invasion, and angiogenesis) to inhibit tumor growth and metastasis. NVP-AUY922 has entered phase I clinical trials.

The strategy of 'drugging the cancer kinome' has led to the successful development and regulatory approval of several novel molecular targeted agents. The spotlight is now shifting to the phosphatidylinositide 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) pathway as a key potential target. This review details the role of the pathway in oncogenesis and the rationale for inhibiting its vital components. The focus will be on the progress made in the development of novel therapies for cancer treatment, with emphasis placed on agents that have entered clinical development. Strategies involving horizontal and vertical blockade of the pathway, as well as the use of biomarkers to select appropriate patients and to provide proof of target modulation will also be highlighted. Finally, we discuss the issues and limitations involved with targeting the PI3K-AKT-mTOR pathway, and predict what the future may hold for these novel anticancer therapeutics.

Heat-shock protein 70 (HSP70) isoforms contribute to tumorigenesis through their well-documented antiapoptotic activity and via their role as cochaperones for the HSP90 molecular chaperone. HSP70 expression is induced following treatment with HSP90 inhibitors, which may attenuate the cell death effects of this class of inhibitor. Here we show that silencing either heat-shock cognate 70 (HSC70) or HSP72 expression in human cancer cell lines has no effect on HSP90 activity or cell proliferation. However, simultaneously reducing the expression of both of these isoforms induces proteasome-dependent degradation of HSP90 client proteins, G1 cell-cycle arrest, and extensive tumor-specific apoptosis. Importantly, simultaneous silencing of HSP70 isoforms in nontumorigenic cell lines does not result in comparable growth arrest or induction of apoptosis, indicating a potential therapeutic window.

Activation of protein kinase clients by the Hsp90 system is mediated by the cochaperone protein Cdc37. Cdc37 requires phosphorylation at Ser13, but little is known about the regulation of this essential posttranslational modification. We show that Ser13 of uncomplexed Cdc37 is phosphorylated in vivo, as well as in binary complex with a kinase (C-K), or in ternary complex with Hsp90 and kinase (H-C-K). Whereas pSer13-Cdc37 in the H-C-K complex is resistant to nonspecific phosphatases, it is efficiently dephosphorylated by the chaperone-targeted protein phosphatase 5 (PP5/Ppt1), which does not affect isolated Cdc37. We show that Cdc37 and PP5/Ppt1 associate in Hsp90 complexes in yeast and in human tumor cells, and that PP5/Ppt1 regulates phosphorylation of Ser13-Cdc37 in vivo, directly affecting activation of protein kinase clients by Hsp90-Cdc37. These data reveal a cyclic regulatory mechanism for Cdc37, in which its constitutive phosphorylation is reversed by targeted dephosphorylation in Hsp90 complexes.

To determine the safety, maximum tolerated dose, and pharmacokinetic-pharmacodynamic profile of a histone deacetylase inhibitor, LAQ824, in patients with advanced malignancy. Patients and Methods: LAQ824 was administered i.v. as a 3-h infusion on days 1, 2, and 3 every 21 days. Western blot assays of peripheral blood mononuclear cell lysates and tumor biopsies pretherapy and posttherapy evaluated target inhibition and effects on heat shock protein-90 (HSP90) client proteins and HSP72.

The PPM1D gene is aberrantly amplified in a range of common cancers and encodes a protein phosphatase that is a potential therapeutic target. However, the issue of whether inhibition of PPM1D in human tumour cells that overexpress this protein compromises their viability has not yet been fully addressed. We show here, using an RNA interference (RNAi) approach, that inhibition of PPM1D can indeed reduce the viability of human tumour cells and that this effect is selective; tumour cell lines that overexpress PPM1D are sensitive to PPM1D inhibition whereas cell lines with normal levels are not. Loss of viability associated with PPM1D RNAi in human tumour cells occurs via the activation of the kinase P38. To identify chemical inhibitors of PPM1D, a high-throughput screening of a library of small molecules was performed. This strategy successfully identified a compound that selectively reduces viability of human tumour cell lines that overexpress PPM1D. As expected of a specific inhibitor, the toxicity to PPM1D overexpressing cell lines after inhibitor treatment is P38 dependent. These results further validate PPM1D as a therapeutic target and identify a proof-of-principle small molecule inhibitor.

Inhibitors of the Hsp90 molecular chaperone are showing considerable promise as potential chemotherapeutic agents for cancer. Here, we describe the structure-based design, synthesis, structure-activity relationships and pharmacokinetics of potent small-molecule inhibitors of Hsp90 based on the 4,5-diarylisoxazole scaffold. Analogues from this series have high affinity for Hsp90, as measured in a fluorescence polarization (FP) competitive binding assay, and are active in cancer cell lines where they inhibit proliferation and exhibit a characteristic profile of depletion of oncogenic proteins and concomitant elevation of Hsp72. Compound 40f (VER-52296/NVP-AUY922) is potent in the Hsp90 FP binding assay (IC50 = 21 nM) and inhibits proliferation of various human cancer cell lines in vitro, with GI50 averaging 9 nM. Compound 40f is retained in tumors in vivo when administered i.p., as evaluated by cassette dosing in tumor-bearing mice. In a human colon cancer xenograft model, 40f inhibits tumor growth by approximately 50%.

The aim of this work was to use phosphorus magnetic resonance spectroscopy (P-31 MRS) to investigate the pharmacodynamic effects of LAQ824, a histone deacetylase (HDAC) inhibitor. Human HT29 colon carcinoma cells were examined by 31P MRS after treatment with LAQ824 and another HDAC inhibitor, suberoylanilide hydroxamic acid. HT29 xenografts and tumor extracts were also examined using P-31 MRS, pre- and post-LAQ824 treatment. Histone H3 acetylation was determined using Western blot analysis, and tumor microvessel density by immunohistochemical staining of CD31. Phosphocholine showed a significant increase in HT29 cells after treatment with LAQ824 and suberoylanilide hydroxamic acid. In vivo, the ratio of phosphomonoester/total phosphorus (TotP) signal was significantly increased in LAQ824-treated HT29 xenografts, and this ratio was inversely correlated with changes in tumor volume. Statistically significant decreases in intracellular pH, beta-nucleoside triphosphate (beta-NTP)/TotP, and beta-NTP/inorganic phosphate (Pi) and an increase in Pi/TotP were also seen in LAQ824-treated tumors. Tumor extracts showed many significant metabolic changes after LAQ824 treatment, in parallel with increased histone acetylation and decreased microvessel density. Treatment with LAQ824 resulted in altered phospholipid metabolism and compromised tumor bioenergetics. The phosphocholine and phosphomonoester increases may have the potential to act as pharmacodynamic markers for noninvasively monitoring tumor response after treatment with LAQ824 or other HDAC inhibitors.

Advances in surgery and chemotherapy have improved the 5-year survival for patients with epithelial ovarian cancer, but have not impacted on the ultimate rate of cure in a disease that is diagnosed in late stage and that recurs in the majority of patients. "Omic" technologies promise to define genetically driven aberrant signaling pathways in malignant cells, provided that bioinformatic expertise can be focused on a cancer that is neither common nor rare. Molecular therapeutics must be linked to molecular diagnostics to permit individualized therapy. Not only epithelial cancer cells but also stroma, vasculature and the immune response must be targeted. Closer collaboration between academic institutions, biotech and pharma will be required to facilitate this process and to interest the private sector in an orphan disease. New preclinical models may permit more efficient development of drugs and siRNA that can target dormant drug resistant stem cells. Strategies must be developed to deal with the heterogeneity of different grades and histotypes. Identification of women at increased risk will facilitate prevention and early detection in subsets of patients. BRCA1/2 might be sequenced in all ovarian cancer patients to identify new kindreds. Epidemiologic algorithms are being developed and validated. Awareness must be raised that oral contraceptives can reduce risk of developing ovarian cancer by 50%. Early detection is likely to require panels of complementary biomarkers, analyzed by sophisticated statistical techniques, to improve sensitivity while maintaining extremely high specificity. As ovarian cancer becomes a chronic disease, greater emphasis will be placed on the challenges facing survivors.

Phosphoinositides have crucial roles in cellular controls, many of which have been established through the use of small-molecule inhibitors. Here, we describe YM201636, a potent inhibitor of the mammalian class III phosphatidylinositol phosphate kinase PIKfyve, which synthesizes phosphatidylinositol 3,5-bisphosphate. Acute treatment of cells with YM201636 shows that the PIKfyve pathway is involved in the sorting of endosomal transport, with inhibition leading to the accumulation of a late endosomal compartment and blockade of retroviral exit. Inhibitor specificity is shown by the use of short interfering RNA against the target, as well as by rescue with the drug-resistant yeast orthologue Fab1. We concluded that the phosphatidylinositol 3,5-bisphosphate pathway is integral to endosome formation, determining morphology and cargo flux.

The mitogenic extracellular kinase 1/2 (MEK1/2) inhibitor, PD0325901, has potent activity in a number of cancer cell types in vitro. In SKMEL-28 human melanoma cells (BRAF mutant), the drug rapidly decreased phosphorylated extracellular signal-regulated kinase 1/2, cyclin 131, and thymidine kinase 1 protein levels. We investigated if 3'-deoxy-3'-[F-18]fluorothymidine-positron emission tomography ([F-18]FLT-PET) could be used to image changes in cell proliferation following MEK1/2 inhibition in vivo. Mice bearing SKMEL-28 and human colon cancer HCT116 (K-RAS mutant) xenografts were treated daily with PD0325901 at 25 mg/kg and imaged by dynamic [18F]FLT-PET after 1 and 10 days of initiating treatment. The drug decreased tumor [F-18]FLT uptake after 1 and 10 days of treatment compared with control animals. The normalized (maximal) [F-18]FLT uptake in SKMEL-28 xenografts (at 60 minutes; NUVmax) after 1 day of vehicle or PD0325901 therapy was 1.81 +/- 0.18 versus 1.23 +/- 0.10, respectively (P = 0.03). In this model, NUVmax after 10 days was 2.07 +/- 0.40 versus 1.08 +/- 0.14, respectively (P = 0.03). The corresponding values for HCT116 tumors were 2.30 +/- 0.84 versus 1.88 +/- 0.36 (P = 0.045) after 1 day, and 1.97 +/- 0.13 versus 1.00 +/- 0.03 (P = 0.03) after 10 days. Similar changes were found for other [F-18]FLT retention variables. The drug decreased phosphorylated extracellular signal-regulated kinase 1/2, cyclin D1, and thymidine kinase 1 protein. Tumor [F-18]FLT-PET variables correlated with proliferation as measured by Ki67 labeling index (r >= 0.6; P >= 0.003). In summary, [F-18]FLT-PET is a sensitive imaging biomarker for detecting the antiproliferative effect of MEK1/2 inhibition by PD0325901.

Phosphatidylinositol-3-kinase (PI3K) is an important target in cancer due to the deregulation of the PI3K/ Akt signaling pathway in a wide variety of tumors. A series of thieno[3,2-d]pyrimidine derivatives were prepared and evaluated as inhibitors of PI3 kinase p110alpha. The synthesis, biological activity, and further profiling of these compounds are described. This work resulted in the discovery of 17, GDC-0941, which is a potent, selective, orally bioavailable inhibitor of PI3K and is currently being evaluated in human clinical trials for the treatment of cancer.

Overexpression of genes, through genomic amplification and other mechanisms, can critically affect the behavior of tumor cells. Genomic amplification of the 13q31-32 region is reported in many tumors, including rhabdomyosarcomas that are primarily pediatric sarcomas resembling developing skeletal muscle. The minimum overlapping region of amplification at 13q31-32 in rhabdomyosarcomas was defined as containing two genes: Glypican-5 (GPC5) encoding a cell surface proteoglycan and C13orf25 encompassing the miR-17-92 micro-RNA cluster. Genomic copy number and gene expression analyses of rhabdomyosarcomas indicated that GPC5 was the only gene consistently expressed and up-regulated in all cases with amplification. Constitutive overexpression and knockdown of GPC5 expression in rhabdomyosarcoma cell lines increased and decreased cell proliferation, respectively. A correlation between expression levels of nascent pre-rRNA and GPC5 (P = 0.001), but not a C13orf25 transcript containing miR-17-92, in primary samples supports an association of GPC5 with proliferative capacity in vivo. We show that GPC5 increases proliferation through potentiating the action of the growth factors fibroblast growth factor 2 (FGF2), hepatocyte growth factor (HGF), and Wnt1A. GPC5 enhanced the intracellular signaling of FGF2 and HGF and altered the cellular distribution of FGF2. The mesoderm-inducing effect of FGF2 and FGF4 in Xenopus blastocysts was also enhanced. Our data are consistent with a role of GPC5, in the context of sarcomagenesis, in enhancing FGF signaling that leads to mesodermal cell proliferation without induction of myogenic differentiation. Furthermore, the properties of GPC5 make it an attractive target for therapeutic intervention in rhabdomyosarcomas and other tumors that amplify and/or overexpress the gene.

Rational and efficient development of new molecular cancer therapeutics requires discovery, validation, and implementation of informative biomarkers. Measurement of molecular target status, pharmacokinetic (PK) parameters of drug exposure, and pharmacodynamic (PD) endpoints of drug effects on target, pathway, and downstream biological processes are extremely important. These can be linked to therapeutic effects in what we term a "pharmacological audit trail." Using biomarkers in preclinical drug discovery and development facilitates optimization of PK, PD, and therapeutic properties so that the best agent is selected for clinical evaluation. Applying biomarkers in early clinical trials helps identify the most appropriate patients; provides proof of concept for target modulation; helps test the underlying hypothesis; informs the rational selection of dose and schedule; aids decision making, including key go/no go questions; and may explain or predict clinical outcomes. Despite many successes such as trastuzumab and imatinib, exemplifying the value of targeting specific cancer defects, only 5% of oncology drugs that enter the clinic make it to marketing approval. Use of biomarkers should reduce this high level of attrition and bring forward key decisions (e.g., "fail fast"), thereby reducing the spiraling costs of drug development and increasing the likelihood of getting innovative and active drugs to cancer patients. In this chapter, we focus primarily on PD endpoints that demonstrate target modulation, including both invasive molecular assays and functional imaging technology. We also discuss related clinical trial design issues. Implementation of biomarkers in trials remains disappointingly low and we emphasize the need for greater cooperation between various stakeholders to improve this.

Seliciclib (CYC202; R-roscovitine) is the first selective, orally bioavailable inhibitor of cyclin-dependent kinases 1, 2, 7 and 9 to enter clinical trial. Preclinical studies showed antitumour activity in a broad range of human tumour xenografts. A phase I trial was performed with a 7-day b.i.d. p.o. schedule. Twenty-one patients (median age 62 years, range: 39-73 years) were treated with doses of 100, 200 and 800 b.i.d. Dose-limiting toxicities were seen at 800 mg b.i.d.; grade 3 fatigue, grade 3 skin rash, grade 3 hyponatraemia and grade 4 hypokalaemia. Other toxicities included reversible raised creatinine (grade 2), reversible grade 3 abnormal liver function and grade 2 emesis. An 800 mg portion was investigated further in 12 patients, three of whom had MAG3 renograms. One patient with a rapid increase in creatinine on day 3 had a reversible fall in renal perfusion, with full recovery by day 14, and no changes suggestive of renal tubular damage. Further dose escalation was precluded by hypokalaemia. Seliciclib reached peak plasma concentrations between 1 and 4 h and elimination half-life was 2-5 h. Inhibition of retinoblastoma protein phosphorylation was not demonstrated in peripheral blood mononuclear cells. No objective tumour responses were noted, but disease stabilisation was recorded in eight patients; this lasted for a total of six courses (18 weeks) in a patient with ovarian cancer.

Pharmacokinetic evaluation is an essential component of drug discovery and should be conducted early in the process so that those compounds with the best chance of success are prioritized and progressed. However, pharmacokinetic analysis has become a serious bottleneck during the 'hit-to-lead' and lead optimization phases due to the availability of new targets and the large numbers of compounds resulting from advances in synthesis and screening technologies. Cassette dosing, which involves the simultaneous administration of several compounds to a single animal followed by rapid sample analysis by liquid chromatography/tandem mass spectrometry, was developed to increase the throughput of in vivo pharmacokinetic screening. Although cassette dosing is advantageous in terms of resources and throughput, there are possible complications associated with this approach, such as the potential for compound interactions. Following an overview of the cassette dosing literature, this article focuses on the application of the technique in anticancer drug discovery. Specific examples are discussed, including the evaluation of cassette dosing to assess pharmacokinetic properties in the development of cyclin-dependent kinase and heat shock protein 90 inhibitors. Subject to critical analysis and validation in each case, the use of cassette dosing is recommended in appropriate chemical series to enhance the efficiency of drug discovery and reduce animal usage.

The molecular chaperone heat shock protein 90 (HSP90) has emerged as an exciting molecular target. Derivatives of the natural product geldanamycin, such as 17-allylamino-17-demethoxy-geldanamycin (17-AAG), were the first HSP90 ATPase inhibitors to enter clinical trial. Synthetic small-molecule HSP90 inhibitors have potential advantages. Here, we describe the biological properties of the lead compound of a new class of 3,4-diaryl pyrazole resorcinol HSP90 inhibitor (CCT018159), which we identified by high-throughput screening. CCT018159 inhibited human HSP90beta with comparable potency to 17-AAG and with similar ATP-competitive kinetics. X-ray crystallographic structures of the NH(2)-terminal domain of yeast Hsp90 complexed with CCT018159 or its analogues showed binding properties similar to radicicol. The mean cellular GI(50) value of CCT018159 across a panel of human cancer cell lines, including melanoma, was 5.3 mumol/L. Unlike 17-AAG, the in vitro antitumor activity of the pyrazole resorcinol analogues is independent of NQO1/DT-diaphorase and P-glycoprotein expression. The molecular signature of HSP90 inhibition, comprising increased expression of HSP72 protein and depletion of ERBB2, CDK4, C-RAF, and mutant B-RAF, was shown by Western blotting and quantified by time-resolved fluorescent-Cellisa in human cancer cell lines treated with CCT018159. CCT018159 caused cell cytostasis associated with a G(1) arrest and induced apoptosis. CCT018159 also inhibited key endothelial and tumor cell functions implicated in invasion and angiogenesis. Overall, we have shown that diaryl pyrazole resorcinols exhibited similar cellular properties to 17-AAG with potential advantages (e.g., aqueous solubility, independence from NQO1 and P-glycoprotein). These compounds form the basis for further structure-based optimization to identify more potent inhibitors suitable for clinical development.

Histone deacetylases (HDACs), histone acetyltransferases (HATs), and the molecular chaperone heat shock protein 90 (HSP90) are attractive anticancer drug targets. High-throughput screening plays a pivotal role in modern molecular mechanism-based drug discovery. Cell-based screens are particularly useful in that they identify compounds that are permeable and active against the selected target or pathway in a cellular context. We have previously developed time-resolved fluorescence cell immunosorbent assays (TRF-Cellisas) for compound screening and pharmacodynamic studies. These assays use a primary antibody to the single protein of interest and a matched secondary immunoglobulin labeled with an europium chelate (Eu). The availability of species-specific secondary antibodies labeled with different lanthanide chelates provides the potential for multiplexing this type of assay. The approach has been applied to the development of a 384-well duplexed cell-based screen to simultaneously detect compounds that induce the co-chaperone HSP70 as a molecular marker of potential inhibitors of HSP90 together with those that modulate cellular acetylation (i.e., potential inhibitors of histone deacetylase or histone acetyltransferase activity). The duplexed assay proved reliable in high-throughput format and approximately 64,000 compounds were screened. Following evaluation in secondary assays, 3 of 13 hits from the HSP70 arm were confirmed. Two of these directly inhibited the intrinsic ATPase activity of HSP90 whereas the third seems to have a different mechanism of action. In the acetylation arm, two compounds increased cellular acetylation, one of which inhibited histone deacetylase activity. A third compound decreased cellular histone acetylation, potentially through a novel mechanism of action.

The promising antitumor activity of 17-allylamino-17-demethoxygeldanamycin (17AAG) results from inhibition of the molecular chaperone heat shock protein 90 (HSP90) and subsequent degradation of multiple oncogenic client proteins. Gene expression microarray and proteomic analysis were used to profile molecular changes in the A2780 human ovarian cancer cell line treated with 17AAG. Comparison of results with an inactive analogue and an alternative HSP90 inhibitor radicicol indicated that increased expression of HSP72, HSC70, HSP27, HSP47, and HSP90beta at the mRNA level were on-target effects of 17AAG. HSP27 protein levels were increased in tumor biopsies following treatment of patients with 17AAG. A group of MYC-regulated mRNAs was decreased by 17AAG. Of particular interest and novelty were changes in expression of chromatin-associated proteins. Expression of the heterochromatin protein 1 was increased, and expression of the histone acetyltransferase 1 and the histone arginine methyltransferase PRMT5 was decreased by 17AAG. PRMT5 was shown to be a novel HSP90-binding partner and potential client protein. Cellular protein acetylation was reduced by 17AAG, which was shown to have an antagonistic interaction on cell proliferation with the histone deacetylase inhibitor trichostatin A. This mRNA and protein expression analysis has provided new insights into the complex molecular pharmacology of 17AAG and suggested new genes and proteins that may be involved in response to the drug or be potential biomarkers of drug action.

Although the heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) shows clinical promise, potential limitations encourage development of alternative chemotypes. We discovered the 3,4-diarylpyrazole resorcinol CCT018159 by high-throughput screening and used structure-based design to generate more potent pyrazole amide analogues, exemplified by VER-49009. Here, we describe the detailed biological properties of VER-49009 and the corresponding isoxazole VER-50589. X-ray crystallography showed a virtually identical HSP90 binding mode. However, the dissociation constant (K(d)) of VER-50589 was 4.5 +/- 2.2 nmol/L compared with 78.0 +/- 10.4 nmol/L for VER-49009, attributable to higher enthalpy for VER-50589 binding. A competitive binding assay gave a lower IC(50) of 21 +/- 4 nmol/L for VER-50589 compared with 47 +/- 9 nmol/L for VER-49009. Cellular uptake of VER-50589 was 4-fold greater than for VER-49009. Mean cellular antiproliferative GI(50) values for VER-50589 and VER-49009 for a human cancer cell line panel were 78 +/- 15 and 685 +/- 119 nmol/L, respectively, showing a 9-fold potency gain for the isoxazole. Unlike 17-AAG, but as with CCT018159, cellular potency of these analogues was independent of NAD(P)H:quinone oxidoreductase 1/DT-diaphorase and P-glycoprotein expression. Consistent with HSP90 inhibition, VER-50589 and VER-49009 caused induction of HSP72 and HSP27 alongside depletion of client proteins, including C-RAF, B-RAF, and survivin, and the protein arginine methyltransferase PRMT5. Both caused cell cycle arrest and apoptosis. Extent and duration of pharmacodynamic changes in an orthotopic human ovarian carcinoma model confirmed the superiority of VER-50589 over VER-49009. VER-50589 accumulated in HCT116 human colon cancer xenografts at levels above the cellular GI(50) for 24 h, resulting in 30% growth inhibition. The results indicate the therapeutic potential of the resorcinylic pyrazole/isoxazole amide analogues as HSP90 inhibitors.

The molecular chaperone HSP90 has emerged as an exciting target for cancer treatment. We review the potential advantages of HSP90 inhibitors, particularly the simultaneous combinatorial depletion of multiple oncogenic "client" proteins, leading to blockade of many cancer-causing pathways and the antagonism of all of the hallmark pathological traits of malignancy. Cancer selectivity is achieved by exploiting cancer "dependencies," including oncogene addiction and the stressed state of malignant cells. The multiple downstream effects of HSP90 inhibitors should make the development of resistance more difficult than with agents having more restricted effects. We review the various classes of HSP90 inhibitor that have been developed, including the natural products geldanamycin and radicicol and also the purine scaffold and pyrazole/isoxazole class of synthetic small molecule inhibitors. A first-in-class HSP90 drug, the geldanamycin analog 17-AAG, has provided proof of concept for HSP90 inhibition in patients at well tolerated doses and therapeutic activity has been seen. Other inhibitors show promise in preclinical and clinical development. Opportunities and challenges for HSP90 inhibitors are discussed, including use in combination with other agents. Most of the current HSP90 inhibitors act by blocking the essential nucleotide binding and ATPase activity required for chaperone function. Potential new approaches are discussed, for example, interference with cochaperone binding and function in the superchaperone complex. Biomarkers for use with HSP90 inhibitors are described. We stress how basic and translational research has been mutually beneficial and indicate future directions to enhance our understanding of molecular chaperones and their exploitation in cancer and other diseases.

Plasma cells producing high levels of paraprotein are dependent on the unfolded protein response (UPR) and chaperone proteins to ensure correct protein folding and cell survival. We hypothesized that disrupting client-chaperone interactions using heat shock protein 90 (Hsp90) inhibitors would result in an inability to handle immunoglobulin production with the induction of the UPR and myeloma cell death. To study this, myeloma cells were treated with Hsp90 inhibitors as well as known endoplasmic reticulum stress inducers and proteasome inhibitors. Treatment with thapsigargin and tunicamycin led to the activation of all 3 branches of the UPR, with early splicing of XBP1 indicative of IRE1 activation, upregulation of CHOP consistent with ER resident kinase (PERK) activation, and activating transcription factor 6 (ATF6) splicing. 17-AAG and radicicol also induced splicing of XBP1, with the induction of CHOP and activation of ATF6, whereas bortezomib resulted in the induction of CHOP and activation of ATF6 with minimal effects on XBP1. After treatment with all drugs, expression levels of the molecular chaperones BiP and GRP94 were increased. All drugs inhibited proliferation and induced cell death with activation of JNK and caspase cleavage. In conclusion, Hsp90 inhibitors induce myeloma cell death at least in part via endoplasmic reticulum stress and the UPR death pathway.

A number of human diseases can be linked to aberrations in protein folding which cause an imbalance in protein homeostasis. Molecular chaperones, including heat shock proteins, act to assist protein folding, stability and activity in the cell. Attention has begun to focus on modulating the expression and/or activity of this group of proteins for the treatment of a wide variety of human diseases. This review will describe the progress made to date in developing pharmacological modulators of the heat shock response, including both agents which affect the entire heat shock response and those that specifically target the HSP70 and HSP90 chaperone families.

Extensive evidence implicates activation of the lipid phosphatidylinositide 3-kinase (PI3K) pathway in the genesis and progression of various human cancers. PI3K inhibitors thus have considerable potential as molecular cancer therapeutics. Here, we detail the pharmacologic properties of a prototype of a new series of inhibitors of class I PI3K. PI103 is a potent inhibitor with low IC50 values against recombinant PI3K isoforms p110alpha (2 nmol/L), p110beta (3 nmol/L), p110delta (3 nmol/L), and p110gamma (15 nmol/L). PI103 also inhibited TORC1 by 83.9% at 0.5 micromol/L and exhibited an IC50 of 14 nmol/L against DNA-PK. A high degree of selectivity for the PI3K family was shown by the lack of activity of PI103 in a panel of 70 protein kinases. PI103 potently inhibited proliferation and invasion of a wide variety of human cancer cells in vitro and showed biomarker modulation consistent with inhibition of PI3K signaling. PI103 was extensively metabolized, but distributed rapidly to tissues and tumors. This resulted in tumor growth delay in eight different human cancer xenograft models with various PI3K pathway abnormalities. Decreased phosphorylation of AKT was observed in U87MG gliomas, consistent with drug levels achieved. We also showed inhibition of invasion in orthotopic breast and ovarian cancer xenograft models and obtained evidence that PI103 has antiangiogenic potential. Despite its rapid in vivo metabolism, PI103 is a valuable tool compound for exploring the biological function of class I PI3K and importantly represents a lead for further optimization of this novel class of targeted molecular cancer therapeutic.

In this age of molecularly targeted drug discovery, robust techniques are required to measure pharmacodynamic (PD) responses in tumors so that drug exposures can be associated with their effects on molecular biomarkers and efficacy. Our aim was to develop a rapid screen to monitor PD responses within xenografted human tumors as an important step towards a clinically applicable technology. Currently there are various methods available to measure PD end points, including immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction, gene expression profiling, and western blotting. These may require relatively large samples of tumor, surrogate tissue, or peripheral blood lymphocytes with subsequent analyses taking several days. The phosphoinositide 3-kinase (PI3-kinase) pathway is frequently deregulated in cancer and is also important in diabetes and autoimmune conditions. In this paper, optimization of the Meso Scale Discovery (MSD) (Gaithersburg, MD) platform to quantify changes in phospho-AKT and phospho-glycogen synthase kinase-3beta in response to a PI3-kinase inhibitor, LY294002, is described, initially in vitro and then within xenografted solid tumors. This method is highly practical with high throughput since large number of samples can be run simultaneously in 96-well format. The assays are robust (coefficient of variation for phospho-AKT 13.4%) and offer significant advantages (in terms of speed and quantitation) over western blots. This optimized procedure can be used for both in vitro and in vivo analysis, unlike an established fixed-cell ELISA with a time-resolved fluorescent end point.

A hit generation and exploration approach led to the discovery of 31 (2-(4-(6-chloro-2-(4-(dimethylamino)phenyl)-3H-imidazo[4,5-b]pyridin-7-yl)piperazin-1-yl)-N-(thiazol-2-yl)acetamide), a potent, novel inhibitor of Aurora-A, Aurora-B and Aurora-C kinases with IC(50) values of 0.042, 0.198 and 0.227microM, respectively. Compound 31 inhibits cell proliferation and has good microsomal stability.

Carcinogens induce complex transcriptional responses in cells that may hold key mechanistic information. Benzo(a)pyrene (BaP) modulation of transcription may occur through the activation of the aryl hydrocarbon receptor (AHR) or through responses to DNA damage. To characterize further the expression profiles induced by BaP in HepG2 and MCF-7 cells obtained in our previous study, they were compared to those induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which activates AHR but does not bind to DNA, and anti-benzo(a)pyrene- trans-7,8-dihydrodiol-9,10-epoxide (BPDE), which binds directly to DNA but does not activate AHR. A total of 22 genes had altered expression in MCF-7 cells after both BaP and TCDD exposure, and a total of 29 genes had altered expression in HepG2 cells. In both cell lines, xenobiotic metabolism was upregulated through induction of NQO1, MGST1, and CYP1B1. A total of 78 expression changes were induced by both BaP and BPDE in MCF-7 cells, and a total of 29 expression changes were induced by both BaP and BPDE in HepG2 cells. These genes were predominantly involved in cell cycle regulation, apoptosis, and DNA repair. BaP and BPDE caused the repression of histone genes in both cell lines, suggesting that regulation of these genes is an important component of the DNA damage response. Interestingly, overlap of the BPDE and TCDD gene expression profiles was also observed. Furthermore, some genes were modulated by BaP but not by TCDD or BPDE, including induction of CRY1 and MAK, which may represent novel signaling pathways that are independent of both AHR activation and DNA damage. Promoter analysis identified candidate genes for direct transcriptional regulation by either AHR or p53. These analyses have further dissected and characterized the complex cellular response to BaP.

The cyclin-dependent kinase (CDK) inhibitor seliciclib (R-roscovitine, CYC202) shows promising antitumor activity in preclinical models and is currently undergoing phase II clinical trials. Inhibition of the CDKs by seliciclib could contribute to cell cycle arrest and apoptosis seen with the drug. However, it is common for drugs to exert multiple effects on gene expression and biochemical pathways. To further our understanding of the molecular pharmacology of seliciclib, we employed cDNA microarrays to determine changes in gene expression profiles induced by the drug in HT29 human colon cancer cells. Concentrations of seliciclib were used that inhibited RB phosphorylation and cell proliferation. An increase in the mRNA expression for CJUN and EGR1 was confirmed by Western blotting, consistent with activation of the ERK1/2 MAPK pathway by seliciclib. Transcripts of key genes required for the progression through mitosis showed markedly reduced expression, including Aurora-A/B (AURK-A/B), Polo-like kinase (PLK), cyclin B2 (CCNB2), WEE1 and CDC25C. Reduced expression of these mitotic genes was also seen at the protein level. siRNA-mediated depletion of Aurora-A protein led to an arrest of cells in the G(2)/M phase, consistent with the effects of seliciclib treatment. Inhibition of mitotic entry following seliciclib treatment was indicated by a reduction of histone H3 phosphorylation, which is catalyzed by Aurora-B, and by decreased expression of mitotic markers, including phospho-protein phosphatase 1 alpha. The results indicate a potential mechanism through which seliciclib prevents entry into mitosis. Gene expression profiling has generated hypotheses that led to an increase in our knowledge of the cellular effects of seliciclib and could provide potential pharmacodynamic or response biomarkers for use in animal models and clinical trials.

The Aurora family of serine/threonine kinases is important for the regulation of centrosome maturation, chromosome segregation, and cytokinesis during mitosis. Overexpression of Aurora kinases in mammalian cells leads to genetic instability and transformation. Increased levels of Aurora kinases have also been linked to a broad range of human tumors. Here, we describe the properties of CCT129202, a representative of a structurally novel series of imidazopyridine small-molecule inhibitors of Aurora kinase activity. This compound showed high selectivity for the Aurora kinases over a panel of other kinases tested and inhibits proliferation in multiple cultured human tumor cell lines. CCT129202 causes the accumulation of human tumor cells with >or=4N DNA content, leading to apoptosis. CCT120202-treated human tumor cells showed a delay in mitosis, abrogation of nocodazole-induced mitotic arrest, and spindle defects. Growth of HCT116 xenografts in nude mice was inhibited after i.p. administration of CCT129202. We show that p21, the cyclin-dependent kinase inhibitor, is induced by CCT129202. Up-regulation of p21 by CCT129202 in HCT116 cells led to Rb hypophosphorylation and E2F inhibition, contributing to a decrease in thymidine kinase 1 transcription. This has facilitated the use of 3'-deoxy-3'[(18)F]fluorothymidine-positron emission tomography to measure noninvasively the biological activity of the Aurora kinase inhibitor CCT129202 in vivo.

The process of drug development in oncology has struggled to alter at a pace in keeping with the rapid discovery and testing of agents that act on a wide variety of molecular targets. The rational development of such agents requires an understanding of drug effect(s) on their purported target. It is likely that testing these drugs in a framework designed to examine cytotoxic agents will fail to establish their full potential. We discuss how data gained from biomarker investigation might impact on drug development and provide examples that highlight the development, validation and use of pharmacokinetic, and especially pharmacodynamic biomarkers as drug development moves from the laboratory into clinical testing. The challenges of performing assays to satisfy regulatory requirements have been the subject of much debate. We recommend the implementation of appropriate, fit-for-purpose biomarkers in clinical trials of all new cancer drugs.

A series of ansa-quinones has been prepared by chemical synthesis, and evaluated by biological techniques. Thus, 19-membered ansa-lactams, simplified analogues of the naturally occurring Hsp90 molecular chaperone inhibitor geldanamycin, were obtained by concise routes, the key steps being the combination of a ring-closing metathesis to give a 17-membered ring followed by Claisen rearrangement to effect ring expansion. The methodology was also used to prepare an "unnatural" 18-membered ring analogue. In ATPase enzyme assays, the synthetic ansa-quinones were weak inhibitors of Hsp90.

3-{1-[(4-Fluorophenyl)sulfonyl]-1H-pyrazol-3-yl}-2-methylimidazo[1,2-a]pyridine, 2a, was discovered in our chemical library as a novel p110alpha inhibitor with an IC(50) of 0.67microM, through screening in a scintillation proximity assay. Optimization of the substituents of 2a increased the p110alpha inhibitory activity by more than 300-fold (2g: IC(50)=0.0018microM). Further structural modification of 2g afforded thiazole derivative 12, which has potent p110alpha inhibitory activity (IC(50) of 0.0028microM) and is highly selective for p110alpha over other PI3K isoforms. Compound 12 also inhibited serum-induced cell proliferation of A375 and HeLa cells in vitro with IC(50) values of 0.14microM and 0.21microM, respectively, and suppressed tumor growth by 37% in a mouse HeLa xenograft model when dosed intraperitoneally at 25mg/kg. These results suggest that selective p110alpha inhibitors may have potential as cancer therapeutic agents.

Although the crystal structure of the anti-cancer target protein kinase B (PKBbeta/Akt-2) has been useful in guiding inhibitor design, the closely related kinase PKA has generally been used as a structural mimic due to its facile crystallization with a range of ligands. The use of PKB-inhibitor crystallography would bring important benefits, including a more rigorous understanding of factors dictating PKA/PKB selectivity, and the opportunity to validate the utility of PKA-based surrogates. We present a "back-soaking" method for obtaining PKBbeta-ligand crystal structures, and provide a structural comparison of inhibitor binding to PKB, PKA, and PKA-PKB chimera. One inhibitor presented here exhibits no PKB/PKA selectivity, and the compound adopts a similar binding mode in all three systems. By contrast, the PKB-selective inhibitor A-443654 adopts a conformation in PKB and PKA-PKB that differs from that with PKA. We provide a structural explanation for this difference, and highlight the ability of PKA-PKB to mimic the true PKB binding mode in this case.

Pharmacokinetic evaluation is an essential component of drug discovery and should be conducted early in the process so that those compounds with the best chance of success are prioritized and progressed. However, pharmacokinetic analysis has become a serious bottleneck during the 'hit-to-lead' and lead optimization phases due to the availability of new targets and the large numbers of compounds resulting from advances in synthesis and screening technologies. Cassette dosing, which involves the simultaneous administration of several compounds to a single animal followed by rapid sample analysis by liquid chromatography/tandem mass spectrometry, was developed to increase the throughput of in vivo pharmacokinetic screening. Although cassette dosing is advantageous in terms of resources and throughput, there are possible complications associated with this approach, such as the potential for compound interactions. Following an overview of the cassette dosing literature, this article focuses on the application of the technique in anticancer drug discovery. Specific examples are discussed, including the evaluation of cassette dosing to assess pharmacokinetic properties in the development of cyclin-dependent kinase and heat shock protein 90 inhibitors. Subject to critical analysis and validation in each case, the use of cassette dosing is recommended in appropriate chemical series to enhance the efficiency of drug discovery and reduce animal usage.

4-Morpholin-4-ylpyrido[3',2':4,5]thieno[3,2-d]pyrimidine 2a was discovered in our chemical library as a novel p110alpha inhibitor with an IC(50) of 1.4 microM. By structural modification of 2a, the 2-aryl-4-morpholinopyrido[3',2':4,5]furo[3,2-d]pyrimidine derivative 10e was discovered as a p110alpha inhibitor with approximately 400-fold greater potency than 2a. Evaluation of isoform selectivity showed that 10e is a potent inhibitor of p110beta. Furthermore, 10e showed anti-proliferative activity in various cell lines, including multi-drug resistant MCF7/ADR-res cells, and was effective against HeLa human cervical tumor xenografts in nude mice.

We have previously reported the imidazo[1,2-a]pyridine derivative 4 as a novel p110alpha inhibitor; however, although 4 is a potent inhibitor of p110alpha enzymatic activity and tumor cell proliferation in vitro, it is unstable in solution and ineffective in vivo. To increase stability the pyrazole of 4 was replaced with a hydrazone and a moderately potent p110alpha inhibitor 7a was obtained. Subsequent optimization of 7a afforded exceptionally potent p110alpha inhibitors, including 8c and 8h, with IC(50) values of 0.30 nM and 0.26 nM, respectively; to the best of our knowledge, these compounds are the most potent PI3K p110alpha inhibitors reported to date. Compound 8c was also stable in solution and exhibited significant anti-tumor effectiveness in vivo.

Chemical probes reveal Hsp90 to be a key molecule for the control of apoptosis in small-cell lung cancer - with important implications for Hsp90 biology and cancer treatment.

Structure-based drug design of novel isoquinoline-5-sulfonamide inhibitors of PKB as potential antitumour agents was investigated. Constrained pyrrolidine analogues that mimicked the bound conformation of linear prototypes were identified and investigated by co-crystal structure determinations with the related protein PKA. Detailed variation in the binding modes between inhibitors with similar overall conformations was observed. Potent PKB inhibitors from this series inhibited GSK3beta phosphorylation in cellular assays, consistent with inhibition of PKB kinase activity in cells.

MN58b is a novel anticancer drug that inhibits choline kinase, resulting in inhibition of phosphocholine synthesis. The aim of this work was to develop a noninvasive and robust pharmacodynamic biomarker for target inhibition and, potentially, tumor response following MN58b treatment. Human HT29 (colon) and MDA-MB-231 (breast) carcinoma cells were examined by proton (1H) and phosphorus (31P) magnetic resonance spectroscopy (MRS) before and after treatment with MN58b both in culture and in xenografts. An in vitro time course study of MN58b treatment was also carried out in MDA-MB-231 cells. In addition, enzymatic assays of choline kinase activity in cells were done. A decrease in phosphocholine and total choline levels (P < 0.05) was observed in vitro in both cell lines after MN58b treatment, whereas the inactive analogue ACG20b had no effect. In MDA-MB-231 cells, phosphocholine fell significantly as early as 4 hours following MN58b treatment, whereas a drop in cell number was observed at 48 hours. Significant correlation was also found between phosphocholine levels (measured by MRS) and choline kinase activities (r2 = 0.95, P = 0.0008) following MN58b treatment. Phosphomonoesters also decreased significantly (P < 0.05) in both HT29 and MDA-MB-231 xenografts with no significant changes in controls. 31P-MRS and 1H-MRS of tumor extracts showed a significant decrease in phosphocholine (P < or = 0.05). Inhibition of choline kinase by MN58b resulted in altered phospholipid metabolism both in cultured tumor cells and in vivo. Phosphocholine levels were found to correlate with choline kinase activities. The decrease in phosphocholine, total choline, and phosphomonoesters may have potential as noninvasive pharmacodynamic biomarkers for determining tumor response following treatment with choline kinase inhibitors.

Phosphoinositide 3-kinase (PI3K) is an attractive target for novel mechanism-based anticancer treatment. We used magnetic resonance (MR) spectroscopy (MRS) to detect biomarkers of PI3K signaling inhibition in human breast cancer cells. MDA-MB-231, MCF-7, and Hs578T cells were treated with the prototype PI3K inhibitor LY294002, and the (31)P MR spectra of cell extracts were monitored. In every case, LY294002 treatment was associated with a significant decrease in phosphocholine levels by up to 2-fold (P < 0.05). In addition, a significant increase in glycerophosphocholine levels by up to 5-fold was also observed (P <or= 0.05), whereas the content of glycerophosphoethanolamine, when detectable, did not change significantly. Nucleotide triphosphate levels did not change significantly in MCF-7 and MDA-MB-231 cells but decreased by approximately 1.3-fold in Hs578T cells (P = 0.01). The changes in phosphocholine and glycerophosphocholine levels seen in cell extracts were also detectable in the (31)P MR spectra of intact MDA-MB-231 cells following exposure to LY294002. When treated with another PI3K inhibitor, wortmannin, MDA-MB-231 cells also showed a significant decrease in phosphocholine content by approximately 1.25-fold relative to the control (P < 0.05), whereas the levels of the remaining metabolites did not change significantly. Our results indicate that PI3K inhibition in human breast cancer cells by LY294002 and wortmannin is associated with a decrease in phosphocholine levels.

Heat shock protein 90 (Hsp90) is a molecular chaperone involved in maintaining the correct conformation and stability of its client proteins. This study investigated the effects of Hsp90 inhibitors on client protein expression and key cellular functions required for tumor angiogenesis. The benzoquinone ansamycin Hsp90 inhibitors geldanamycin and/or its derivatives 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin inhibited production of vascular endothelial growth factor (VEGF)-A by tumor cells and blocked proliferative responses of human endothelial cells at nanomolar concentrations. 17-AAG also significantly reduced endothelial cell migration, tubular differentiation, invasion through Matrigel, and secretion of urokinase-type plasminogen activator at concentrations at or below those that inhibited proliferation. 17-AAG significantly reduced expression of VEGF receptor (VEGFR)-2 and established Hsp90 client proteins in human endothelial cells in vitro as well as in mouse vena cava, mesenteric vessels, and blood vessels within human tumor xenografts in vivo; this was associated with decreased tumor microvessel density. Finally, we showed for the first time that Hsp90 inhibitors also reduce expression of VEGFR-1 on human vascular endothelial cells, VEGFR-3 on lymphatic endothelial cells in vitro, and all three VEGFRs on mouse vasculature in vivo. Thus, we identify Hsp90 inhibitors as important regulators of many aspects of tumor angiogenesis (and potentially lymphangiogenesis) and suggest that they may provide therapeutic benefit not only via direct effects on tumor cells but also indirectly by inhibiting the production of angiogenic cytokines and responses of activated endothelial cells that contribute to tumor progression and metastasis.

Clinical trials of new cancer drugs should ideally include measurements of parameters such as molecular target expression, pharmacokinetic (PK) behavior, and pharmacodynamic (PD) endpoints that can be linked to measures of clinical effect. Appropriate PK/PD biomarkers facilitate proof-of-concept demonstrations for target modulation; enhance the rational selection of an optimal drug dose and schedule; aid decision-making, such as whether to continue or close a drug development project; and may explain or predict clinical outcomes. In addition, measurement of PK/PD biomarkers can minimize uncertainty associated with predicting drug safety and efficacy, reduce the high levels of drug attrition during development, accelerate drug approval, and decrease the overall costs of drug development. However, there are many challenges in the development and implementation of biomarkers that probably explain their disappointingly low implementation in phase I trials. The Pharmacodynamic/Pharmacokinetic Technologies Advisory committee of Cancer Research UK has found that submissions for phase I trials of new cancer drugs in the United Kingdom often lack detailed information about PK and/or PD endpoints, which leads to suboptimal information being obtained in those trials or to delays in starting the trials while PK/PD methods are developed and validated. Minimally invasive PK/PD technologies have logistic and ethical advantages over more invasive technologies. Here we review these technologies, emphasizing magnetic resonance spectroscopy and positron emission tomography, which provide detailed functional and metabolic information. Assays that measure effects of drugs on important biologic pathways and processes are likely to be more cost-effective than those that measure specific molecular targets. Development, validation, and implementation of minimally invasive PK/PD methods are encouraged.

Activation of the phosphatidylinositol-3-kinase (PI3K)/AKT survival pathway is a mechanism of cytotoxic drug resistance in ovarian cancer, and inhibitors of this pathway can sensitize to cytotoxic drugs. The HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) depletes some proteins involved in PI3K/AKT signaling, e.g., ERBB2, epidermal growth factor receptor (EGFR), and phosphorylated AKT (p-AKT). 17-AAG and paclitaxel were combined (at a fixed 1:1 ratio of their IC(50)) in four ovarian cancer cell lines that differ in expression of p-AKT, EGFR, and ERBB2. The EGFR-overexpressing A431 and KB epidermoid cell lines were also included. Combination indices (CI) were calculated using the median-effect equation and interpreted in the context of 17-AAG-mediated inhibition of PI3K signaling. Synergy was observed in IGROV-1- and ERBB2-overexpressing SKOV-3 ovarian cancer cells that express a high level of constitutively activated p-AKT [CI at fraction unaffected (fu)(0.5) = 0.50 and 0.53, respectively]. Slight synergy was observed in A431 cells (moderate p-AKT/overexpressed EGFR; CI at fu(0.5) = 0.76) and antagonism in CH1 (moderate p-AKT), HX62 cells (low p-AKT), and KB cells (low p-AKT/overexpressed EGFR; CI at fu(50) = 3.0, 3.5, and 2.0, respectively). The observed effects correlated with changes in the rate of apoptosis induction. 17-AAG induced a decrease in HSP90 client proteins (e.g., C-RAF, ERBB2, and p-AKT) or in downstream markers of their activity (e.g., phosphorylated extracellular signal-regulated kinase or p-AKT) in SKOV-3, IGROV-1, and CH1 cells at IC(50) concentrations. A non-growth-inhibitory concentration (6 nmol/L) reduced the phosphorylation of AKT (but not extracellular signal-regulated kinase) and sensitized SKOV-3 cells to paclitaxel. In conclusion, 17-AAG may sensitize a subset of ovarian cancer to paclitaxel, particularly those tumors in which resistance is driven by ERBB2 and/or p-AKT.

CCT018159 was recently identified as a novel inhibitor of heat shock protein (Hsp) 90, a promising target for cancer therapy. Pharmacokinetic and metabolic properties are likely to be important for efficacy and need to be optimized during drug development. Here, we define the preclinical metabolism and pharmacokinetics of CCT018159 and some early derivatives. In addition, we assess in vitro metabolic stability screening and in vivo cassette dosing (simultaneous administration of several compounds to a single animal) as approaches to investigate these compounds. The plasma clearance following individual i.v. administration to mice was rapid (0.128-0.816 L/h), exceeding hepatic blood flow. For CCT066950 and CCT066952, this could be attributed in part to extensive (>80%) blood cell binding. Oral bioavailability ranged from 1.8% to 29.6%. Tissue distribution of CCT066952 was rapid and moderate, and renal excretion of the compounds was minimal (<1% of dose excreted). Compounds underwent rapid glucuronidation both in vivo and following incubation with mouse liver microsomes. However, whereas CCT066965 was metabolized to the greatest extent in vitro, this compound displayed the slowest plasma clearance. The rank order of the compounds from the highest to lowest area under the curve was the same following discrete and cassette dosing. Furthermore, pharmacokinetic variables were similar whether the compounds were dosed alone or in combination. We conclude that the pharmacokinetics of CCT018159 are complex. Cassette dosing is currently the best option available to assess the pharmacokinetics of this promising series of compounds in relatively high throughput and is now being applied to identify compounds with optimal pharmacokinetic properties during structural analogue synthesis.

The heat shock protein 90 (HSP90) chaperones represent some 1-2% of all cellular protein and are key players in protein quality control in cells. They are over expressed in many human cancers and the fact that many oncogenic proteins are clients has prompted much recent research on HSP90 inhibitors as new cancer therapeutics. A brief introduction is followed by a detailed review of the various classes of inhibitors, both natural product-based and synthetic, that have emerged over the last decade. The natural products geldanamycin, radicicol and novobiocin have provided the start points for new drugs in this area and their medicinal chemistry is reviewed, including the exciting recent results emerging from clinical trials using geldanamycin analogues. The detailed understanding of the binding mode of these compounds to HSP90 has been significantly enhanced by X-ray crystallography of HSP90 constructs co-crystallised with various ligands. Efforts to replace the natural product inhibitors with more drug-like synthetic compounds have mushroomed over the last 4 years. The purines and the 3,4-diarylpyrazoles have proven to be the most successful and their medicinal chemistry is reviewed with particular emphasis on structure-based design. Protein/ligand co-crystal structures have shown that conserved water molecules in the active site are a vital part of the hydrogen-bonding network established on binding both natural product and synthetic inhibitors. Medicinal chemists have used this information to develop high affinity lead compounds. Recent research provides the platform for exciting developments in the area of HSP90 inhibition over the next few years.

This review explains why the chaperone Hsp90 is an exciting protein target for the discovery of new drugs to treat cancer in the clinic, and summarises the properties of natural product derived inhibitors before relating the discovery and current state of development of synthetic pyrazole compounds. Blockade of Hsp90 results in reduced cellular levels of several proteins implicated in cancer including CDK4, ERBB2 and C-RAF, and causes simultaneous inhibition of cancer cell proliferation in culture and of tumor xenograft growth in vivo. Hsp90 has an ATPase domain that is necessary for its Hsp chaperone function, and X-ray crystallography has shown that natural product inhibitors (geldanamycin, radicicol) of Hsp90 function bind to this domain. High throughput assays focusing on the ATPase activity of Hsp90 were developed and used to discover novel chemical starting points for cancer drug discovery. The discovery, synthesis and SAR of 3,4-diaryl pyrazoles is described. X-Ray crystallography of protein-inhibitor complexes revealed important interactions involving the resorcinol substituent at C-3, and these X-ray structures strongly influenced subsequent medicinal chemistry research that has resulted in highly potent inhibitors with sub-micromolar activity in cells. SAR and X-ray data are summarised for analogues in which the 4-phenyl substituent is replaced by amides or piperazine derivatives. Prospects for the pyrazoles as they progress towards clinical development are discussed in relation to current Phase I trials with derivatives of geldanamycin.

The molecular chaperone heat shock protein 90 (HSP90) has emerged as an exciting molecular target for cancer therapy. It operates as part of a multichaperone complex and is essential for the conformation, stability, and function of several key oncogenic client proteins such as mutant p53, ERBB2, B-RAF, C-RAF, and CDK4. The HSP90-based chaperone machine is driven by the hydrolysis of ATP and ADP/ATP nucleotide exchange. Many of the inhibitors of HSP90 interrupt the intrinsic ATPase activity, causing degradation of the client proteins via the ubiquitin-proteasome pathway. The first-in-class HSP90 inhibitor in clinical trials is the geldanamycin analog, 17-allylamino, 17-demethoxygeldanamycin (17-AAG). The results that have emerged from these trials have been encouraging, with stable disease observed in two melanoma patients. Pharmacodynamic endpoints, such as induction of HSP70 and downregulation of C-RAF and CDK4 in peripheral blood mononuclear cells and tumor biopsies from treated patients, provided evidence of HSP90 inhibition at well-tolerated doses. The toxicity of 17-AAG has been mild. Several preclinical studies have shown that 17-AAG may enhance the efficacy of a variety of chemotherapeutic agents. Phase II clinical trials in various cancers have been initiated as well as Phase I trials of combined therapy with 17-AAG. However, there are several limitations with 17-AAG such as solubility, stability, and hepatotoxicity. Thus, it is not surprising that new HSP90 agents are under development against this novel target for cancer therapy and several show promise.

An accurate, sensitive, robust and selective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin hydrochloride (17-DMAG) in human plasma has been developed and validated. Plasma samples were prepared by liquid/liquid extraction with ethyl acetate. The chromatographic separation was achieved within 9 min on a Synergy Polar column with a linear gradient and a mobile phase consisting of methanol and 0.1% formic acid in water. Detection of 17-DMAG and the internal standard (IS), olomoucine, was achieved by MS/MS with electrospray ionisation in positive ion mode. The calibration curve, ranging from 1.89 to 1890 nM, was linear r > 0.994 using a 1/y2 weighted linear regression. The assay showed no significant interferences from endogenous compounds. The lower limit of quantitation (LLOQ) was 1.89 nM, using 250 microL of plasma, with inter-assay precision (%RSD) and accuracy (%RE) values of 11.6% and -5.8%, respectively. Intra-assay precision ranged from 7.8-13.6%. The method described here is being used to evaluate the pharmacokinetic profiles of 17-DMAG given as a once weekly infusion in patients with advanced solid tumours.

Cancer drug development is leading the way in exploiting molecular biological and genetic information to develop "personalized" medicine. The new paradigm is to develop agents that target the precise molecular pathology driving the progression of individual cancers. Drug developers have benefited from decades of academic cancer research and from investment in genomics, genetics and automation; their success is exemplified by high-profile drugs such as Herceptin (trastuzumab), Gleevec (imatinib), Tarceva (erlotinib) and Avastin (bevacizumab). However, only 5% of cancer drugs entering clinical trials reach marketing approval. Cancer remains a high unmet medical need, and many potential cancer targets remain undrugged. In this review we assess the status of the discovery and development of small-molecule cancer therapeutics. We show how chemical biology approaches offer techniques for interconnecting elements of the traditional linear progression from gene to drug, thereby providing a basis for increasing speed and success in cancer drug discovery.

The last decade has seen the molecular chaperone heat shock protein 90 (HSP90) emerge as an exciting target for cancer therapy. This is because HSP90 is involved in maintaining the conformation, stability, activity and cellular localisation of several key oncogenic client proteins. These include, amongst others, ERBB2, C-RAF, CDK4, AKT/PKB, steroid hormone receptors, mutant p53, HIF-1alpha , survivin and telomerase hTERT. Therefore, modulation of this single drug target offers the prospect of simultaneously inhibiting all the multiple signalling pathways and biological processes that have been implicated in the development of the malignant phenotype. The chaperone function of HSP90 requires the formation of a multichaperone complex, which is dependent on the hydrolysis of ATP and ADP/ATP exchange. Most current inhibitors of HSP90 act as nucleotide mimetics, which block the intrinsic ATPase activity of this molecular chaperone. The first-in-class inhibitor to enter and complete phase I clinical trials was the geldanamycin analogue, 17-allylamino-17-demethoxygeldanamycin. The results of these trials have demonstrated that HSP90 is a valid drug target. Evidence of clinical activity has been seen in patients with melanoma, breast and prostate cancer. This article provides a personal perspective of the present efforts to increase our understanding of the molecular and cellular consequences of HSP90 inhibition, with examples from work in our own laboratory. We also review the discovery and development of novel small-molecule inhibitors and discuss alternative approaches to inhibit HSP90 activity, both of which offer exciting prospects for the future.

Novel piperazinyl, morpholino and piperidyl derivatives of the pyrazole-based Hsp90 inhibitor CCT018159 are described. Structure-activity relationships have been elucidated by X-ray co-crystal analysis of the new Compounds bound to the N-terminal domain of human Hsp90. Key features of the binding mode are essentially identical to the recently reported potent analogue VER-49009. The most potent of the new compounds has a methylsulfonylbenzyl substituent appended to the piperazine nitrogen, possesses an IC50 of less than 600 nM binding against the enzyme and demonstrates low micromolar inhibition of tumour cell proliferation. (C) 2006 Elsevier Ltd. All rights reserved.

Short routes are reported to novel macrolides (e.g., 9, 12, 20) related to the HSP90 inhibitor radicicol. (c) 2006 Elsevier Ltd. All rights reserved.

Novel resorcinylic macrolides, for example, 17, 24, were prepared via ring-closing metathesis as analogues of the HSP90 inhibitor radicicol. (c) 2006 Elsevier Ltd. All rights reserved.

The Cancer Genome Project has identified several oncogenic mutations in BRAT that represent important opportunities for cancer drug discovery. The (V600E)BRAF mutation accounts for approximately 90% of the mutations identified. A strong case has emerged from molecular, cellular, and structural studies for the identification and development of inhibitors of this mutated BRAF protein. The authors have developed and run a high-throughput screen to find inhibitors of (V600E)BRAF using an enzyme cascade assay in which oncogenic BRAF activates MEK1, which in turn activates ERK2, which then phosphorylates the transcription factor ELK1. A phosphospecific antibody, Europium-labeled secondary antibody, and a time-resolved fluorescent readout were used to measure phosphorylation of ELK1. Overall assay variation was 12.4%. The assay was used to screen 64,000 compounds with an overall Z' factor of 0.58 +/- 0.12. A series of 3,5,di-substituted pyridines were identified as inhibitors of the cascade assay. These compounds did not inhibit a shortened activated MEK1 to ELK1 cascade but were active (0.5-27.9 mu M) in a (V600E)BRAF assay and represent a potential starting point for future drug discovery and development.

Hsp90 encodes a ubiquitous molecular chaperone protein conserved among species which acts on multiple substrates, many of which are important cell-signaling proteins. Inhibition of Hsp90 function has been promoted as a mechanism to degrade client proteins involved in tumorigenesis and disease progression. Several assays to monitor inhibition of Hsp90 function currently exist but are limited in their use for a drug discovery campaign. Using data from the crystal structure of an initial hit compound, we have developed a fluorescence polarization assay to monitor binding of compounds to the ATP-binding site of Hsp90. This assay is very robust (Z' > 0.9) and can detect affinity of compounds with IC(50)s to 40 nM. We have used this assay in conjunction with cocrystal structures of small molecules to drive a structure-based design program aimed at the discovery and optimization of a novel class of potent Hsp90 inhibitors. (c) 2006 Elsevier Inc. All rights reserved.

HM74 and HM74a have been identified as receptors for niacin. HM74a mediates the pharmacological anti-lipolytic effects of niacin in adipocytes by reducing intracellular cyclic AMP (cAMP) and inhibiting release of free fatty acids into the circulation. In macrophages, niacin induces peroxisome proliferator-activated receptor gamma (PPAR gamma)-dependent and cAMP-dependent expression of genes mediating reverse cholesterol transport, although via an unidentified receptor. We describe constitutive expression of HM74a mRNA and hypoxia-and IFN gamma-inducible expression of HM74 and HM74a in human monocytic cell lines and primary cells in culture. In U937 cells niacin-induced expression of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), the most potent endogenous ligand of PPAR gamma. Both niacin and the structurally distinct HM74/HM74a ligand acifran-induced nuclear expression of PPAR gamma protein and enhanced PPAR gamma transcriptional activity. Niacin-induced PPAR gamma transcriptional activity was pertussis toxin sensitive and required activity of phospholipase A(2) (EC 3.1.1.4), cyclo-oxygenase (EC 1.14.99.1) and prostaglandin D-2 synthase (EC 5.3.99.2). Niacin also induced PPAR gamma transcriptional activity in HM74 and HM74a CHO cell transfectants, although not in vector-only control cells. This was sensitive to pertussis toxin and to inhibition of phoshoplipase A(2) and cyclo-oxygenase activity. Additionally, niacin increased intracellular cAMP in U937 via a pertussis toxin and cyclo-oxygenase-sensitive mechanism. These results indicate that HM74 and HM74a can mediate macrophage responses to niacin via activation of the prostaglandin synthesis pathway and induction and activation of PPAR gamma. This suggests a novel mechanism(s) mediating the clinical effects of pharmacological doses of niacin. (c) 2005 Elsevier Inc. All rights reserved.

Histone deacetylase inhibitors (HDACI) are emerging as growth inhibitory compounds that modulate gene expression and inhibit tumor cell proliferation. We assessed whether 3'-deoxy-3'-[F-18]fluorothymidine-positron emission tomography ([F-18]FLT-PET) could he used to noninvasively measure the biological activity of a novel HDACI LAQ824 in vivo. We initially showed that thymidine kinase 1 (TK1; EC2.7.1.21), the enzyme responsible for [F-18]FLT retention in cells, was regulated by LAQ824 in a drug concentration-dependent manner in vitro. In HCT116 colon carcinoma xenograft-bearing mice, LAQ824 significantly decreased tumor [F-18]FLT uptake in a dose-dependent manner. At day 4 of treatment, [F-18]FLT tumor-to-heart ratios at 60 minutes (NUV60) were 2.16 +/- 0.15, 1.86 +/- 0.13, and 1.45 +/- 0.20 in vehicle, and 5 and 25 mg/kg LAQ824 treatment groups, respectively (P <= 0.05). LAQ825 at 5 mg/kg also significantly reduced both TK1 levels and [F-18]FLT uptake at day 10 but not at day 2 (P <= 0.05). [F-18]FLT NUV60 correlated significantly with cellular proliferation (r = 0.68; P = 0.0019) and was associated with drug-induced histone H4 hyperacetylation. Of interest to [F-18]FLT-PET imaging, both TK1 mRNA copy numbers and protein levels decreased in the order vehicle > 5 mg/kg LAQ824 > 25 mg/kg LAQ824, providing a rationale for the use of [F-18]FLT-PET in this setting. We also observed increases in Rb hypophosphorylation and p21 levels, factors that could have contributed to the alteration in TK1 transcription in vivo. In conclusion, we have shown the utility of [F-18]FLT-PET for monitoring the biological activity of the HDACI, LAQ824. Drug-induced changes in tumor [F-18]FLT uptake were due, at least in part, to reductions in TK1 transcription and translation.

A series of benzo-macrolactones of varying ring size and conformation has been prepared by chemical synthesis and evaluated by structural and biological techniques. Thus, 12- to 16-membered lactones were obtained by concise routes, involving ring-closing metathesis as a key step. In enzyme assays, the 13-, 15-, and 16-membered analogs are good inhibitors, suggesting that they can adopt the required conformation to fit in the ATP-binding site. This was confirmed by cocrystallization of 13-, 14-, and 15-membered lactones with the N-terminal domain of yeast Hsp90, showing that they bind similarly to the "natural" 14-membered radicicol. The most active compounds in the ATPase assays also showed the greatest growth-inhibitory potency in HCT116 human colon cancer cells and the established molecular signature of Hsp90 inhibition, i.e., depletion of client proteins with upregulation of Hsp70.

Protein kinase B (PKB/AKT) has been identified as a promising cancer drug target downstream of PI3 kinase. To find novel inhibitors of PKB/AKT kinase activity for progression as anticancer agents, the authors have used a high-throughput screen based on AlphaScreen (TM) technology. A known kinase inhibitor, the isoquinoline H8, was used as a positive control with mean inhibition in the screen of 43.4% +/- 13.1%. The performance of the screen was highly acceptable with Z' and Z factors of 0.83 +/- 0.07 and 0.75 +/- 0.04, respectively. A number of confirmed hits (similar to 0.1% hit rate) were identified from 63,500 compounds screened. Five compounds have previously been described as PKB inhibitors, demonstrating the ability of the assay to find authentic inhibitors of the enzyme. Five hits had the potential to interfere with the assay signal and were deemed to be false positives. Two compounds were nonspecific inhibitors of PKB as enzyme inhibition in a filter-based assay was markedly reduced in the presence of 0.01% Triton X100. The authors now include an interference assay during hit confirmation procedures and check compound activity in the presence of Triton X100 in an attempt to eliminate nonspecific aggregators at an early stage.

A series of 4-morpholino-2-phenylquinazolines and related derivatives were prepared and evaluated as inhibitors of PI3 kinase p110 alpha. In this series, the thieno[3,2-d]pyrimidine derivative 15e showed the strongest inhibitory activity against p110 alpha, with an IC50 value of 2.0 nM, and inhibited proliferation of A375 melanoma cells with an IC50 value of 0.58 mu M. Moreover, 15e was found to be selective for p110 alpha over other PI3K isoforms and protein kinases, making it the first example of a selective PI3K p110 alpha inhibitor. (c) 2006 Elsevier Ltd. All rights reserved.

The authors have designed high-throughput screens to identify compounds that promote or inhibit terminal differentiation of primary human epidermal keratinocytes. Eleven known inhibitors of signaling pathways and approximately 4000 compounds of diverse structure were screened using an In-Cell Western system based on immunofluorescent staining of the terminal differentiation marker, involucrin. Staurosporine, a nonspecific protein kinase C inhibitor, and H89, a protein kinase A inhibitor, promoted expression of involucrin. Conversely, U0126, a MEK inhibitor, and SAHA or SBHA, 2 histone deacetylase inhibitors, reduced the expression of involucrin during calcium-induced stratification. In addition, the authors found 1 novel compound that induced keratinocyte differentiation and 2 novel compounds that were inhibitory to calcium-induced differentiation. The differentiation-inducing compound also inhibited growth of a human squamous cell carcinoma line by stimulating both differentiation and apoptosis. Because the compound affected the tumor cells at a lower concentration than primary keratinocytes, it may have potential as an antitumor therapy.

The last several years have seen major progress towards the goal of translating our growing understanding of the molecular basis of cancer into drugs with improved therapeutic activity and selectivity. Tremendous advances have been made but significant obstacles remain. In this review we assess our experience in the design and development of signal transduction drugs for cancer treatment, with a specific focus on small molecule kinase inhibitors. The drugg-ability of cancer kinome targets is exemplified by imatinib, gefitinib, erlotinib and many other emerging agents. We assess the current status of the design of potent and selective kinase inhibitors, which has benefited greatly from high throughput screening and structure-based approaches. A diverse range of kinase inhibitory scaffolds is now available based on these methods. Multi-parameter optimisation now focuses as much on pharmacokinetic and metabolic properties as it does on target potency and selectivity. Development of a 'molecular audit trail' requiring assays to demonstrate mechanism of action in vitro and in vivo is essential. Current issues include our relatively poor ability to predict the level of kinase selectivity in the intact cell, uncertainties around the most desirable selectivity profile, and the emergence of drug resistance.

R-roscovitine (seliciclib, CYC202) is a cyclin-dependent kinase inhibitor currently in phase II clinical trials in patients with cancer. Here, we describe its mouse metabolism and pharmacokinetics as well as the identification of the principal metabolites in hepatic microsomes, plasma, and urine. Following microsomal incubation of R-roscovitine at 10 microg/mL (28 micromol/L) for 60 minutes, 86.7% of the parent drug was metabolized and 60% of this loss was due to formation of one particular metabolite. This was identified as the carboxylic acid resulting from oxidation of the hydroxymethyl group of the amino alcohol substituent at C2 of the purine core present in R-roscovitine. Identification was confirmed by chemical synthesis and comparison of an authentic sample of the R-roscovitine-derived carboxylate metabolite (COOH-R-roscovitine). Other minor metabolites were identified as C8-oxo-R-roscovitine and N9-desisopropyl-R-roscovitine; these accounted for 4.9% and 2.6% of the parent, respectively. The same metabolic pattern was observed in vivo, with a 4.5-fold lower AUC(infinity) for R-roscovitine (38 micromol/L/h) than for COOH-R-roscovitine (174 micromol/L/h). Excretion of R-roscovitine in the urine up to 24 hours post-dosing accounted for an average of only 0.02% of the administered dose of 50 mg/kg, whereas COOH-R-roscovitine represented 65% to 68% of the dose irrespective of the route of administration (i.v., i.p., or p.o.). A partially deuterated derivative (R-roscovitine-d9) was synthesized to investigate if formation of COOH-R-roscovitine could be inhibited by replacement of metabolically labile protons with deuterium. After 60 minutes of incubation of R-roscovitine-d9 or R-roscovitine with mouse liver microsomes, formation of COOH-R-roscovitine-d9 was decreased by approximately 24% compared with the production of COOH-R-roscovitine. In addition, the levels of R-roscovitine-d9 remaining were 33% higher than those of R-roscovitine. However, formation of several minor R-roscovitine metabolites was enhanced with R-roscovitine-d9, suggesting that metabolic switching from the major carbinol oxidation pathway had occurred. Synthetic COOH-R-roscovitine and C8-oxo-R-roscovitine were tested in functional cyclin-dependent kinase assays and shown to be less active than R-roscovitine, confirming that these metabolic reactions are deactivation pathways.

Several mitogen-activated protein kinase (MAPK) signaling inhibitors are currently undergoing clinical trial as part of novel mechanism-based anticancer treatment strategies. This study was aimed at detecting biomarkers of MAPK signaling inhibition in human breast and colon carcinoma cells using magnetic resonance spectroscopy. We investigated the effect of the prototype MAPK kinase inhibitor U0126 on the (31)P-MR spectra of MDA-MB-231, MCF-7 and Hs578T breast, and HCT116 colon carcinoma cells. Treatment of MDA-MB-231 cells with 50 micromol/L U0126 for 2, 4, 8, 16, 24, 32, and 40 hours caused inhibition of extracellular signal-regulated kinases (ERK1/2) phosphorylation from 2 hours onwards. (31)P-MR spectra of extracted cells indicated that this was associated with a significant drop in phosphocholine levels to 78 +/- 8% at 8 hours, 74 +/- 8% at 16 hours, 66 +/- 7% at 24 hours, 71 +/- 10% at 32 hours, and 65 +/- 10% at 40 hours post-treatment. In contrast, the lower concentration of 10 micromol/L U0126 for 40 hours had no significant effect on either P-ERK1/ 2 or phosphocholine levels in MDA-MB-231 cells. Depletion of P-ERK1/2 in MCF-7 and Hs578T cells with 50 micromol/L U0126 also produced a drop in phosphocholine levels to 51 +/- 17% at 40 hours and 23 +/- 12% at 48 hours, respectively. Similarly, in HCT116 cells, inhibition with 30 micromol/L U0126 caused depletion of P-ERK1/2 and a decrease in phosphocholine levels to 80 +/- 9% at 16 hours and 61 +/- 4% at 24 hours post-treatment. The reduction in phosphocholine in MDA-MB-231 and HCT116 cells correlated positively with the drop in P-ERK1/2 levels. Our results show that MAPK signaling inhibition with U0126 is associated with a time-dependent decrease in cellular phosphocholine levels. Thus, phosphocholine has potential as a noninvasive pharmacodynamic marker for monitoring MAPK signaling blockade.

Vascular and angiogenic processes provide an important target for novel cancer therapeutics. Dynamic contrast-enhanced magnetic resonance imaging is being used increasingly to noninvasively monitor the action of these therapeutics in early-stage clinical trials. This publication reports the outcome of a workshop that considered the methodology and design of magnetic resonance studies, recommending how this new tool might best be used.

Hypoxia-inducible factor-1 (HIF-1) is a transcriptional complex that is activated in response to hypoxia and growth factors. HIF-1 plays a central role in tumor progression, invasion, and metastasis. Overexpression of the HIF-1alpha subunit has been observed in many human cancers and is associated with a poor prognostic outcome with conventional treatments. Targeting HIF-1 using novel small molecule inhibitors is, therefore, an attractive strategy for therapeutic development. We have generated U2OS human osteosarcoma cells stably expressing a luciferase reporter construct under the control of a hypoxia response element (U2OS-HRE-luc). The U2OS-HRE-luc cells were robustly and reproducibly sensitive to hypoxic stress in a HIF-1-dependent manner. We developed an automated U2OS-HRE-luc cell-based assay that was used in a high-throughput screen to identify compounds that inhibited HIF-1 activity induced by treatment with the hypoxia mimetic, deferoxamine mesylate. We performed a pilot screen of the National Cancer Institute Diversity Set of 2,000 compounds. We identified eight hit compounds, six of these were also identified by Rapisarda et al. in an independent hypoxia screen. However, there were two novel hit compounds, NSC-134754 and NSC-643735, that did not significantly inhibit constitutive luciferase activity in U2OS cells (U2OS-luc). We showed that both NSC-134754 and NSC-643735 significantly inhibited HIF-1 activity and HIF-1alpha protein induced by deferoxamine mesylate. Interestingly, NSC-134754 but not NCS-643735 inhibited HIF-1 activity and HIF-1alpha protein induced by hypoxia and significantly inhibited Glut-1 expression. Finally, we showed that both NCS-134754 and NCS-643735 inhibited HIF-1alpha protein induced by insulin-like growth factor-1. Our cell-based assay approach has successfully identified novel compounds that differentially target hypoxia and/or growth factor-mediated induction of HIF-1alpha.

High-throughput screening identified the 3,4-diarylpyrazole CCT018159 as a novel and potent (7.1 microM) inhibitor of Hsp90 ATPase activity. Here, we describe the synthesis of CCT018159 and a number of close analogues together with data on their biochemical properties. Some initial structure-activity relationships are discussed, as well as the crystal structure of CCT018159 bound to Hsp90.

To study the toxicity and pharmacokinetic-pharmacodynamic profile of 17-allylamino, 17- demethoxygeldanamycin (17-AAG) and to recommend a dose for phase II trials.

High-throughput screening of chemical libraries and the subsequent rapid progress of hit compounds through an iterative developmental test cascade are essential parts of modern molecular mechanism-based drug discovery. These processes depend on the use of efficient assay technologies and equipment. Enzyme-linked immunosorbent assays have historically been carried out in 96-well microtitre plates. Improvements in reagents and assay technologies mean that solid-phase immunoassays can be adapted for higher throughput to play an important role in modern drug discovery. The molecular chaperone heat-shock protein (Hsp) 90 is an important anticancer drug target because it maintains the conformation, stability, and function of many important oncogenic client proteins, including those involved with signal transduction, cell proliferation, survival, differentiation, motility angiogenesis, and metastasis. Using the standard inhibitors of the adenosine triphosphatase (ATPase) activity of Hsp90, geldanamycin (GA) and 17-allylamino-17- demethoxygeldanamycin (17AAG), novel solid-phase immunoassays have been validated using a time-resolved fluorescence (TRF) end point. Their utility for confirming the mechanism of action of Hsp90 inhibition in secondary cell-based assays has been shown and applied to the novel Hsp90 inhibitor CCT018159. Adaptation of these assays for later studies using human tumour xenografts and samples obtained from a Phase 1 trial of 17AAG is also described. Finally, comparison is made between the use and applicability of this type of immunoassay and other techniques such as western blotting, immunohistochemistry, and flow cytometry analysis.

A LC-tandem mass spectrometry method to quantify the quinazoline-based thymidylate synthase inhibitors BGC945 and BGC638 in mouse plasma was developed. BGC945 and BGC638 were extracted from mouse plasma using protein precipitation with acetonitrile. Chromatography was performed on a Fluophase RP 5 microm, 100 mmx2.0mm i.d. column using a gradient of ammonium acetate and acetonitrile as a mobile phase with a flow rate of 0.2 mLmin(-1). The injection volume for each sample was 20 microL with a total run time of 7.5 min. This method was validated in the range 25-4000 nM (r2=0.99). The analytical assay performance showed that the method was accurate (mean intra- and inter-day assay R.E. were below 12% and 11%, respectively), reproducible (mean intra- and inter-day R.S.D. were less than 13% and 5% for all quality control levels, respectively) and sensitive (lower limit of quantification was 25 nM) in the range studied. This validated method has been used to define the first pharmacokinetic report of BGC945 and BGC638 in mice.

The small-molecule compound pifithrin-alpha (PFT-alpha) has been reported to inhibit p53 function and protect against a variety of genotoxic agents. We show here that PFT-alpha is unstable in tissue culture medium and is rapidly converted to its condensation product PFT-beta. Both compounds showed limited solubility with PFT-alpha precipitating out of tissue culture medium at concentrations >30 micromol/L. PFT-alpha and -beta exhibited cytotoxic effects in vitro towards two human wild-type p53-expressing tumor cell lines, A2780 ovarian and HCT116 colon (IC(50) values for both cell lines were 21.3 +/- 8.1 micromol/L for PFT-alpha and 90.3 +/- 15.5 micromol/L for PFT-beta, mean +/- SD, n = 4). There was no evidence of protection by clonogenic assay with either compound in combination with ionizing radiation. Indeed, there was some evidence that PFT-alpha enhanced cytotoxicity, particularly at higher concentrations of PFT-alpha. Neither compound had any effect on p53, p21, or MDM-2 protein expression following ionizing radiation exposure and there was no evidence of any abrogation of p53-dependent, ionizing radiation-induced cell cycle arrest. Similarly, there was no evidence of cellular protection, or of effects on p53-dependent gene transcription, or on translation of MDM-2 or p21 following UV treatment of these human tumor cell lines. In addition, there was no effect on p53 or p21 gene transactivation or p38 phosphorylation after UV irradiation of NIH-3T3 mouse fibroblasts. In conclusion, neither PFT-alpha nor -beta can be regarded as a ubiquitous inhibitor of p53 function, and caution should be exercised in the use of these agents as specific p53 inhibitors.

Histone acetylation plays an important role in regulating the chromatin structure and is tightly regulated by two classes of enzyme, histone acetyltransferases (HAT) and histone deacetylases (HDAC). Deregulated HAT and HDAC activity plays a role in the development of a range of cancers. Consequently, inhibitors of these enzymes have potential as anticancer agents. Several HDAC inhibitors have been described; however, few inhibitors of HATs have been disclosed. Following a FlashPlate high-throughput screen, we identified a series of isothiazolone-based HAT inhibitors. Thirty-five N-substituted analogues inhibited both p300/cyclic AMP-responsive element binding protein-binding protein-associated factor (PCAF) and p300 (1 to >50 micromol/L, respectively) and the growth of a panel of human tumor cell lines (50% growth inhibition, 0.8 to >50 micromol/L). CCT077791 and CCT077792 decreased cellular acetylation in a time-dependent manner (2-48 hours of exposure) and a concentration-dependent manner (one to five times, 72 hours, 50% growth inhibition) in HCT116 and HT29 human colon tumor cell lines. CCT077791 reduced total acetylation of histones H3 and H4, levels of specific acetylated lysine marks, and acetylation of alpha-tubulin. Four and 24 hours of exposure to the compounds produced the same extent of growth inhibition as 72 hours of continuous exposure, suggesting that growth arrest was an early event. Chemical reactivity of these compounds, as measured by covalent protein binding and loss of HAT inhibition in the presence of DTT, indicated that reaction with thiol groups might be important in their mechanism of action. As one of the first series of small-molecule inhibitors of HAT activity, further analogue synthesis is being pursued to examine the potential scope for reducing chemical reactivity while maintaining HAT inhibition.

A major goal of cancer research is to translate our understanding of the causation of malignancy at the level of the genome and biochemical pathways into the development of drugs with improved activity and cancer selectivity. This paper provides a personal perspective of the current status of efforts to achieve this goal, with a particular focus on drugging the cancer kinome. Remarkable progress has been made in this area, but many challenges remain. The value of cancer kinome sequencing is emphasized. Three projects in which the author's laboratory is involved are reviewed in detail. These involve the discovery and development of inhibitors of cyclin-dependent kinases, phosphoinositide 3-kinases, and the Hsp90 molecular chaperone.

To investigate pharmacokinetic-pharmacodynamic relationships for the trisubstituted aminopurine cyclin-dependent kinase inhibitors olomoucine, bohemine, and CYC202 (R-roscovitine; seliciclib) in the HCT116 human colon carcinoma model.

The crystal structure of a previously reported screening hit 1 (CCT018159) bound to the N terminal domain of molecular chaperone Hsp90 has been used to design 5-amide analogues. These exhibit enhanced potency against the target in binding and functional assays with accompanying appropriate cellular pharmaeodynamic changes. Compound 11 (VER-49009) compares favorably with the clinically evaluated 17-AAG.

Information from X-ray crystal structures of Hsp90 inhibitors bound to the human Hsp90 molecular chaperone was used to assist in the design of 3-(5-chloro-2,4-dihydroxyphenyl)-pyrazole-4-carboxamides as novel inhibitors of Hsp90. Accessing an extra interaction with the protein via Phe138 gave a significant increase in binding potency compared to similar analogues that do not make this interaction. (c) 2005 Elsevier Ltd. All rights reserved.

In this study we investigated the in vitro time dependence of radiosensitisation, pharmacokinetics and metabolism of NU7026, a novel inhibitor of the DNA repair enzyme DNA-dependent protein kinase (DNA-PK). At a dose of 10 muM, which is nontoxic to cells per se, a minimum NU7026 exposure of 4 h in combination with 3 Gy radiation is required for a significant radiosensitisation effect in CH1 human ovarian cancer cells. Following intravenous administration to mice at 5 mg kg(-1), NU7026 underwent rapid plasma clearance (0.108 l h(-1)) and this was largely attributed to extensive metabolism. Bioavailability following interperitoneal (i.p.) and p.o. administration at 20 mg kg(-1) was 20 and 15%, respectively. Investigation of NU7026 metabolism profiles in plasma and urine indicated that the compound undergoes multiple hydroxylations. A glucuronide conjugate of a bis-hydroxylated metabolite represented the major excretion product in urine. Identification of the major oxidation site as C-2 of the morpholine ring was confirmed by the fact that the plasma clearance of NU7107 (an analogue of NU7026 methylated at C-2 and C-6 of the morpholine ring) was four-fold slower than that of NU7026. The pharmacokinetic simulations performed predict that NU7026 will have to be administered four times per day at 100 mg kg(-1) i.p. in order to obtain the drug exposure required for radiosensitisation.

SU5416 (Z-3-[(2,4-dimethylpyrrol-5-yl) methylidenyl]-2-indolinone; semaxanib) is a small molecule inhibitor of the vascular endothelial growth factor receptor (VEGFR2). A Phase I dose escalation study was performed. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was used as a pharmacodynamic assessment tool. In all, 27 patients were recruited. SU5416 was administered twice weekly by fixed rate intravenous infusion. Patients were treated in sequential cohorts of three patients at 48, 65, 85 110 and 145 mg m(-2). A further dose level of 190 mg m(-2) after a 2-week lead in period at a lower dose was completed; thereafter, the cohort at 145 mg m(-2) was expanded. SU5416 showed linear pharmacokinetics to 145 mg m(-2) with a large volume of distribution and rapid clearance. A significant degree of interpatient variability was seen. SU5416 was well tolerated, by definition a maximum-tolerated dose was not defined. No reproducible changes were seen in DCE-MRI end points. Serial assessments of VEGF in a cohort of patients treated at 145 mg m(-2) did not show a statistically significant treatment-related change. Parallel assessments of the impact of SU5416 on coagulation profiles in six patients showed a transient effect within the fibrinolytic pathway. Clinical experience showed that patients who had breaks of therapy longer than a week could not have treatment reinitiated at a dose of 190 mg m(-2) without unacceptable toxicity. The 145 mg m(-2) dose level is thus the recommended dose for future study.

To establish the pharmacokinetic and pharmacodynamic profile of the heat shock protein 90 (HSP90) inhibitor 17-allylamino, 17-demethoxygeldanamycin (17-AAG) in ovarian cancer xenograft models.

The first golden era of cancer drug development was initiated in the 1940s and gave rise to the cytotoxic agents that dominate current cancer medicine. The second golden era is now underway in which cancer genomics will direct drug development.

Hsp90 is a ubiquitously expressed molecular chaperone that folds, stabilizes, and functionally regulates many cellular proteins. The benzoquinone ansamysin 17-allylamino-17demethoxygeldanamycin (17-AAG) is an anticancer drug that disrupts Hsp90 binding to its clients, causing their degradation through the ubiquitin-dependent proteasomal pathway. The protein kinase B-RAF is mutated in similar to 7% of human cancers. The most common mutation (similar to 90%) is B-V600E-RAF, which has constitutively elevated kinase activity, stimulates cancer cell proliferation, and promotes survival. Here, we show that B-V600E-RAF is an Hsp90 client protein that requires Hsp90 for its folding and stability. (V600E)BRAF is more sensitive to degradation by 17-AAG treatment than B-WT-RAF and we show that the majority of the other mutant forms of B-RAF are also sensitive to 17-AAG-mediated proteasomal degradation. Our data show that B-RAF is an important target for 17-AAG in human cancer. (Cancer Res 2005; 65(23): 10686-91).

Deregulated expression of the Wilms' tumor gene (WT1) has been implicated in the maintenance of a malignant phenotype in leukemias and a wide range of solid tumors through interference with normal signaling in differentiation and apoptotic pathways. Expression of high levels of WT1 is associated with poor prognosis in leukemias and breast cancer. Using real-time (Taqman) reverse transcription-PCR and RNase protection assay, we have shown up-regulation of WT1 expression following cytotoxic treatment of cells exhibiting drug resistance, a phenomenon not seen in sensitive cells. WT1 is subject to alternative splicing involving exon 5 and three amino acids (KTS) at the end of exon 9, producing four major isoforms. Exon 5 splicing was disrupted in all cell lines studied following a cytotoxic insult probably due to increased exon 5 skipping. Disruption of exon 5 splicing may be a proapoptotic signal because specific targeting of WT1 exon 5-containing transcripts using a nuclease-resistant antisense oligonucleotide (ASO) killed HL60 leukemia cells, which were resistant to an ASO targeting all four alternatively spliced transcripts simultaneously. K562 cells were sensitive to both target-specific ASOs. Gene expression profiling following treatment with WT1 exon 5-targeted antisense showed up-regulation of the known WT1 target gene, thrombospondin 1, in HL60 cells, which correlated with cell death. In addition, novel potential WT1 target genes were identified in each cell line. These studies highlight a new layer of complexity in the regulation and function of the WT1 gene product and suggest that antisense directed to WT1 exon 5 might have therapeutic potential.

Deregulated expression of the Wilms' tumor gene (WT1) has been implicated in the maintenance of a malignant phenotype in leukemias and a wide range of solid tumors through interference with normal signaling in differentiation and apoptotic pathways. Expression of high levels of WT1 is associated with poor, prognosis in leukemias and breast cancer. Using real-time (Taqman) reverse transcription-PCR and RNase protection assay, we have shown up-regulation of WT1 expression following cytotoxic treatment of cells exhibiting drug resistance, a phenomenon not seen in sensitive cells. WT1 is subject to alternative splicing involving exon 5 and three amino acids (KTS) at the end of exon 9, producing four major isoforms. Exon 5 splicing was disrupted in all cell lines studied following a cytotoxic insult probably due to increased exon 5 skipping. Disruption of exon 5 splicing may be a proapoptotic signal because specific targeting of WT1 exon 5-containing transcripts using a nuclease-resistant antisense oligonucleotide (ASO) killed HL60 leukemia cells, which were resistant to an ASO targeting all four alternatively spliced transcripts simultaneously. K562 cells were sensitive to both target-specific ASOs. Gene expression profiling following treatment with WT1 exon 5-targeted antisense showed up-regulation of the known WT1 target gene, thrombospondin 1, in HL60 cells, which correlated with cell death. In addition, novel potential WT1 target genes were identified in each cell line. These studies highlight a new layer of complexity in the regulation and function of the WT1 gene product and suggest that antisense directed to WT1 exon 5 might have therapeutic potential.

Deregulation of the cell cycle commonly occurs during tumorigenesis, resulting in unrestricted cell proliferation and independence from mitogens. Cyclin-dependent kinase inhibitors have the potential to induce cell cycle arrest and apoptosis in cancer cells. CYC202 (R-roscovitine) is a potent inhibitor of CDK2/cyclin E that is undergoing clinical trials. Drugs selected to act on a particular molecular target may exert additional or alternative effects in intact cells. We therefore studied the molecular pharmacology of CYC202 in human colon cancer cells. Treatment of HT29 and KM12 colon carcinoma cell lines with CYC202 decreased both retinoblastoma protein phosphorylation and total retinoblastoma protein. In addition, an increase in the phosphorylation of extracellular signal-regulated kinases 1/2 was observed. As a result, downstream activation of the mitogen-activated protein kinase pathway occurred, as demonstrated by an increase in ELK-1 phosphorylation and in c-FOS expression. Use of mitogen-activated protein kinase kinases 1/2 inhibitors showed that the CYC202-induced extracellular signal-regulated kinases 1/2 phosphorylation was mitogen-activated protein kinase kinases 1/2 dependent but did not contribute to the cell cycle effects of the drug, which included a reduction of cells in G(1), inhibition of bromodeoxyuridine incorporation during S-phase, and a moderate increase in G(2)-M phase. Despite activation of the mitogen-activated protein kinase pathway, cyclin D1 protein levels were decreased by CYC202, an effect that occurred simultaneously with loss of retinoblastoma protein phosphorylation and inhibition of cell cycle progression. The reduced expression of cyclin D1 protein was independent of the p38(SAPK) and phosphatidylinositol 3-kinase pathways, which are known regulators of cyclin D1 protein. Interestingly, CYC202 caused a clear reduction in cyclins D1, A, and B1 mRNA, whereas c-FOS mRNA increased by 2-fold. This was accompanied by a loss of RNA polymerase II phosphorylation and total RNA polymerase II protein, suggesting that CYC202 was inhibiting transcription, possibly via inhibition of CDK7 and CDK9 complexes. It can be concluded that although CYC202 can act as a CDK2 inhibitor, it also has the potential to inhibit CDK4 and CDK1 activities in cancer cells through the down-regulation of the corresponding cyclin partners. This provides a possible mechanism by which CYC202 can cause a reduction in retinoblastoma protein phosphorylation at multiple sites and cell cycle arrest in G(1), S, and G(2)-M phases. In addition to providing useful insights into the molecular pharmacology of CYC202 in human cancer cells, the results also suggest potential pharmacodynamic end points for use in clinical trials with the drug.

The impact of the presence of a germ-line BRCA1 mutation on gene expression in normal breast fibroblasts after radiation-induced DNA damage has been investigated.

The molecular chaperone Hsp90 is not only of major current interest in fundamental biological research but also recognised as an exciting new target for the treatment of cancer and other diseases. In addition to playing an important role in response to proteotoxic heat shock and others stresses, Hsp90 is also critical for maintaining normal cellular homeostasis. Hsp90 is responsible for ensuring the conformational stability, shape and function of a selected range of key proteins, including many kinases and transcription factors. Furthermore, recent studies show that Hsp90 plays a key role in development and evolution. Hsp90 is overexpressed in cancer cells and is thought to be involved in dealing with the cellular stress associated with malignancy, as well as being essential for a range of key oncogenic proteins, including ErbB2, Raf-1, Akt/PKB, mutant p53 and many others. A major attraction of Hsp90 inhibitors is their potential to inhibit a range of 'mission critical' cancer pathways, thereby blocking all of the 'hallmark traits' of malignancy and exhibiting broad-spectrum antitumour activity. The first-in-class Hsp90 inhibitor 17AAG has entered clinical trials with promising early results and a range of other agents is under investigation and preclinical development. This article reviews the current status and future prospects for the exploitation of Hsp90 as a new molecular target for cancer treatment.

Determination of pharmacokinetic properties in the intact animal remains a major bottleneck in drug discovery. Cassette dosing involves administration of a cocktail of drugs to individual animals. Here we describe the cassette dosing properties of a 107-membered library of 2,6,9-trisubstituted purine cyclin-dependent kinase 2 (CDK2) inhibitors. A three-step parallel synthesis approach produced compounds with purity ranging from 63% to 100%. Cassette dosing was validated by comparing the pharmacokinetic parameters obtained following i.v. administration of a mixture of olomoucine, R-roscovitine (CYC202), and bohemine, each at 16.6 mg/kg, with results for administration of single agents at 50 mg/kg. No significant difference was observed between the pharmacokinetic parameters of agents when dosed in combination compared with those of individual compounds. CYC202 showed the highest area under the curve (AUC) and the longest elimination half-life (t(1/2)). Further cassettes evaluated the library of trisubstituted purines with CYC202 and purvalanol A included as pharmacokinetic standards in a validated limited sampling strategy. The ratios of pharmacokinetic parameters to that of CYC202 [AUC, maximum concentration (C(max)), and t(1/2)] remained similar when compounds were tested in two different cassettes or as individual compounds. Following dosing of the same cassette on three different days, there was less than 20% variation in pharmacokinetic parameters between days. The structure-pharmacokinetics relationship showed that the favored purine substituents are benzylamine and veratrylamine at position 6, amino-2 propanol at position 2, and methylpropyl or hydroxyethyl at position 9. Without cassette dosing, this study would have used 3 times as many animals and would have taken 4 times longer, illustrating the power of this method in lead optimization.

There is extensive evidence from the molecular and genomic analysis of human cancers that the PI 3-kinase (phosphoinositide 3-kinase)-Akt/PKB (protein kinase B) pathway is deregulated in malignant progression. Furthermore, the causal involvement of PI 3-kinase is supported by gene-knockout mouse models. Prototype inhibitors show evidence of anticancer activity in vitro and in vivo animal models. The recent development of isoform-selective inhibitors shows considerable promise for cancer treatment.

The molecular chaperone heat-shock protein 90 (HSP90) plays a key role in the cell by stabilizing a number of client proteins, many of which are oncogenic. The intrinsic ATPase activity of HSP90 is essential to this activity. HSP90 is a new cancer drug target as inhibition results in simultaneous disruption of several key signaling pathways, leading to a combinatorial approach to the treatment of malignancy. Inhibitors of HSP90 ATPase activity including the benzoquinone ansamycins, geldanamycin and 17-allylamino-17-demethoxygeldanamycin, and radicicol have been described. A high-throughput screen has been developed to identify small-molecule inhibitors that could be developed as therapeutic agents with improved pharmacological properties. A colorimetric assay for inorganic phosphate, based on the formation of a phosphomolybdate complex and subsequent reaction with malachite green, was used to measure the ATPase activity of yeast HSP90. The Km for ATP determined in the assay was 510+/-70 microM. The known HSP90 inhibitors geldanamycin and radicicol gave IC(50) values of 4.8 and 0.9 microM respectively, which compare with values found using the conventional coupled-enzyme assay. The assay was robust and reproducible (2-8% CV) and used to screen a compound collection of approximately 56,000 compounds in 384-well format with Z' factors between 0.6 and 0.8.

The molecular chaperone Hsp90 is an exciting cancer drug target. The first Hsp90 inhibitor to enter clinical trials--the geldanamycin derivative 17AAG--has recently demonstrated proof-of-concept for successful target modulation, with sighs of therapeutic benefit. An important property of Hsp90 inhibitors is their ability to cause simultaneous, combinatorial blockade of multiple cancer-causing pathways by promoting the degradation of many oncogenic client proteins. However, the reason for therapeutic selectivity in cancer cells versus normal cells is unclear. New research now shows that Hsp90 exists in cancer cells in a heightened, activated state that is highly susceptible to inhibition by 17AAG.

In eukaryotes, genomic DNA is packaged with histone proteins into the cell nucleus as chromatin, condensing the DNA > 10,000-fold. Chromatin is highly dynamic and exerts profound control on gene expression. Localised chromatin decondensation facilitates access of nuclear machinery. Chromatin displays epigenetic inheritance, in that changes in its structure can pass to the next generation independently of the DNA sequence itself. It is now clear that the post-translational modification of histones, for example, acetylation, methylation and phosphorylation, plays a crucial role in the regulation of nuclear function through the 'histone code'. There has been significant progress in identifying and understanding the enzymes that control these complex processes, in particular histone acetyltransferases and histone deacetylases. The exciting discovery that compounds inhibiting histone deacetylase activity also have antitumour properties has focused attention on their use as anticancer drugs. As a consequence, there is ongoing evaluation of several histone deacetylase inhibitor compounds in Phase I and II clinical trials with promising early results. It is likely that many of the enzymes involved in the control of histone modification will provide therapeutic opportunities for the treatment of cancer, including histone methyltransferases and Aurora kinases.

The detailed molecular basis and determinants of in vivo tumour sensitivity to conventional anticancer agents remain unclear. We examined the cellular and molecular consequences of cisplatin treatment using two ovarian tumour xenograft models that had not been previously adapted to culture in vitro. Both xenografts were curable with clinically relevant multiple doses of cisplatin. Following a single dose of cisplatin (6 mg kg(-1) i.p.) growth delays of 25 and 75 days were obtained for pxn100 and pxn65, respectively. This difference in response was not due to differences in DNA damage. Pxn100 tumours had a functional p53 response and a wild-type p53 sequence, whereas pxn65 harboured a mutant p53 and lacked a functional p53 response. Microarray analysis revealed the induction of p53-regulated genes and regulators of checkpoint control and apoptosis in pxn100 tumours following cisplatin-treatment. By contrast, there was no p53-dependent response and only limited changes in gene expression were detected in the pxn65 tumours. TUNEL analysis demonstrated high levels of apoptosis in the pxn100 tumours following cisplatin treatment, but there was no detectable apoptosis in the pxn65 tumours. Our observations show that a marked in vivo response to cisplatin can occur via p53-dependent apoptosis or independently of p53 status in human ovarian xenografts.

We review in detail how gene expression microarray technology is benefiting all phases of the discovery, development and subsequent use of new cancer therapeutics. Global gene expression profiling is valuable in cancer classification, elucidation of biochemical pathways and the identification of potential targets for novel molecular therapeutics. We exemplify the value in tissue culture and animal models of cancer, as well as in clinical studies. The power of expression profiling alongside gene knockout or knockdown methods such as RNA interference is illustrated. The use of basal or constitutive gene expression profiling to understand and predict drug sensitivity or resistance is described. The ability of expression profiling to define detailed molecular signatures of drug action is emphasised. The approach can identify on-target and off-target effects. It can be used to identify molecular biomarkers for proof of concept studies, pharmacodynamic endpoints and prognostic markers for predicting outcome and patient selection.

The development of new molecular therapeutics for cancer treatment is based on the identification of molecular abnormalities that drive the malignant process. The discovery of novel molecular targets is made quicker and more efficient by use of powerful high throughput technologies such as genome sequencing and gene expression microarrays. Similarly, the drug discovery process is being accelerated by a range of other valuable technologies. Using these approaches, we can expect to see a range of personalised molecular therapeutics becoming available for cancer treatment.

Inhibition of the ATPase activity of the chaperone protein HSP90 is a potential strategy for treatment of cancers. We have determined structures of the HSP90alpha N-terminal domain complexed with the purine-based inhibitor, PU3, and analogs with enhanced potency both in enzyme and cell-based assays. The compounds induce upregulation of HSP70 and downregulation of the known HSP90 client proteins Raf-1, CDK4, and ErbB2, confirming that the molecules inhibit cell growth by a mechanism dependent on HSP90 inhibition. We have also determined the first structure of the N-terminal domain of HSP90beta, complexed with PU3. The structures allow a detailed rationale to be developed for the observed affinity of the PU3 class of compounds for HSP90 and also provide a structural framework for design of compounds with improved binding affinity and drug-like properties.

Development of hypoxia-targeted therapies has stimulated the search for clinically applicable noninvasive markers of tumour hypoxia. Here, we describe the validation of [F-18]fluoroetanidazole ([F-18]FETA) as a tumour hypoxia marker by positron emission tomography (PET). Cellular transport and retention of [F-18]FETA were determined in vitro under air vs nitrogen. Biodistribution and metabolism of the radiotracer were determined in mice bearing MCF-7, RIF-1, EMT6, HT1080/26.6, and HT1080/1-3C xenografts. Dynamic PET imaging was performed on a dedicated small animal scanner. [F-18]FETA, with an octanol-water partition coefficient of 0.16+/-0.01, was selectively retained by RIF-1 cells under hypoxia compared to air (3.4- to 4.3-fold at 60-120 min). The radiotracer was stable in the plasma and distributed well to all the tissues studied. The 60-min tumour/muscle ratios positively correlated with the percentage of pO(2) values <5 mmHg (r = 0.805, P = 0.027) and carbogen breathing decreased [F-18]FETA-derived radioactivity levels (P = 0.028). In contrast, nitroreductase activity did not influence accumulation. Tumours were sufficiently visualised by PET imaging within 3060 min. Higher fractional retention of [F-18]FETA in HT1080/1-3C vs HT1080/26.6 tumours determined by dynamic PET imaging (P = 0.05) reflected higher percentage of pO(2) values <1 mmHg (P = 0.023), lower vessel density (P = 0.026), and higher radiobiological hypoxic fraction (P = 0.008) of the HT1080/1-3C tumours. In conclusion, [F-18]FETA shows hypoxia-dependent tumour retention and is, thus, a promising PET marker that warrants clinical evaluation. (C) 2004 Cancer Research UK.

Molecular chaperones are proteins that ensure the appropriate folding, stability and function of other proteins in the cell. There is increasing evidence that molecular chaperones are not only important in normal cell homeostasis and response to stresses, such as heat shock, but can also be involved in disease pathology. in particular, HSP90 has emerged as an important potential drug target in oncology. This is because it is essential for the stability of a long list of 'client proteins', including ErbB2/human epithelial growth factor receptor (HER)2, Raf-1, Akt/protein kinase B (PKB), Polo-1, Met, mutant p53 and human telomerase reverse transcriptase (hTERT), as well as the oestrogen and androgen receptor. Inhibition of HSP90 leads to depletion of these oncogenic clients by the ubiquitin proteasome pathway, thereby providing a simultaneous combinatorial attack on all of the hallmark phenotypic traits of cancer cells. Because of the rapid progress made, this review focuses on the patents dealing with the discovery and application of small molecule inhibitors of HSP90, although limited coverage of other applications in the area of HSP90 and additional chaperones is also included. Pioneering work with natural products, namely geldanamycin and related ansamycin antibiotics together with radicicol macrocycle derivatives, established the therapeutic potential of inhibiting the essential N-terminal ATPase of HSP90 by competing at the nucleotide binding site. A geldanamycin analogue, 17-(desmethoxy), 17-allylamino geldanamycin (17AAG), is completing Phase I clinical trials with promising initial results. The use of high-throughput screening and rational design based on X-ray crystal structures of HSP90 has led to the discovery of small molecule HSP90 inhibitors based on purine and pyrazole scaffolds. The continued progression of these various compound classes into clinical trials should help to establish proof of concept for inhibition of HSP90 as a Viable therapeutic approach for the treatment of cancer and potentially other diseases. This would in turn validate protein folding as a strategy for drug development and encourage additional chaperones to be explored as molecular targets.

There is currently much interest in developing analogues of the benzoquinone ansamycin geldanamycin that may overcome the limitations of 17-(allylamino)-17-demethoxygeldanamycin (17AAG), which is the first known inhibitor of heat shock protein 90 (Hsp90) to enter clinical trials. Studies were performed to assess whether cassette dosing, the coadministration of several compounds to a single animal, is a suitable approach to evaluate the preclinical pharmacokinetics of geldanamycin analogues in high throughput.

Tumor resistance to current drugs prevents curative treatment of human colon cancer. A pressing need for effective, tumor-specific chemotherapies exists. The non-receptor-tyrosine kinase c-Src is overexpressed in >70% of human colon cancers and represents a tractable drug target. KM12L4A human metastatic colon cancer cells were stably transfected with two distinct kinase-defective mutants of c-src. Their response to oxaliplatin, to SN38, the active metabolite of irinotecan ( drugs active in colon cancer), and to activation of the death receptor Fas was compared with vector control cells in terms of cell cycle arrest and apoptosis. Both kinase-defective forms of c-Src co-sensitized cells to apoptosis induced by oxaliplatin and Fas activation but not by SN38. Cells harboring kinase-defective forms of c-Src carrying function blocking point mutations in SH3 or SH2 domains were similarly sensitive to oxaliplatin, suggesting that reduction in kinase activity and not a Src SH2-SH3 scaffold function was responsible for the observed altered sensitivity. Oxaliplatin-induced apoptosis, potentiated by kinase-defective c-Src mutants, was dependent on activation of caspase 8 and associated with Bid cleavage. Each of the stable cell lines in which kinase-defective mutants of c-Src were expressed had reduced levels of Bcl-x(L). However, inhibition of c-Src kinase activity by PP2 in vector control cells did not alter the oxaliplatin response over 72 h nor did it reduce Bcl-x(L) levels. The data suggest that longer term suppression of Src kinase activity may be required to lower Bcl-x(L) levels and sensitize colon cancer cells to oxaliplatin-induced apoptosis.

STK15/Aurora2 is a centrosome-associated serine/threonine kinase, the protein levels and kinase activity of which rise during G2 and mitosis. STK15 overexpression induces tumorigenesis and is amplified in various human cancers and tumor cell lines. Thus, STK15 represents an important therapeutic target for small molecule inhibitors that would disrupt its activity and block cell proliferation. The availability of a robust and selective small molecule inhibitor would also provide a useful tool for identification of the potential role of STK15 in cell cycle regulation and tumor development. The authors report the development of a novel, fast, simple microplate assay for STK15 activity suitable for high-throughput screening. In the assay, gamma-(33)P-ATP and STK15 were incubated in a myelin basic protein (MBP)-coated FlashPlate(R) to generate a scintillation signal. The assay was reproducible, the signal-to-noise ratio was high (11) and the Z' factor was 0.69. The assay was easily adapted to a robotic system for drug discovery programs targeting STK15. The authors also demonstrate that STK15 is regulated by phosphorylation and the N-amino terminal domain of the protein. Treatment with phosphatase inhibitors (okadaic acid) or deletion of the N-amino terminal domain results in a significant increase in the enzymatic activity.

Drug discovery is an expensive, slow and high risk enterprise. Only one in ten of the agents that enter clinical development is successful, with an average cost of US dollars 500-800 million and a typical time-scale of 10-15 years from preclinical discovery research to regulatory approval. On the other hand, many new targets are emerging from genome sequencing and the improved understanding of molecular pathology. Also, new technologies are increasing the speed and improving the efficiency of drug discovery. These new advances should facilitate progress towards the development of personalised therapies that are targeted to the genetics and molecular pathology of individual patients. The availability of pharmacokinetic (PK) and pharmacodynamic (PD) endpoints is absolutely critical to modern drug development. They allow us to understand how much of the drug gets there (into the body and ideally to the target cells) and what it does (with respect to modulation of the molecular target and the cognate biochemical pathways and downstream biological effects). PK and PD endpoints allow us to construct a pharmacological audit trail, so that all of the successive stages from drug administration through to biological effects and clinical outcome can be monitored and interpreted. This in turn provides a rational basis for decision making, e.g. stop/go, during development. An understanding of PK/PD relationships also gives us s basis for selecting the optimal drug dose and schedule. Better, less invasive methods are required. Developments in molecular/functional imaging show promise and current examples are provided.

There are now unprecedented opportunities for the development of improved drugs for cancer treatment. Following on from the Human Genome Project, the Cancer Genome Project and related activities will define most of the genes in the majority of common human cancers over the next 5 years. This will provide the opportunity to develop a range of drugs targeted to the precise molecular abnormalities that drive various human cancers and opens up the possibility of personalized therapies targeted to the molecular pathology and genomics of individual patients and their malignancies. The new molecular therapies should be more effective and have less-severe side effects than cytotoxic agents. To develop the new generation of molecular cancer therapeutics as rapidly as possible, it is essential to harness the power of a range of new technologies. These include: genomic and proteomic methodologies (particularly gene expression microarrays); robotic high-throughput screening of diverse compound collections, together with in silico and fragment-based screening techniques; new structural biology methods for rational drug design (especially high-throughput X-ray crystallography and nuclear magnetic resonance); and advanced chemical technologies, including combinatorial and parallel synthesis. Two major challenges to cancer drug discovery are: (1) the ability to convert potent and selective lead compounds with activity by the desired mechanism on tumor cells in culture into agents with robust, drug-like properties, particularly in terms of pharmacokinetic and metabolic properties; and (2) the development of validated pharmacodynamic endpoints and molecular markers of drug response, ideally using noninvasive imaging technologies. The use of various new technologies will be exemplified. A major conceptual and practical issue facing the development and use of the new molecular cancer therapeutics is whether a single drug that targets one of a series of key molecular abnormalities in a particular cancer (e.g. BRAF) will be sufficient on its own to deliver clinical benefit ("house of cards" and tumor addiction models). The alternative scenario is that it will require either a combination of agents or a class of drug that has downstream effects on a range of oncogenic targets. Inhibitors of the heat-shock protein (HSP) 90 molecular chaperone are of particular interest in the latter regard, because they offer the potential of inhibiting multiple oncogenic pathways and simultaneous blockade of all six "hallmark traits" of cancer through direct interaction with a single molecular drug target. The first-in-class HSP90 inhibitor 17AAG exhibited good activity in animal models and is now showing evidence of molecular and clinical activity in ongoing clinical trials. Novel HSP90 inhibitors are also being sought. The development of HSP90 inhibitors is used to exemplify the application of new technologies in drug discovery against a novel molecular target, and in particular the need for innovative pharmacodynamic endpoints is emphasized as an essential component of hypothesis-testing clinical trials.

The tumor suppressor protein, pRb, regulates progression through the G1 phase of the cell cycle by its ability to bind to and regulate the activity of a variety of transcription factors. This function of pRb is disabled through its phosphorylation by the cyclin-dependent kinase (CDK) family of serine/threonine kinases. In many human cancers, genetic alteration such as loss of CDK inhibitor function and deregulated G1 cyclin expression leads to inappropriate phosphorylation and hence inactivation of this tumor suppressor. Identification of cell-permeable small molecules that block pRb phosphorylation in these tumors could therefore lead to development of an effective anticancer treatment. As a result, we have developed a high-throughput assay to detect changes in the level of pRb phosphorylation in cells. Signal detection is by a time-resolved fluorescence-based cellular immunosorbant assay on a fixed monolayer of cells. This comprises a mouse monoclonal antibody that recognizes the phosphorylated form of serine 608 on pRb, a known site of CDK phosphorylation, and a Europium-labeled secondary antibody for signal detection. The assay is reproducible and amenable to automation and has been used to screen 2000 compounds in a search for cell-permeable small molecules that will block pRb phosphorylation.

The Hsp90 molecular chaperone has emerged as one of the most exciting targets for cancer drug development. Hsp90 is overexpressed in many malignancies, very likely as a result of the stress that is induced both by the hostile cancer microenvironment and also by the mutation and abberant expression of oncoproteins. A particularly attractive feature of Hsp90 as a cancer drug target is that it is required for the conformational stability and function of a wide range of oncogenic 'client' proteins, including c-Raf-1, Cdk4, ErbB2, mutant p53, c-Met, Polo-1 and telomerase hTERT. Inhibition of Hsp90 should therefore block multiple mission critical oncogenic pathways in the cancer cell, leading to inhibition of all the hallmark traits of malignancy. This combinatorial blockade of oncogenic targets should give rise to board spectrum antitumour activity across multiple cancer types. The 'druggability' of Hsp90 was confirmed by the discovery that the natural products geldanamycin and radicicol, which have anticancer activity, exert their biological effects by inhibiting the essential ATPase activity associated with the N-terminal domain of the protein. The first-in-class Hsp90 inhibitor has entered clinical trial and provided proof of concept that Hsp90 can be inhibited and clinical benefit seen at non-toxic doses. Further development is underway and a related analogue 17DMAG also shows promise in preclinical models. In addition, novel Hsp90 inhibitors have been identified using methods such as high throughput screening and x-ray crystallography. The opportunities and challenges involved in translating the fast moving biology of Hsp90 into patient benefit is discussed.

HSP90 inhibitors such as 17AAG have the major therapeutic advantage that they exert downstream inhibitory effects on multiple oncogenic client proteins. They therefore block several mission critical cancer-causing pathways and have the potential to modulate all of the hallmark biological features of malignancy. Consistent with this combinatorial anti-oncogenic profile, 17AAG exhibits broad-spectrum antitumour activity against cultured cancer cell lines and in vivo animal models. However, there are clear differences in sensitivity between various cancer cell lines and it is quite possible that some tumour types or individual patients will be more responsive in the clinic than others. We describe the methods used to investigate the genes and proteins involved in the mechanism of action of HSP90 inhibitors and discuss the significance of these for cellular sensitivity. Methods used involve the conventional cell and molecular biology techniques, together with the more recent application of high throughput global technologies such as gene expression microarrays and proteomics. Selected examples that seem to play a role in sensitivity to HSP90 inhibitors are highlighted and the potential relevance to the response of cancer patients is discussed. Important determinants of response include: 1) Dependence upon key HSP90 client proteins such as ERBB2, steroid hormone receptors and AKT/PKB; 2) Levels of HSP90 family members and co-chaperones, such as HSP70 and AHA1; and 3) expression of various cell cycle and apoptotic regulators. In the case of 17AAG, metabolic enzymes such as NQO1 and membrane efflux pumps are also important for sensitivity.

The potential clinical applications of the prototype first-in-class Hsp90 inhibitor 17AAG and other emerging Hsp90 drugs are very exciting. Rigorously planned and executed clinical trials, incorporating measurement of appropriate biomarkers and pharmocodynamic endpoints are critical for selecting the optimal dose and schedule. A detailed understanding of the molecular mode of action of Hsp90 inhibitors alongside the elucidation of the molecular pathology of individual cancers will help us to identify tumour types and individual patients that will benefit most from treatment. Careful in vitro and in vivo experiments are needed to choose the most potentially advantageous combination studies. It is important to construct a pharmacologic audit trail linking molecular biomarkers and pharmacokinetic and pharmacodynamic parameters to tumour response endpoints. Phase I clinical studies with 17AAG have shown that the drug can be given at does that are well tolerated and that also achieve active pharmacokinetic exposures and elicit molecular signatures of gene and protein expression that are indicative of Hsp90 inhibition. Furthermore, examples of disease stabilisation have been documented, consistent with the generally cytostatic responses that are seen in animal models. Selecting tumour types for Phase II clinical trials must involve balancing 1) our knowledge of molecular response determinants, such as the expression of and dependence upon key client proteins and 2) more pragmatic evidence of antitumour activity in the relevant preclinical models. Examples of likely disease targets include chronic myeloid leukaemia, melanoma, breast, ovarian, brain, thyroid, colorectal and prostate cancer.

Global gene expression profiling has potential for elucidating the complex cellular effects and mechanisms of action of novel targeted anticancer agents or existing chemotherapeutics for which the precise molecular mechanism of action may be unclear. In this study, decreased expression of genes required for RNA and protein synthesis, and for metabolism were detected in rectal cancer biopsies taken from patients during a 5-fluorouracil infusion. Our observations demonstrate that this approach is feasible and can detect responses that may have otherwise been missed by conventional methods. The results suggested new mechanism-based combination treatments for colorectal cancer and demonstrated that expression profiling could provide valuable information on the molecular pharmacology of established and novel drugs.

We are in a new era of drug discovery, in which it is feasible to develop therapeutic agents targeted at a particular protein or biological activity in a living cell. This has been made possible by major advances in our understanding of cell and molecular biology, epitomized by the 2001 Nobel prize award for Physiology or Medicine to Lee Hartwell, Tim Hunt and Paul Nurse, who were recognised for their work on key regulators of the cell cycle. Technological advances have also played a decisive role, leading to the sequencing of the human genome and increased throughput at many stages of the drug discovery and development process. For example, developments in high throughput screening, structural biology and microarray technology are increasing the speed of drug discovery. In this chapter we focus on the long, and often difficult, pathway which leads from identification of a hit in a screen to regulatory approval of a drug for disease treatment. The emphasis in this chapter is on the development of anticancer drugs, as this is our own area of expertise and also because cancer is a disease in which the cell cycle is already a major target for therapeutic intervention. However, many of the concepts, approaches and issues are generally common to other therapeutic areas.

To perform a Phase I study of SR-4554, a fluorinated 2-nitroimidazole noninvasive probe of tumor hypoxia detected by (19)F magnetic resonance spectroscopy (MRS).

The epidemiology, genetics/genomics and molecular biology of cancer all point to the involvement of a large number of genes in the malignant progression of the vast majority of human cancers. Our current conceptual models of cancer are discussed here and are integrated with an assessment of the strategies required for treating and potentially curing human cancers driven by multiple genome abnormalities. There are settings in which excellent responses will be seen in cancers driven primarily by single genomic abnormalities, e.g., imatinib in chronic myeloid leukemia and gastrointestinal stromal tumors. Other multigenic cancers will require drug cocktails or single drugs acting on multiple downstream targets.

Studies of pharmacokinetics (which is what the body does to the drug) and pharmacodynamics (which is what the drug does to the body) are essential components of the modern process of cancer drug discovery and development. Defining the precise relationship between pharmacokinetics and pharmacodynamics is critical. It is especially important to establish a well understood pharmacological "audit trail" that links together all of the essential parameters of drug action, from the molecular target to the clinical effects. The pharmacological audit trail allows us to answer two absolutely crucial questions: (1) how much gets there; and (2) what does it do? During the pre-clinical drug discovery phase, it is essential that pharmacokinetic/pharmacodynamic (PK/PD) properties are optimized, so that the best candidate can be selected for clinical development. As part of contemporary mechanistic, hypothesis-testing clinical trials, construction of the pharmacological PK/PD audit trail facilitates rational decision-making. However, PK/PD endpoints frequently require invasive sampling of body fluids and tissues. Non-invasive molecular measurements, e.g. using MRI or spectroscopy, or positron emission tomography, are therefore very attractive. This review highlights the need for PK/PD endpoints in modern drug design and development, illustrates the value of PK/PD endpoints, and emphasises the importance of non-invasive molecular imaging in drug development. Examples cited include the use of PK/PD endpoints in the development of molecular therapeutic drugs such as the Hsp90 molecular chaperone inhibitor 17AAG, as well as the development of SR-4554 as a non-invasive probe for the detection of tumour hypoxia.

17-allylamino,17-demethoxygeldanamycin (17AAG) is a novel anticancer drug that inhibits heat shock protein 90 (Hsp90), resulting in proteasomal degradation of several oncogenic proteins. We used phosphorus magnetic resonance spectroscopy (31P-MRS) to determine whether 17AAG treatment leads to alterations in phospholipids that could serve as pharmacodynamic markers for tumor response to 17AAG.

The availability of a noninvasive method to detect and quantify apoptosis in tumours will enable tumour response to several cancer therapies to be assessed. We have synthesised two radiotracers, annexin V and the N-succinimidyl-3-iodobenzoic acid (SIB) derivative of annexin V, labelled with radio-iodine (I-124 and I-125) and provided proof of the concept by assessing specific binding and biodistribution of these probes to apoptotic cells and tumours. We have also assessed the tumour uptake of [I-124] annexin V in a mouse model of apoptosis. RIF-I cells induced to undergo apoptosis in vitro showed a drug concentration-dependent increased binding of [I-125] annexin V and [I-125] SIB-annexin V. In the same model system, there was an increase in terminal deoxynucleotidyl transferase-mediated nick end labelling (TUNEL)-positive cells and a decrease in clonogenic survival. Radiotracer binding was completely inhibited by preincubation with unlabelled annexin V. In RIF-1 tumour-bearing mice, rapid distribution of [I-125]SIB annexin V-derived radioactivity to kidneys was observed and the radiotracer accumulated in urine. The binding of [I-125]SIB-annexin V to RIF-1 tumours increased by 2.3-fold at 48 h after a single intraperitoneal injection of 5-fluorouracil ( 165 mg kg(-1) body weight), compared to a 4.4- fold increase in TUNEL-positive cells measured by immunostaining. Positron emission tomography images with both radiotracers demonstrated intense localisation in the kidneys and bladder. Unlike [I-124]SIB-annexin V, [I-124] annexin V also showed localisation in the thyroid region presumably due to deiodination of the radiolabel. [I-124]SIB-annexin V is an attractive candidate for in vivo imaging of apoptosis by PET.

Current anticancer drug development strategies involve identifying novel molecular targets which are crucial for tumourigenesis. The molecular chaperone heat shock protein (HSP) 90 is of interest as an anticancer drug target because of its importance in maintaining the conformation, stability and function of key oncogenic client proteins involved in signal transduction pathways leading to proliferation, cell cycle progression and apoptosis, as well as other features of the malignant phenotype such as invasion, angiogenesis and metastasis. The natural product HSP90 inhibitors geldanamycin and radicicol exert their antitumour effect by inhibiting the intrinsic ATPase activity of HSP90, resulting in degradation of HSP90 client proteins via the ubiquitin proteosome pathway. Anticancer selectivity may derive from the simultaneous combinatorial effects of HSP90 inhibitors on multiple cancer targets and pathways. 17-allylamino, 17-demethoxygeldanamycin (17AAG), a geldanamycin derivative, showed good activity and cancer selectivity in preclinical models and has now progressed to Phase I clinical trial in cancer patients with encouraging initial results. Phase II trials including combination studies with cytotoxic agents are now being planned and these should allow the therapeutic activity of 17AAG to be determined. Second generation HSP90 inhibitors may be designed to overcome some of the drawbacks of 17AAG, including limited oral bioavailability and solubility. They could also be engineered to target specific functions of HSP90, which may not only provide greater molecular selectivity and clinical benefit but may also increase understanding of the complex functions of this molecular chaperone. HSP90 inhibitors provide proof of concept for drugs directed at HSP90 and protein folding and this principle may be applicable to other medical conditions involving protein aggregation and stability.

High-throughput screening is an essential component of the toolbox of modern technologies that improve speed and efficiency in contemporary cancer drug development. This is particularly important as we seek to exploit, for maximum therapeutic benefit, the large number of new molecular targets emerging from the Human Genome Project and cancer genomics. Screening of diverse collections of low molecular weight compounds plays a key role in providing chemical starting points for iterative optimisation by medicinal chemistry. Examples of successful drug discovery programmes based on high-throughput screening are described, and these offer potential in the treatment of breast cancer and other malignancies.

Tumor hypoxia is associated with poor prognosis and a more malignant tumor phenotype. SR-4554, a fluorinated 2-nitroimidazole, is selectively bioreduced and bound in hypoxic cells. We present validation studies of SR-4554 as a noninvasive hypoxia marker detected by fluorine-19 magnetic resonance spectroscopy ((19)F MRS) in the P22 carcinosarcoma, a tumor with clinically relevant hypoxia levels.

Drug discovery is being revolutionised by a number of technological developments. These include high throughput screening, combinatorial chemistry and genomics. The impact of the new technologies is to accelerate the pace of anticancer discovery. The completion of the Human Genome Project and the ongoing high throughput sequencing of cancer genomes will facilitate the identification of a range of new molecular targets for drug discovery. Over the next few years we will have a complete molecular understanding of the various combinations of genes and cognate pathways that drive the malignant phenotype and tumour progression. The vision for postgenomic cancer drug discovery must be to identify therapeutic agents that correct or exploit each of these molecular abnormalities. In this way, it will be possible to develop personalised drug combinations that are targeted to the molecular make up of individual tumours. It is anticipated that these therapies will be more effective and less toxic than current approaches, although combinations of novel agents with existing cytotoxic therapies are likely to continue for some time. Examples of postgenomic, mechanism-based drugs include Glivec, Herceptin and Iressa, with many more agents undergoing preclinical and clinical development. An interesting new approach involves the development of inhibitors of heat shock protein (Hsp90) molecular chaperone. Because Hsp90 is required for the correct folding, stability and function of a range of oncoproteins that are mutated or over expressed in cancer, Hsp90 inhibitors have the potential to provide a simultaneous, combinatorial attack on multiple oncogenic pathways. By depleting the levels of multiple oncoproteins in cancer cells and blocking a wide range of oncogenic pathways, Hsp90 inhibitors have the potential to inhibit all of the hallmark characteristics of cancer cells. Progress in the preclinical and clinical development of Hsp90 inhibitors will be described, including an update on clinical studies with the first-in-class agent 17AAG. The use of the postgenomic technology of gene expression microarrays in cancer pharmacology and drug development will be exemplified.

ErbB2 is overexpressed in 25-30% of breast and ovarian cancers, correlates with poor prognosis and lower survival and has also been associated with chemoresistance. We have established an isogenic pair of human ovarian cells that differ only in the expression of erbB2 protein in order to elucidate the role of the protein in determining cellular sensitivity to various drugs and agents. These included cisplatin and paclitaxel, the main drugs used in the treatment of ovarian cancer, and also various signal transduction inhibitors affecting the ras and PI3K pathways. Transfection of erbB2 resulted in cells stably overexpressing the protein and showing increased motility compared to the empty vector control cells. In cells overexpressing erbB2, the most notable effect on chemosensitivity was that of significantly increased (5-fold) sensitivity to the heat shock protein 90 (HSP90) molecular chaperone inhibitor geldanamycin. In contrast, erbB2-overexpressing cells showed statistically significant resistance to cisplatin, the PI3K inhibitor LY294002 and the tyrosine kinase inhibitor emodin. No significant difference in growth inhibition was observed after exposure to paclitaxel, two additional HSP90 inhibitors radicicol and 17AAG, the cyclin-dependent kinase inhibitor flavopiridol, the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor PD153035, the mek inhibitor UO126 or the farnesyl transferase inhibitor R115777. Exposure of cells to geldanamycin, 17AAG, emodin, LY294002 and cisplatin led to depletion of erbB2 in the transfected cells. These data suggest that erbB2 status in ovarian cancr may contribute to chemosensitivity, in some cases leading to increased sensitivity (as with geldanamycin) but in other cases leading to resistance (as with cisplatin).

R115777 (Zarnestra) is a farnesyl protein transferase inhibitor currently undergoing worldwide clinical trials. As acquired drug resistance may limit the efficacy of the drug, a model of acquired resistance has been established in vitro by continuous drug exposure of the human colon cancer cell line KM12. A stably resistant cell line possessing 13-fold resistance to R115777 was generated. The resistant cells showed cross-resistance to another, structurally different farnesyl transferase inhibitor-277, but not to GGTI-298. A lack of cross-resistance was observed to a variety of other agents, which included clinically used drugs, such as doxorubicin, etoposide, cisplatin, and paclitaxel, as well as signal transduction blockers, such as the mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor UO126, the phosphatidylinositol 3'-kinase inhibitor LY294002, and the epidermal growth factor receptor tyrosine kinase inhibitor PD153035. Resistance did not appear to be related to differences in drug efflux pumps, such as P-glycoprotein or in drug accumulation. Total levels of farnesyl transferase protein subunits were similar in the parent and resistant cells, but, notably, the enzyme activity was markedly reduced in the resistant cell line compared with the parent cells. This was not because of a mutation in the enzyme or a difference in activation of the a-subunit of farnesyl transferase by phosphorylation. Hence, resistance to R115777 was generated; the mechanism of resistance in this model may be associated with the enzyme target of the inhibitor. The results suggest that the development of clinical resistance may occur with farnesyl protein transferase inhibitors.

There is presently enormous interest in the function and regulatory roles of histone acetyltransferase enzymes. Along with deacetylases it is now evident that these enzymes play a key role in many cellular processes including chromatin remodeling and gene transcription. As such, effective small molecule enzyme inhibitors would be useful tools for molecular pharmacology and may also be suitable for further development into agents for the treatment of diseases such as cancer, A high-throughput assay based on the use of scintillating microplates (FlashPlates) suitable for screening libraries of compounds for inhibitors of acetylase activity is described here. Confirmation of activity of selected compounds is achieved with a conventional filter assay, the details of which are also described. In addition, an assay suitable for confirming that cellular protein acetylation has been altered by inhibition of acetylases or deacetylases is also presented. On the same plate, cells are grown, exposed to compound, fixed, and permeabilized, and protein acetylation is determined using standard ELISA methodology and a europium-labeled second antibody. This latter method provides a medium-throughput alternative to the use of immunoblotting for mechanistic studies. (C) 2002 Elsevier Science (USA). All rights reserved.

There is an urgent need to develop non-invasive pharmacodynamic endpoints for the evaluation of new molecular therapeutics that inhibit signal transduction. We hypothesised that, when labelled appropriately, changes in choline kinetics could be used to assess geldanamycin pharmacodynamics, which involves inhibition of the HSP90 molecular chaperone-->Rafl -->Mitogenic Extracellular Kinase-->Extracellular Signal-Regulated Kinase I and 2 signal transduction pathway. Towards identifying a potential pharmacodynamic marker response, we have studied radiolabelled choline metabolism in HT29 human colon carcinoma cells following treatment with geldanamycin. We studied the effects of geldanamycin, on net cellular accumulation of (methyl-C-14) choline and (methyl-C-14) phosphocholine production. In parallel experiments, the effects of geldanamycin on extracellular signal-regulated kinase I and 2 phosphorylation and cell viability were also assessed. Additional validation studies were carried out with the mitogenic extracellular kinase inhibitor U0126 as a positive control; a cyclin-dependent kinase-2 inhibitor roscovitine and the phosphatidylinositol 3-kinase inhibitor LY294002 as negative controls. Hemicholinium-3, an inhibitor of choline transport and choline kinase activity was included as an additional control. In exponentially growing HT29 cells, geldanamycin inhibited extracellular signal-regulated kinase I and 2 phosphorylation in a concentration- and time-dependent manner. These changes were associated with a reduction in (methyl-C-14) choline uptake, (methyl-C-14) phosphocholine production and cell viability. Brief exposure to U0126, suppressed phosphocholine production to the same extent as Hemicholinium-3. In contrast to geldanamycin and U0126, which act upstream of extracellular signal-regulated kinase I and 2, roscovitine and LY294002 failed to suppress phosphocholine production. Our results suggest that when labelled with carbon-11 isotope, (methyl- C)choline may be a useful pharmacodynamic marker for the non-invasive evaluation of geldanamycin analogues. (C) 2002 Cancer Research UK.

Client protein activation by Hsp90 involves a plethora of cochaperones whose roles are poorly defined. A ubiquitous family of stress-regulated proteins have been identified (Aha1, activator of Hsp90 ATPase) that bind directly to Hsp90 and are required for the in vivo Hsp90-dependent activation of clients such as v-Src, implicating them as cochaperones of the Hsp90 system. In vitro, Aha1 and its shorter homolog, Hch1, stimulate the inherent ATPase activity of yeast and human Hsp90. The identification of these Hsp90 cochaperone activators adds to the complex roles of cochaperones in regulating the ATPase-coupled conformational changes of the Hsp90 chaperone cycle.

Antitumor and pharmacodynamic studies were performed in MCF-7 human breast cancer cells and companion xenografts with the farnesyl protein transferase inhibitor, R115777, presently undergoing Phase II clinical trials, including in breast cancer. R115777 inhibited growth of MCF-7 cells in vitro with an IC(50) of 0.31 +/- 0.25 microM. Exposure of MCF-7 cells to increasing concentrations of R115777 for 24 h resulted in the inhibition of protein farnesylation, as indicated by the appearance of prelamin A at concentrations >1 microM. After continuous exposure to 2 microM R115777, prelamin A levels peaked at 2 h post drug exposure and remained high for up to 72 h. R115777 administered p.o. twice daily for 10 consecutive days to mice bearing established s.c. MCF-7 xenografts induced tumor inhibition at a dose of 25 mg/kg [percentage of treated versus control (% T/C) = 63% at day 21]. Greater inhibition was observed at doses of 50 mg/kg (% T/C at day 21 = 38%) or 100 mg/kg (% T/C at day 21 = 43%). The antitumor effect appeared to be mainly cytostatic with little evidence of tumor shrinkage to less than the starting volume. Tumor response correlated with an increase in the appearance of prelamin A, but no changes in the prenylation of lamin B, heat shock protein 40, or N-Ras were detectable. In addition, significant increases in apoptotic index and p21(WAF1/CIP1) expression were observed, concomitant with a decrease in proliferation as measured by Ki-67 staining. An increase in prelamin A was also observed in peripheral blood lymphocytes in a breast cancer patient who responded to R115777. These data show that R115777 possesses preclinical antitumor activity against human breast cancer and that the appearance of prelamin A may provide a sensitive and convenient pharmacodynamic marker of inhibition of prenylation and/or response.

Hypoxia is important in tumor biology and therapy. This study compared the novel luminescence fiber-optic OxyLite sensor with the Eppendorf polarographic electrode in measuring tumor oxygenation. Using the relatively well-oxygenated P22 tumor, oxygen measurements were made with both instruments in the same individual tumors. In 24 air-breathing animals, pooled electrode pO(2) readings lay in a range over twice that of sensor pO(2(5min)) values (-3.2 to 80 mm Hg and -0.1 to 34.8 mm Hg, respectively). However, there was no significant difference between the means +/- 2 SE of the median pO(2) values recorded by each instrument (11.0 +/- 3.3 and 8.1 +/- 1.9 mm Hg, for the electrode and sensor respectively, P = 0.07). In a group of 12 animals treated with carbon monoxide inhalation to induce tumor hypoxia, there was a small but significant difference between the means +/- 2 SE of the median pO(2) values reported by the electrode and sensor (1.7 +/- 0.9 and 2.9 +/- 0.7 mm Hg, respectively, P = 0.009). A variable degree of disparity was seen on comparison of pairs of median pO(2) values from individual tumors in both air-breathing and carbon monoxide-breathing animals. Despite the differences between the sets of readings made with each instrument from individual tumors, we have shown that the two instruments provide comparable assessments of tumor oxygenation in groups of tumors, over the range of median pO(2) values of 0.6 to 28.1 mm Hg.

17-Allylamino-17-demethoxygeldanamycin (17AAG) is a first-in-class heat shock protein 90 (Hsp90) molecular chaperone inhibitor to enter clinical trials. The downstream molecular and cellular consequences of Hsp90 inhibition are not well defined. 17AAG has shown activity against human colon cancer in cell culture and xenograft models. In this study, we demonstrated that in addition to depleting c-Raf-1 and inhibiting ERK-1/2 phosphorylation in human colon adenocarcinoma cells, 17AAG also depleted N-ras, Ki-ras, and c-Akt and inhibited phosphorylation of c-AKT: A consequence of these events was the induction of cell line-dependent cytostasis and apoptosis, although the latter did not result from dephosphorylation of proapoptotic BAD: One cell line, KM12, did not exhibit apoptosis and in contrast to the other cell lines overexpressed Bag-1, but did not express BAX: Taken together with other determinants of 17AAG sensitivity, these results should contribute to a more complete understanding of the molecular pharmacology of 17AAG, which in turn should aid the future rational clinical development and use of the drug in colon and other tumor types.

Flavopiridol is a broad-spectrum inhibitor of cyclin-dependent kinases (cdks) and represents the first in this anticancer class to enter clinical trials. In anticipation of the likelihood that, as with other cancer drugs, acquired resistance may limit the drug's efficacy, an acquired resistance model has been established by in vitro drug exposure of the human colon carcinoma cell line HCT116. This stably resistant line, possessing 8-fold resistance to flavopiridol, showed a lack of cross-resistance to the anticancer agents etoposide, doxorubicin, paclitaxel, topotecan, and cisplatin, and notably to other chemical classes of cdk inhibitors: the aminopurines roscovitine and purvalanol A, 9-nitropaullone, and hymenialdisine. Resistance did not seem to be related to differences in the levels of multidrug resistance drug efflux proteins, P-glycoprotein, and MRP1. Moreover, there were no changes in overall drug accumulation between the resistant and sensitive cell lines. Flavopiridol induced cell cycle arrest, apoptosis, and inhibition of retinoblastoma gene product phosphorylation on serine 780 in both parental and resistant lines, but the latter required 8-fold higher concentrations to achieve these effects. Cyclin E protein levels and cyclin E-associated kinase activity were increased in the resistant line, suggesting that overexpression of cyclin E may be the mechanism of resistance to flavopiridol. However, transfection of cyclin E to increase expression of this protein did not result in an increase in resistance to flavopiridol. Thus, up-regulation of cyclin E alone does not seem to cause resistance to this cdk inhibitor.

The role of histone acetyltransferases (HATs) in the regulation of crucial cellular functions, e.g., gene transcription, differentiation, and proliferation, has recently been documented and there is increasing evidence that aberrant expression of these enzymes may have a role to play in the development of the malignant phenotype. The availability of potent and selective small molecule inhibitors of HATs would provide useful proof of principle probes for further validation of these enzymes as drug discovery targets and may also provide lead molecules for clinical drug development. We have developed a microplate assay for HAT activity suitable for high-throughput screening. In the assay, following incubation of histone H3, [3H]acetylCoA, and enzyme (recombinant p300/CBP-associated factor expressed as a glutathione S-transferase fusion protein), radiolabeled histone was captured onto the walls of a scintillating microplate (FlashPlate) generating a scintillation signal. The assay was reproducible, amenable to automation, and generated a wide signal to noise ratio. Although antiacetylated histone antibodies were initially used to capture the radiolabeled product, it was subsequently shown that a signal was effectively produced by histone passively binding to the walls of the FlashPlate. This resulted in a simple "mix and measure" assay that is currently being used for the identification of HAT inhibitors.

With the imminent completion of the Human Genome Project, biomedical research is being revolutionised by the ability to carry out investigations on a genome wide scale. This is particularly important in cancer, a disease that is caused by accumulating abnormalities in the sequence and expression of a number of critical genes. Gene expression microarray technology is gaining increasingly widespread use as a means to determine the expression of potentially all human genes at the level of messenger RNA. In this commentary, we review developments in gene expression microarray technology and illustrate the progress and potential of the methodology in cancer biology, pharmacology, and drug development. Important applications include: (a) development of a more global understanding of the gene expression abnormalities that contribute to malignant progression; (b) discovery of new diagnostic and prognostic indicators and biomarkers of therapeutic response; (c) identification and validation of new molecular targets for drug development; (d) provision of an improved understanding of the molecular mode of action during lead identification and optimisation, including structure-activity relationships for on-target versus off-target effects; (e) prediction of potential side-effects during preclinical development and toxicology studies; (f) confirmation of a molecular mode of action during hypothesis-testing clinical trials; (g) identification of genes involved in conferring drug sensitivity and resistance; and (h) prediction of patients most likely to benefit from the drug and use in general pharmacogenomic studies. As a result of further technological improvements and decreasing costs, the use of microarrays will become an essential and potentially routine tool for cancer and biomedical research.

New anticancer drugs are increasingly targeted to specific abnormalities in the sequence and expression of a series of genes that operate in a stepwise, combinatorial manner to drive the progression of human cancer. These new-generation molecular therapeutics are expected to be more effective and less toxic than the broadly antiproliferative cytotoxic drugs of the previous era, which still dominate medical treatment of cancer today. Molecular and genomic technologies, particularly the availability of the human genome sequence and the ongoing sequencing of cancer genomes, are now having a major impact on target discovery and validation. Over the past year, the progress of three novel anticancer agents in particular (Herceptin, Glivec and Iressa) has exemplified the potential utility of innovative molecular therapeutics in the clinic. Drugs acting on a range of new genome-based molecular targets are now in preclinical and clinical development.

Cancer drug therapy is undergoing a major transition from the previous pregenomic cytotoxic era to the new postgenomic era. Future mechanism-based therapeutic agents will increasingly be designed to act on molecular targets that are causally involved in the malignant progression of human cancers. Such agents are predicted to show greater therapeutic selectivity for cancer versus normal cells. New cancer drug targets are identified and validated in various ways. The determination of the normal human genome sequence, followed by that of multiple cancer genomes, is accelerating target discovery. Other new technologies, particularly high throughput screening, combinatorial chemistry and gene expression microarrays, are increasing the speed and efficiency of drug development. Examples of new molecular therapeutics showing promising activity in the clinic include Herceptin, Glivec and Iressa. However, many challenges remain as we test the vision of individualised combinatorial genome-based therapy, using drugs targeted to every significant molecular abnormality in cancer.

Kirsten-ras is frequently mutated in colorectal cancers and may be an important therapeutic target, particularly because we have previously shown that acquisition of a mutation is associated with a poorer outcome. Understanding the role of Kirsten-ras and the consequences of inhibiting its activity or expression will contribute to our comprehension of colorectal cancer biology and may help to rationalize the choice of molecular targets suitable for therapeutic manipulation. Therefore we undertook a simple screen, incubating a library of oligonucleotides with Kirsten-ras mRNA and RNase H to identify an antisense oligonucleotide that effectively inhibited Kirsten-ras expression. We show for the first time in a human colon cancer cell line that inhibition of Kirsten-ras expression inhibits constitutive phosphorylation of Erk1/2, but not c-Akt, suggesting that in these cells constitutive phosphorylation of Erk 1/2 is dependent upon Kirsten-ras. Successful inhibition of Kirsten-ras had little effect on cell number or cell death and there was no evidence for accumulation of cells in any particular phase of the cell cycle. Kirsten-ras inhibition significantly reduced secretion of VEGF-A165 into the culture medium. Gene expression profiling by microarray detected altered expression of a number of genes. Of particular interest for future studies was the altered expression of genes encoding products involved in protein trafficking and the potential effects of these changes on cell adhesion. Our results suggest that, at least in this model, Kirsten-ras may contribute to malignancy predominantly through effects on angiogenesis, invasion, and metastasis, and that therapies directed at Kirsten-ras, including antisense approaches, may have particular utility through these mechanisms.

Evidence suggests that DT-diaphorase is involved in the activation and mechanism of cytotoxicity of the investigational indoloquinone anticancer drug EO9 under aerobic conditions. Data also implicate a role for other enzymes including NADPH: cytochrome P450 reductase, especially in low DT-diaphorase tumour cells and under hypoxic conditions. Here, we used purified rat NADPH: cytochrome P450 reductase to provide additional evidence in support of a role for this enzyme in activation of EO9 to generate free radical and DNA-damaging species. Electron spin resonance spectrometry studies showed that NADPH: cytochrome P450 reductase reduced EO9 to a fret: radical species, including a drug radical (most likely the semiquinone) and reactive oxygen species. Plasmid DNA experiments showed that reduction of EO9 catalysed by NADPH: cytochrome P450 reductase results in single-strand breaks in DNA. The information obtained may contribute to the understanding of the molecular mechanism of DNA damage and cytotoxicity exerted by EO9 and may be useful in the design of future bioreductive drugs. (C) 2001 Elsevier Science Inc. All rights reserved.

Antiangiogenic and antivascular agents provide new approaches to treating tumours. These may avoid many of the problems experienced with current approaches such as inherent and acquired resistance to treatment. Tumours do not grow beyond 1-2 mm(3) in size without the development of new vessels (Folkman, 1971). Such neo-vascularization (angiogenesis) allows tumour cells to increase their nutrient supply, survive and proliferate despite the new vessels often having structural and functional differences compared to normal tissue vasculature. Treatments targeted at tumour vasculature have produced impressive results in animal models (Lindsay et al, 1996; Watson et al, 1996; O'Reilly, 1997; Horsman et al, 1998). These therapies are now entering clinical trials, However, the successful introduction of these therapies into clinical practice will require the development of reliable ways to assess angiogenesis and its modification or inhibition in vivo. Here we discuss some of the emerging imaging techniques that may be useful. (C) 2001 Cancer Research Campaign.

A number of molecular therapeutic agents, derived from exploiting our knowledge of the oncogenic pathways that are frequently deregulated in cancer, are now entering clinical trials. One of these is the novel agent 17-allylamino-17-demethoxygeldanamycin that acts to inhibit the hsp90 molecular chaperone. Treatment of four human colon cancer cell lines with iso-effective concentrations of this agent resulted in depletion of c-raf-1 and akt and inhibition of signal transduction. We have used gene expression array analysis to identify genes responsive to treatment with this drug. The expression of hsp90 client protein genes was not affected, but hsc hsp70, hsp90beta, keratin 8, keratin 18 and caveolin-1 were deregulated following treatment. These observations were consistent with inhibition of signal transduction and suggested a possible mechanism of resistance or recovery from 17-allylamino-17-demethoxygeldanamycin treatment. The results shed light on the molecular mode of action of the hsp90 inhibitors, and suggest possible molecular markers of drug action for use in hypothesis testing clinical trials. Oncogene (2000) 19, 4125 - 4133

Many tumors overexpress the NQO1 gene, which encodes DT-diaphorase (NADPH:quinone oxidoreductase; EC 1.6.99.2). This obligate two-electron reductase deactivates toxins and activates bioreductive anticancer drugs. We describe the establishment of an isogenic human tumor cell model for DT-diaphorase expression. An expression vector was used in which the human elongation factor 1alpha promoter produces a bicistronic message containing the genes for human NQO1 and puromycin resistance. This was transfected into the human colon BE tumor line, which has a disabling point mutation in NQO1. Two clones, BE2 and BE5, were selected that were shown by immunoblotting and enzyme activity to stably express high levels of DT-diaphorase. Drug response was determined using 96-h exposures compared with the BE vector control. Functional validation of the isogenic model was provided by the much greater sensitivity of the NQO1-transfected cells to the known DT-diaphorase substrates and bioreductive agents streptonigrin (113- to 132-fold) and indoloquinone EO9 (17- to 25-fold) and the inhibition of this potentiation by the DT-diaphorase inhibitor dicoumarol. A lower degree of potentiation was seen with the clinically used agent mitomycin C (6- to 7-fold) and the EO9 analogs, EO7 and EO2, that are poorer substrates for DT-diaphorase (5- to 8-fold and 2- to 3-fold potentiation, respectively), and there was no potentiation or protection with menadione and tirapazamine. Exposure time-dependent potentiation was seen with the diaziquone analogs methyl-diaziquone and RH1 [2, 5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone], the latter being an agent in preclinical development. In contrast to the in vitro potentiation, there was no difference in the response to mitomycin C when BE2 and BE vector control were treated as tumor xenografts in vivo. This isogenic model should be valuable for mechanistic studies and bioreductive drug development.

To our knowledge, 17-allylamino,17-demethoxygeldanamycin (17AAG) is the first inhibitor of heat shock protein 90 (Hsp90) to enter a phase I clinical trial in cancer. Inhibition of Hsp90, a chaperone protein (a protein that helps other proteins avoid misfolding pathways that produce inactive or aggregated states), leads to depletion of important oncogenic proteins, including Raf-1 and mutant p53 (also known as TP53). Given its ansamycin benzoquinone structure, we questioned whether the antitumor activity of 17AAG was affected by expression of the NQO1 gene, which encodes the quinone-metabolizing enzyme DT-diaphorase.

There is enormous potential for the discovery of innovative cancer drugs with improved efficacy and selectivity for the third millennium. In this review we show how novel mechanism-based agents are being discovered by focusing on the molecular targets and pathways that are causally involved in cancer formation, maintenance and progression. We also show how new technologies, from genomics through high through-put bioscience, combinatorial chemistry, rational drug design and molecular pharmacodynamic and imaging techniques, are accelerating the pace of cancer drug discovery. The process of contemporary small molecule drug discovery is described and progress and current issues are reviewed. New and potential targets and pathways for therapeutic intervention are illustrated. The first examples of a new generation of molecular therapeutics are now entering hypothesis-testing clinical trials and showing activity. The early years of the new millennium will see a range of exciting new agents moving from bench to bedside and beginning to impact on the management and cure of cancer.

DT-diaphorase has been implicated in the activation and mechanism of cytotoxicity of the investigational indoloquinone anticancer drug EO9. Here, we have used a highly purified DT-diaphorase isolated from rat Walker tumour cells to provide unambiguous evidence for the ability of this enzyme to catalyze reduction of EO9 and to provide a more detailed characterization of the reaction. Under the conditions used hypoxia had no effect on the initial rate of this reduction but did effect the nature and stability of metabolites formed. Electron spin resonance (ESR) spectrometry studies showed that DT-diaphorase reduced EO9 to a highly oxygen-sensitive metabolite that is probably the hydroquinone. In the presence of air, this metabolite is auto-oxidized to generate both drug- and oxygen-based radicals. Comproportionation:disproportionation reactions may also be involved in the generation of these radical species. The identification of these metabolites may contribute to the understanding of the molecular mechanism of DNA damage and cytotoxicity exerted by EO9. (C) 1998 Elsevier Science Inc.

Hypoxia occurs to a variable extent in a vast majority of rodent and human solid tumors. It results from an inadequate and disorganized tumor vasculature, and hence an impaired oxygen delivery. A probe for the non-invasive detection of tumor hypoxia could find important utility in the selection of patients for therapy with bioreductive agents, anti-angiogenic/anti-vascular therapies and hypoxia-targeted gene therapy In addition, tumor hypoxia has been shown to predict for treatment outcome following radio- or chemotherapy in human cancers, the underlying mechanism for which may involve hypoxia driving genetic instability and resulting tumor progression. Beyond oncology, utility can also be envisaged in stroke, ischemic heart disease, peripheral vascular disease, arthritis and other disorders. Design, validation, preclinical development and current status of a fluorinated 2-nitroimidazole, N-(2-hydroxy-3,3,3-trifluoropropyl)-2-(2-nitro-1-imidazolyl) acetamide (SR 4554, CRC 94/17), which has been rationally designed for the measurement of tumor hypoxia by magnetic resonance spectroscopy (MRS) and imaging (MRT), are reviewed. Application in positron emission tomography (PET) detection is also proposed. Design goals were: (i) a nitro group with appropriate redox potential for selective reduction and binding in hypoxic tumor cells; (ii) hydrophilic/hydrogen bonding character in the side chain to limit nervous tissue penetration and prevent neurotoxicity; and (iii)three equivalent fluorine atoms to enhance MRS/MRI detection, located in a metabolically stable position. Reduction of SR 4554 by mouse liver microsomes was dependent on oxygen content, with a half-maximal inhibition at 0.48 +/- 0.06%. SR 4554 underwent nitroreduction by hypoxic but not oxic tumor cells in vitro and electron energy loss spectroscopic analysis showed selective retention in the hypoxic regions of multicellular tumor spheroids. Pharmacokinetic design goals were met. In particular, low brain tissue concentrations were seen in contrast to excellent tumor levels, as measured by high performance liquid chromatography. The extent of this restricted entry to brain tumor was surprising given the overall octanol/water partition coefficient and was attributed to the hydrophilic/ hydrogen bonding character of the side chain. Quantitative MRS was used to assess the retention of F-19 signal in murine tumors and human tumor xenografts The 19F retention index (FRI; ratio of F-19 signal levels at 6 h relative to that at 45 min) ranged from 0.5 to 1.0 and 0.2 to 0.9 for murine tumors and human xenografts respectively. The correlation between SR 4554 retention and pO(2) was not a linear one, but when FRI was >0.5, the % pO(2) less than or equal to 5 mmHg was always >60%, indicating that high FRI was associated with low levels of oxygenation. Finally, whole body F-19-MRI in mice demonstrated that SR 4554 and related metabolites localized mainly in tumor, liver and bladder regions A selective MRS signal was readily detectable in tumors at doses at least 7-fold lower than those likely to cause toxicity in mice. We conclude that proof of principle is established for the use of SR 4554 as a non-invasive MRS/MRI probe for the detection of tumor hypoxia. Based on these promising studies, SR 4554 has been selected for clinical development.

The chemistry of the mitomycin C-related drug indoloquinone EO9 would suggest that its mechanism of action is likely to involve DNA damage after reductive activation. The ability of this agent to induce DNA damage in intact cells has been examined using alkaline filter elution. After treatment with pharmacologically relevant concentrations of EO9, both DNA strand breaks and interstrand cross-links were detected in rat Walker tumour cells and human HT29 colon carcinoma cells. These cell lines express relatively high levels of DT-diaphorase (NAD(P)H: quinone acceptor oxidoreductase), which is believed to be involved in EO9 activation. The extent of DNA damage was increased by approximately 30-fold under hypoxia in BE colon carcinoma cells that express non-functional DT-diaphorase, but this dramatic hypoxia enhancement was not seen in HT-29 cells. These data are consistent with cytotoxicity studies that indicate that DT-diaphorase appears to be important in EO9 activation under aerobic conditions, but other enzymes may be more relevant under hypoxia. The involvement of DT-diaphorase in DNA damage induction was further investigated using cell-free assays. DNA cross-links were detectable in plasmid DNA coincubated with EO9, cofactor and DT-diaphorase but not in the absence of this enzyme. In contrast, using a Tao polymerase stop assay, monofunctional alkylation was detected in plasmid DNA without metabolic activation, although the sequence selectivity was altered after reduction catalysed by DT-diaphorase.

Growth factors and certain oncogenes activate a range of phospholipid-mediated signal transduction pathways resulting in cell proliferation. Demethoxyviridin (DMV), a structural analogue of wortmannin and recently reported as a potent inhibitor of phosphoinositide-3-kinase, inhibited bombesin plus insulin-stimulated increase in cell number in Swiss 3T3 cells, a model of cell proliferation. The drug produced cytostatic effects at concentrations below 1 mu M and cytotoxic effects at 10 mu M. In intact Swiss 3T3 cells DMV inhibited insulin-stimulated PI 3- and 4-kinases and bombesin-stimulated phospholipases C, D and A(2) in the nanomolar range. DMV also inhibited bombesin-stimulated tyrosine phosphorylation of a range of proteins at nM concentrations. This study shows that DMV inhibited multiple stimulated signalling pathways which lead to increased Swiss 3T3 cell proliferation. A stable analogue of DMV may have chemotherapeutic potential. (C) 1997 Elsevier Science B.V.

A series of 36 nitrothiophene tyrphostins were synthesized, 32 of which were novel structures. Their ability to inhibit the epidermal growth factor (EGF) receptor tyrosine kinase was assessed in a cell-free assay. Compounds containing a dinitrile, 2-aminoethene-1,1-dinitrile or a thioamide group were good inhibitors of the receptor tyrosine kinase. Although anti-proliferative and cytotoxic activity was seen, no evidence of inhibition of EGF receptor autophosphorylation in intact cells was observed. The compounds showed no preferential inhibition of EGF-dependent proliferation of fibroblasts transfected with the EGF receptor. Furthermore, in a panel of squamous cell carcinoma cell lines with varying levels of EGF receptor expression, there was no selective cell kill of lines with the highest EGF receptor expression. The 2-nitro-5-substituted-thiophenes and the 2-nitro-3-substituted-thiophenes showed reduction potentials falling within the range likely to be reduced by cellular reducing agents, while the 2-nitro-4-substituted-thiophenes and 4-nitro-2-substituted-thiophenes did not. Compounds from the 2-nitro-5-substituted-thiophene series were shown to induce DNA damage, while no evidence of DNA damage was demonstrated with compounds from the 2-nitro-4-substituted-thiophene series. The 2-nitro-5-substituted-thiophene compound 4 showed significant tumour-type selectivity in the US National Cancer Institute human tumour cell line panel. The leukaemia cell lines were particularly sensitive to the compound, as were the majority of the colon cancer, melanoma and breast cancer cell lines, while the central nervous system-derived lines and the non-small cell lung cancer lines were particularly resistant. Further work is required to determine the precise mechanisms involved in these effects.

N-(2-Hydroxy-3,3,3-trifluoropropyl)-2-(2-nitro-1-imidazolyl) acetamide (SR-4554) is a fluorinated 2-nitroimidazole which has been rationally designed as a non-invasive probe for tumor hypoxia. The key selection criteria for this molecule were low central nervous system penetration and toxicity, high metabolic stability other than nitroreduction, good tumor uptake and high sensitivity for detection by magnetic resonance spectroscopy. As part of the pre-clinical development strategy, pharmacokinetic, bioavailability and biodistribution studies were performed in mice. Pharmacokinetic studies in mice demonstrated that SR-4554 was rapidly absorbed into plasma following i.p. administration and eliminated with a half-life of 42 min, similar to other 2-nitroimidazoles. By comparing the areas under the concentration-time curve (AUC), the tumor exposure towards SR-4554 was on average 84% of the value obtained for the plasma exposure. SR-4554 penetrated tumor tissue extremely well but, in contrast to misonidazole and certain other fluorinated analogues, its distribution into brain tissue was poor (AUCbrain/AUCplasma = 0.07), suggesting potentially lower toxicity in spite of its higher lipophilicity (P = 0.43 versus 0.63, respectively). The bioavailability of SR-4554 from i.p. and p.o. routes was 100 and 96% respectively. In non-tumor-bearing mice, SR-4554 was excreted mainly as unchanged drug. The percentage of the injected i.p. dose of SR-4554 excreted unchanged in the urine over 24 h was 68 +/- 8%. Neither SR-4554 nor its metabolites were detected in mouse feces. We propose that these favorable pharmacokinetic properties of SR-4554 are due to the hydrophilic character and hydrogen-bonding capability of the amide and hydroxyl functions in the compound.

NAD(P)H:quinone acceptor oxidoreductase (quinone reductase) (DT-diaphorase, EC 1.6.99.2) is involved in the process of reductive activation of cytotoxic antitumor quinones and nitrobenzenes. In this study, we initially examined the relative abilities of mouse, rat, and human quinone reductases to reduce two prodrugs, CB 1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] and EO9 [5-(1-aziridinyl)-3-(hydroxymethyl)-2-(3-hydroxy-1-propenyl)-1- methyl-1H-indole-4,7-dione]. By using Escherichia coil-expressed quinone reductases and evaluating them under identical conditions, we confirmed previous findings showing that the human enzyme is not as effective as the rat enzyme in reducing CB 1954 and EO9, although the two enzymes have similar NAD(P)H-menadione reductase activities. Interestingly, although the amino acid sequence of mouse quinone reductase is more homologous to that of the rat enzyme, we found that the mouse enzyme behaves similarly to the human enzyme in its ability to reduce these compounds and to generate drug-induced DNA damage. To determine the region of quinone reductase that is responsible for the catalytic differences, two mouse-rat chimeric enzymes were generated, MR-P, a chimeric enzyme that has mouse amino-terminal and rat carboxyl-terminal segments of quinone reductase, was shown to have catalytic properties resembling those of rat quinone reductase, and RM-P, a chimeric enzyme that has rat amino-terminal and mouse carboxyl-terminal segments of quinone reductase, was shown to have catalytic properties resembling those of mouse quinone reductase. In addition, MR-P and RM-P were found to be inhibited by flavones with K-l values similar to those for rat and mouse quinone reductases, respectively. Based on these results, we propose that the carboxyl-terminal portion of the enzyme plays an important role in the reduction of cytotoxic drugs and the binding of flavones.

Aerobic sensitivity to indoloquinone EO9 has been shown to correlate with cellular levels of the two-electron reducing enzyme DT-diaphorase. However, little is known about the relative roles of one- and two-electron reducing enzymes in the hypoxic cytotoxicity of EO9. We have characterised a panel of 23 human tumour cell lines for both bioreductive enzyme activities and aerobic sensitivity to EO9. Eight cell lines were then selected for a comparison of aerobic and hypoxic sensitivities. Activities of DT-diaphorase showed a wide range (> 10,000-fold), while activities of the one-electron reducing cytochrome b5 and cytochrome P450 reductases were generally lower and showed only a 15- and 25-fold range respectively. The aerobic cytotoxicity of EO9 was clearly related to the cellular levels of DT-diaphorase (r = 0.87), with higher levels giving increased sensitivity, but not to the levels of one-electron reducing enzymes. In contrast, there was no relationship between sensitivity to BCNU, cisplatin or the bioreductive agent SR 4233 (tirapazamine) and activities of any of these reducing enzymes. Under hypoxic conditions sensitivity to EO9 was markedly increased in cell lines with low levels of DT-diaphorase activity, while cell lines with high levels show only a small increase in sensitivity. This is reflected by a clear correlation (r = 0.98) between cellular DT-diaphorase activity and the ratio of aerobic to hypoxic sensitivity to EO9. However, we have now for the first time demonstrated an inverse correlation (r = 0.93) between the cellular activity of DT-diaphorase and hypoxic sensitivity to EO9, that is sensitivity decreases with increasing DT-diaphorase activity. Moreover, this correlation was lost when cells were exposed to drug in the presence of dicoumarol, supporting an involvement of DT-diaphorase in this relationship. These observations question the previously straightforward role for DT-diaphorase in the metabolic activation of EO9. Whereas DT-diaphorase is associated with increased toxicity in air, it appears to reduce the cytotoxicity of EO9 in hypoxic conditions. This suggests either that the one-electron reduction product of EO9 metabolism, the semiquinone, is more toxic than the two-electron reduction product, the hydroquinone, or that the hydroquinone is not cytotoxic and aerobic toxicity is due to the transient appearance of the semiquinone upon back oxidation of the hydroquinone.

A series of benzylidenemalononitrile derivatives previously synthesized by condensing aromatic aldehydes with malononitrile derivatives are known as tyrphostins. In this study, 32 tyrphostins were synthesized, 19 of which are novel compounds. Both hydroxylated derivatives and compounds containing heteroaromatic moieties were prepared. We have confirmed and extended the observation that the tyrphostins displayed an enhancement in their ability to inhibit the epidermal growth factor (EGF) receptor tyrosine kinase domain as the number of hydroxyl groups on the aromatic portion was increased. IC50 values of 1-5 microM were readily achieved. Some inhibitory activity was seen with the heteroaromatic structures, with two compounds exhibiting IC50 values of 56 and 77 microM. However, these derivatives were poor inhibitors of the EGF receptor tyrosine kinase activity as compared to the hydroxylated derivatives. The ability of the 32 tyrphostins synthesized in the present study to inhibit proliferation of a human breast adenocarcinoma cell line (MCF-7) was determined using [3H]thymidine incorporation as a measure of DNA synthesis. Some of the compounds containing pyridine, imidazole or thiophene portions displayed antiproliferative activity comparable to that of tyrphostins prepared from 3,4,5-trihydroxybenzaldehyde. The lack of inhibitory effect of these heteroaromatic compounds on the EGF receptor tyrosine kinase activity suggests that their antiproliferative activity is not related to inhibition of EGF receptor function. As the growth of the MCF-7 cell line is governed by other factors, such as the insulin-like growth factors (IGFs) and oestradiol, it is also still to be established whether the antiproliferative activity of the hydroxylated tyrphostins is directly related to inhibition of the EGF receptor tyrosine kinase activity.

The epidermal growth factor (EGF) receptor is overexpressed in squamous cell carcinoma and the EGF receptor has been proposed as a potential target for new therapeutic agents in this tumour type. We have utilized a tyrphostintype inhibitor of the EGF receptor tyrosine kinase domain (RG50864) to study EGF-dependent proliferation and phosphorylation in two human squamous cell carcinoma cell lines. There were selected on the basis that whereas both cell lines have a large number of EGF receptors, one is growth inhibited by EGF (A431) while the proliferation of the other cell line (B2A4) is stimulated by EGF. EGF induced receptor autophosphorylation in each of the two cell lines; however, the level of phosphorylation was greater in the A431 cells than in the B2A4 cells. The pattern of proteins phosphorylated in response to EGF was different in the two squamous cell lines. RG50864 antagonized the EGF-dependent proliferation of B2A4 cells, but was unable to reverse the inhibitory effect of EGF on A431 cell growth. RG50864 partially inhibited EGF receptor autophosphorylation in both cell lines and completely inhibited the EGF-dependent phosphorylation of other cellular proteins, one of which co-migrated with MAP2kinase in both cell lines. Moreover, different dose-response relationships for the inhibition of phosphorylation of various proteins were observed in A431 versus B2A4 cells. As a substrate competitive inhibitor of the EGF receptor tyrosine kinase, the primary mode of action of RG50864 may be to prevent the association and/or phosphorylation of multiple specific substrates of the receptor in a fashion which may be cell line dependent. The precise relationship of these phosphorylation events to tyrphostin sensitivity remains to be established.

3-amino-1,2,4-benzotriazine-1,4-di-N-oxide (tirapazamine, WIN 59075, SR 4233, NSC 130181) has entered phase 1 clinical trials as a bioreductive hypoxic cell cytotoxin because of its novel structure and impressive selective cytotoxicity towards hypoxic cells. Understanding the enzymology and underlying mechanism of oxidative and reductive DNA damage may allow more optimal development and use of this agent and contribute to the rational design of new bioreductive drugs. Here we provide unambiguous evidence that WIN 59075 undergoes one-electron reduction by purified rat liver NADPH:cytochrome P450 oxidoreductase to generate single- and double-strand breaks in plasmid DNA. The DNA damage caused may be important for the therapeutic toxicity of the drug. Enzyme kinetic parameters for this oxidoreductase reaction are in the range 1.01-1.61 mM for Km and 4416-5099 nmol/min/mg for Vmax. The relative levels of expression and cellular localization of target tumour NADPH:cytochrome P450 oxidoreductase may contribute to the therapeutic selectivity of WIN 59075.

We examined the ability of platelet-activating factor (PAF) and its lyso derivative (lyso-PAF) to elicit increases in intracellular free calcium concentration ([Ca2+]i) in HT29 human colon carcinoma cells. Using spectrofluorimetric analysis with indo-1 as the [Ca2+]i reporter molecule, we found that 1-10 microM concentrations of both lipids stimulated substantial, reversible, monophasic [Ca2+]i elevations. Evidence was obtained that the two lipids may act via specific receptors to release Ca2+ from internal stores. Homologous desensitization was observed in both cases and PAF and lyso-PAF were also able to desensitize cells reciprocally (heterologous desensitization). The potent PAF receptor antagonist WEB 2086 (3-[4-(chlorophenyl)-9-methyl-6H-thieno[3,2-f][1,2, 4]triazolo-[4,3-a][1,4]-diazepin-2-yl]-1-(4-morpholinyl)-1-propano ne) successfully blocked PAF-induced [Ca2+]i elevations, but did not affect rises in response to lyso-PAF, suggesting that lyso-PAF may act through a different cellular receptor or mechanism. Higher concentrations (> 10 microM) of PAF resulted in non-reversible [Ca2+]i elevations which were caused by Ca2+ influx following membrane lysis. However, the WEB 2086 insensitivity of these effects and the resultant cellular toxicity clearly showed that such events were mechanistically distinct from the reversible [Ca2+]i elevations apparently operating via WEB 2086-sensitive receptors. Preliminary spatio-temporal observations, using confocal microscopy and fluo-3 as the [Ca2+]i reporter molecule, suggested that PAF can also induce [Ca2+]i elevations in the absence of cell lysis in monolayer HT29 cells. Visual impressions were obtained of cellular and subcellular heterogeneity and of [Ca2+]i oscillations in responding cells. However, these need to be interpreted with caution because of the intrinsic limitations of the methodology, particularly using non-ratiometric dyes. The significance of a receptor-mediated, reversible elevation of [Ca2+]i by sub-toxic concentrations of PAF in HT29 colon cancer cells remains to be elucidated, but it is tempting to speculate that PAF might function as a locally acting signalling mediator in these and possibly other tumour cells.

Although a number of bioreductive agents are substrates for purified DT-diaphorase the role of this enzyme in either activation or detoxification of these agents in the whole cell is unclear. The aim of this study was to determine the role of DT-diaphorase in the metabolic activation of EO9 under both aerobic and hypoxic conditions.

Studies with purified DT-diaphorase have shown that the enzyme is capable of catalyzing a two-electron reduction of the novel indoloquinone EO9 to a DNA-damaging alkylating species. The aim of this study was to determine to what extent DT-diaphorase may be involved in the metabolic activation of EO9 and mitomycin C in both aerobic and hypoxic conditions. Two human colon-carcinoma cell lines were used; HT29 has high levels of DT-diaphorase whilst BE lacks this activity because of a point mutation in the NQOI gene. In aerobic conditions the 2 cell lines show similar sensitivities to a number of cytotoxic drugs including cisplatin, doxorubicin and etoposide. They are equally sensitive to the benzotriazine di-N-oxide SR 4233 but HT29 is more sensitive than BE to mitomycin C and EO9. Sensitivity to SR 4233 is increased by about 100-fold for both cell lines in hypoxic conditions. DT-diaphorase-deficient BE cells show markedly increased sensitivity to mitomycin C and particularly EO9 in hypoxic conditions, whereas DT-diaphorase-rich HT29 cells show little hypoxic sensitization to these agents unless exposed in the presence of dicoumarol. These results suggest that DT-diaphorase can reduce EO9 and mitomycin C to potent cytotoxic species in aerobic conditions, and this activity predominates over the one-electron-reducing enzymes even in hypoxic conditions. In the absence of DT-diaphorase activity, EO9 and mitomycin C are reduced in hypoxic conditions, presumably by one-electron-reducing enzymes, to a similar or greater extent than is achieved with DT-diaphorase.

In order to address the association of enhanced drug efflux with the multidrug-resistant (MDR) phenotype, we have studied the cellular pharmacokinetics of anthracyclines in the P-glycoprotein (Pgp)-positive MDR cell lines H69/LX4 (human small cell lung cancer) and EMT6/AR1.0 (mouse mammary tumour). Both doxorubicin (DOX) and daunorubicin (DNR) were accumulated to a lesser extent and effluxed at a higher rate by MDR cells than by their drug-sensitive counterparts. In contrast, the 9-alkyl substituted compound, aclacinomycin A (ACL), was accumulated and effluxed from parent and MDR cells at an identical rate. In experiments designed to examine energy-dependent efflux, DOX and DNR were shown to be efficiently effluxed against the concentration gradient in the presence of glucose. However, in the same experiments the analogues ACL and Ro 31-3294 (9-alkyl and morpholinyl substituted), which have previously been shown to retain activity against MDR cell lines, were accumulated and effluxed at identical rates in parent and MDR EMT6 cells. Hence, 9-alkyl and morpholinyl substituted compounds appear to behave less favourably as substrates for energy-driven drug efflux by Pgp-positive MDR cells than do DOX or DNR. Resistance modifiers verapamil and cyclosporin A appeared to abolish energy-dependent efflux for DOX and DNR in both the EMT6 and H69 MDR lines whereas they had no effect on the cellular efflux of ACL. The altered cellular pharmacology in MDR cell lines may provide a rational basis for the use of modified anthracycline analogues (e.g. 9-alkyl and morpholinyl (substituted) and resistance of modifying agent in the treatment of tumours expressing a Pgp-mediated phenotype.

This study highlights the usefulness of laser scanning confocal microscopy in the examination of subcellular disposition of anthracyclines in tumour cell lines. The distribution of anthracycline compounds has been studied in two pairs of parental and multidrug resistant (MDR) cell lines. For the parental EMT6 mouse mammary tumour cell line EMT6/P treated with doxorubicin (DOX) the anthracycline fluorescence was shown to be predominantly nuclear but with some particulate cytoplasmic fluorescence and very low levels of plasma membrane staining. In the same experiments much fainter fluorescence was seen for the EMT6/AR1.0 MDR subline which hyperexpresses P-glycoprotein. The loss of nuclear fluorescence was comparatively greater than loss of cytoplasmic fluorescence. For the human large cell lung cancer line COR-L23/P cellular DOX disposition was markedly nuclear with nuclear membrane staining and diffuse cytoplasmic fluorescence. For the MDR line COR-L23/R, which lacks P-glycoprotein expression, DOX fluorescence was reduced in the nucleus compared with the parental line, but an intense area of perinuclear staining was seen consistent with localisation to the Golgi apparatus. The morpholinyl-substituted analogue MR-DOX achieved very similar subcellular distribution in both parental and MDR lines, consistent with its retention of activity in the latter. The presence of verapamil during anthracycline exposure increased the intensity of fluorescence in the MDR lines, particularly in the nucleus. Relatively little effect was seen in the parental lines. Confocal microscopy provides high resolution images of the subcellular distribution of anthracyclines in parent and MDR cell lines. Differences in drug disposition in various cell lines may provide insights into the mechanism of multidrug resistance and suggest strategies for its therapeutic circumvention.

We describe an improved technique which allows the analysis of enzyme reaction kinetics for gamma-glutamyl transpeptidase (gamma-GT) by flow cytometry. This is technically difficult because of the location of the enzyme on the external surface of the cell membrane leading to the rapid escape of the product. The reaction is determined by monitoring the conversion of gamma-glutamyl aminomethylcoumarin to aminomethylcoumarin. Reaction kinetics are described for BL8 hepatocyte and JB1 hepatoma cells lines, together with inhibition kinetics for the active site-directed glutamine analogue L-(alpha-S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid. We show that it is possible to follow the reaction dynamics in a heterogeneous mixture of BL8 and JB1 cells allowing discrimination of the two cell types based on gamma-GT activity. Improvements for further optimizing the assay of this important enzyme are suggested.

Cancer pharmacokinetics has made an enormous contribution to the development and optimization of cancer therapy. While consolidating its position and subjecting itself to prospective clinical trials in areas of established pharmacology, it needs also to keep constantly on the move in order to respond to the challenging demands of more futuristic approaches. Antibodies, cytokines, nucleic acid drugs and therapeutic genes will be metabolized and excreted and will in addition display special difficulties in terms of tissue and cell uptake. Pharmacokinetic considerations must accompany all new experimental therapies for scientific reasons as well as to fulfil regulatory requirements (Peck et al, 1992). The very latest approaches will understandably be seen by many practising clinicians as science fiction--for example obtaining a genetic fingerprint of a patient's tumour in order to guide corrective therapy, whether it be based on a chemical drug or a replacement gene. But it is from such speculative attempts that the real breakthroughs of the future may follow and pharmacokinetic/pharmacodynamic studies are essential to these efforts. In terms of the role of pharmacokinetics and pharmacodynamics in day to day management of individual patients, we do need to be more pragmatic. We must address tough questions such as: Can the assays be conducted on a routine basis? Do the measurements affect treatment outcome? Are they cost effective? Will they actually be used by clinical and nursing staff in a busy hospital environment? There is ample evidence in the following pages that pharmacokinetics will continue to prove vital to support both routine patient management and the exciting new approaches to cancer therapy.

The five examples given here illustrate new cytotoxic agents at different stages of evaluation. In all cases, considerable effort has gone into detailed pharmacokinetic studies conducted before and during the clinical phase I studies. Has this effort contributed significantly to the development of these agents? At present, it has to be said that the contribution made in the case of these particular agents has been modest. For the anthrapyrazoles, the availability of the pharmacokinetic data did not permit a pharmacokinetically guided dose escalation to be performed because of non-linear kinetics, and a similar comment can be made for rhizoxin, since the human plasma AUC values at the MTD were much lower than in the mouse. For the camptothecin analogues, a detailed knowledge of the kinetics of the closed and open forms of the various agents did not influence the way in which the studies were conducted, nor did pharmacokinetic information appreciably do so for EO9, although some comfort was gained by clinical investigators when the short half-life seen in preclinical species was also observed in humans. For suramin, therapeutic drug monitoring is clearly essential, although toxicity remains a problem. Of course, a proper understanding of the pharmacokinetics and metabolism of these agents greatly improves the interpretation of the clinical observations made and is often critical in planning the next stages of development. This is more clearly seen with agents that have unusual forms of toxicity, such as flavone acetic acid, for which the achievement of notional target concentrations is a key element in clinical trials (Kerr et al, 1987; Maughan et al, 1992). Moreover, as reviewed elsewhere (Graham and Workman, 1992; see also Graham and Kaye, this volume), there are several other instances where pharmacokinetically guided dose escalation has greatly improved the conduct of a phase I study. Good examples of this are iododoxorubicin (Gianni et al, 1990), mitotic inhibitor CI-980 (Brodfuehrer et al, 1992) and DNA intercalator CI-958 (Whitfield et al, 1992). Not surprisingly then, pharmacokinetics can help guide early clinical studies of some compounds but not others and whether they will be of value can only be determined by carrying out the pharmacokinetic measurements. The real value of the pharmacokinetic studies for the five compounds reviewed may not yet have been seen. Interpatient variations in drug handling can play a major part in determining levels of anti-tumour activity as well as toxicity.(ABSTRACT TRUNCATED AT 400 WORDS)

Bioreductive drugs undergo metabolic reduction to generate cytotoxic metabolites. This process is facilitated by bioreductive enzymes and the lower oxygen conditions present in solid tumours compared to normal tissues. Because of this specificity, bioreductive drugs have enormous potential to contribute to modern cancer therapy. Examples undergoing clinical trials include N-oxides such as tirapazamine, aziridinylnitroimidazoles RSU 1069/RBU 6145 and quinones such as indoloquinone EO9. Other novel structures are also under study. Here we review the experimental development of bioreductive drugs and their role in cancer therapy.

The novel imidazoisoquinoline SDZ 62-434, originally identified as a platelet-activating factor (PAF) antagonist, has antiproliferative activity in a range of cell lines from human solid and haematological malignancies. Using an MTT cytotoxicity assay, IC50 values of 5 microM - 111 microM were observed following a 24 h exposure. Similar results were obtained using a clonogenic assay. The HT29 colon adenocarcinoma was particularly sensitive while the MCF-7 breast carcinoma was the most resistant in our panel. Only a 2-3 fold cross-resistance was seen in the doxorubicin and cisplatin resistant variants of the A2780 ovarian carcinoma; the drug did not modulate sensitivity to doxorubicin in either parent or resistant lines. No cross-resistance to SDZ 62-434 was seen in a doxorubicin-resistant MCF-7 variant. Cytotoxicity was not due to non-specific membrane lysis. The potent PAF antagonist WEB 2086 did not modulate SDZ 62-434 cytotoxicity, indicating no role for PAF receptors. Precursor incorporation studies in A2780 cells showed that DNA synthesis was inhibited more effectively than protein synthesis while RNA synthesis was unaffected. SDZ 62-434 inhibited both bombesin and platelet-derived growth factor-induced DNA synthesis in quiescent Swiss 3T3 cells. This suggests a possible role for SDZ 62-434 as an inhibitor of signal transduction in cancer cells.

Membrane-active antitumour ether lipids such as ET18-OMe (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) and SRI 62-834 ((+-)-2-(Hydroxy[tetrahydro-2-(octadecyloxy) methylfuran-2-yl] methoxyl phosphinyloxy)-N,N,N-trimethylethaniminium hydroxide) are selectively cytotoxic to tumour cells in vitro. Their precise mechanisms of action are unclear, but they are known to have effects on cell membranes and cell signalling. A previous report suggested that ether lipids cause a biphasic sustained rise in intracellular free calcium [Lazenby et al., Cancer Res 50: 3327-3330, 1990]. We show here that the second phase is an experimental artefact due to cell membrane permeabilization by ether lipids in serum-free buffers. In serum-free medium, the membrane toxicity of antitumour ether lipids was increased 50-60 fold, when compared to medium containing 10% serum. Membrane disruption was neither dependent on extracellular calcium, nor modulated by preloading cells with the calcium chelators bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or 2-[[2-[bis(carboxymethyl)amino]-5-methylphenoxy]methyl]-6- methoxy-8-[bis(carboxymethyl)amino]quinoline. This indicates that the mechanism of membrane damage by ether lipids does not involve changes in calcium homeostasis. Using indo-1 and fura-2 as calcium probes, we established that lower concentrations of antitumour ether lipids do elicit a genuine monophasic and transient rise in intracellular free calcium, predominantly mobilized from internal stores. This acute calcium agonist activity of ether lipids is distinct from the inhibitory effects on cell signalling reported previously after more prolonged exposure. It appears that the calcium elevation induced by antitumour ether lipids is unlikely to be instrumental in their selective and potent antitumour activity.

The benzotriazine di-N-oxide SR 4233 (tirapazamine, WIN 59075) is currently in phase I clinical trials as the lead compound in a series of novel and highly selective antitumour hypoxic cytotoxins. Reductive bioactivation is thought to proceed via a one-electron reduced, oxidizing nitroxide radical and also forms the inactive single N-oxide SR 4317 via radical disproportionation or a second one-electron reduction. In mouse liver microsomes reductive metabolism is catalysed predominantly by cytochrome P450 (70%) and cytochrome P450 reductase (30%). The aim of the present study was to examine which cytochrome P450 isozymes may be involved. Reduction of SR 4233 to SR 4317 was monitored by HPLC analysis. Metabolism by microsomes from both control and dexamethasone-induced BALB/c male mice was 70% inhibited by carbon monoxide. The cytochrome P450 inhibitor SKF 525A, following aerobic preincubation, also inhibited SR 4233 reduction by 58%. Reduction was induced 2-3-fold by dexamethasone and was not accountable by increases in cytochrome P450 reductase or DT-diaphorase. The induction data and the greater degree of inhibition of SR 4233 reduction by metyrapone compared to alpha-naphthoflavone suggested a possible involvement of Cyp2b, Cyp2c and Cyp3a cytochrome P450 subfamilies. Both Cyp3a (7.4-fold) and Cyp2b (1.8-fold) type enzymes were shown by western immunoblot analysis to be induced by dexamethasone, the latter correlating more closely with increased SR 4233 reductase activity and also with the 2-fold induction of benzphetamine N-demethylase, a Cyp2b-type enzyme. No inhibition of SR 4233 reduction was seen with erythromycin or cyclosporin A which act as substrates/inhibitors for Cyp3a-type enzymes, but inhibition was seen with p-nitrophenol and tolbutamide which are substrates for Cyp2el- and Cyp2c-type enzymes, respectively (11% and 25% inhibition in induced microsomes). SR 4233 itself inhibited benzphetamine N-demethylase, which is catalysed by Cyp2b-type enzymes but not erythromycin N-demethylase which is catalysed by Cyp3a-type isoforms. Immunoinhibition studies with epitope specific monoclonal antibodies were consistent with the major involvement of phenobarbitone- and steroid-inducible products of the Cyp2b and Cyp2c subfamilies. These forms contributed at least 53% and 26%, respectively, of the cytochrome P450-associated SR 4233 reductase activity in the induced microsomes. The findings support our earlier conclusion that cytochrome P450 is the major SR 4233 reductase in mouse liver and provides leads as to the possible involvement of specific isoforms in human tumours and normal tissues.

The effect of combining the oxygen-transport-modifying drug BW12C with mitomycin C was investigated in a phase 1 study of 26 patients with advanced gastrointestinal cancer. The dose of BW12C was increased from 20 mg/kg to 60 mg/kg. Dose-limiting toxicity of vomiting was experienced at doses greater than 50 mg/kg. This corresponded to whole blood levels > or = 700 micrograms/ml and to > 50% haemoglobin modification. Whole blood concentrations of BW12C and modification of the haemoglobin oxygen saturation curve were linearly dependent on dose. BW12C whole blood pharmacokinetics were best described by a one-compartment model and were clearly dose-dependent. The half-life increased from 2.1 h at a dose of 20 mg/kg to 7.2 h at a dose of 60 mg/kg. The AUC increased in a similar non-linear fashion with increasing dose. Mitomycin C was given at a fixed dose of 20 mg/m2 at the end of the BW12C infusion. Mitomycin C plasma pharmacokinetics fitted a two-compartment model, giving a mean beta half-life of 50 +/- 7 min and AUC of 1.1 +/- 0.08 micrograms/ml h, and were unaffected by the combined treatment. There was no evidence of increased mitomycin C toxicity.

The novel benzotriazine di-N-oxide SR 4233 (3-amino-1,2,4-benzotriazine-1,4-di-N-oxide) shows high selective cytotoxicity toward hypoxic tumor cells. We investigated its pharmacokinetics and bioreductive metabolism in mouse plasma, brain, liver and tumor in vivo and also tumor metabolism in vitro. Plasma elimination T1/2 increased slightly with dose, and metabolite kinetics were dose-dependent. Peak concentration and area under the curve0-infinity increased linearly with dose from 0.1 to 0.3 mmol kg-1 i.v. After 0.2 mmol kg-1 i.v., elimination was biphasic (T1/2 alpha < 2 min; T1/2 beta, 26.5 min). Peak plasma concentration and area under the curve0-infinity were 26 and 13.6 micrograms ml-1 hr, respectively. Peak plasma concentration for the two-electron reduction product SR 4317 (3-amino-1,2,4-benzotriazine-1-oxide) was 7 to 9 micrograms ml-1 and for the four-electron reduction product SR 4330 (3-amino-1,2,4-benzotriazine) peak plasma concentration was 0.5 to 1.0 micrograms ml-1. Identical results were obtained after i.p. administration. Oral dosing gave lower peak plasma drug concentrations (2-3 micrograms ml-1) but reasonable bioavailability (75%). SR 4233 underwent extensive bioreduction in KHT tumors. Tumor/plasma ratios (percentages) for SR 4233 were 32% compared to 174 (SR 4317) and 196% (SR 4330), respectively. Similar SR 4233 tissue/plasma percentages were obtained in RIF-1 and 16C tumors, but EMT6 tumors were markedly lower at 7%. Reduction also occurred with tumor homogenates in vitro (KHT = EMT6 > RIF-1). Conversion to SR 4317 and SR 4330 was more extensive in liver, with tissue/plasma percentages between 50 to 220 and 500 to 1800%, respectively. The brain showed a similar pattern to tumors. Urinary recoveries (0-8 hr) were low at 4.5% for SR 4233 and 0.4% for the reduced metabolites. A further 30% occurred as a glucuronide. Concentrations of SR 4233 required for effective in vitro cytotoxicity are achieved in vivo, and extensive bioreductive metabolism occurs in tumor and normal tissues.

EO9 is a novel and fully synthetic bioreductive alkylating indoloquinone. Although structurally-related to mitomycin C, EO9 exhibits a distinct preclinical antitumour profile and there are also differences in its biochemical activation. In this study, EO9 was found to demonstrate preferential cytotoxicity against solid tumours in vitro as compared to leukaemia cell fines both in the Corbett two-tumour assay and in the disease-oriented human tumour cell fine panel of the U.S. National Cancer Institute. In the latter system activity was particularly apparent in colon, melanoma and central nervous system fines, together with some renal and non-small cell lung lines. Preferential cytotoxicity towards hypoxic versus aerobic EMT6 mouse mammary tumour cells was observed. In vivo, EO9 was inactive against the P388 murine leukaemia, while exerting significant antiproliferative effects against several murine and human solid tumours, including the generally resistant MAC mouse colon tumours and gastric, ovarian and breast xenografts. These results confirmed in vitro observations of preferential sofid tumour activity. In animal toxicology studies, EO9 induced vascular congestion in the gastrointestinal tract, but no significant bone marrow toxicity. The LD10 value of EO9 after a single intravenous injection into mice was 9 mg/kg(27 mg/m2). A dose of one-tenth of the mouse equivalent LD10 (2.7 Mg/M2), the recommended starting dose for clinical phase I studies, was found to be safe in rats. Considering its distinct mechanism of bioactivation as compared to mitomycin C, its preferential solid tumour activity, its excellent activity against hypoxic cells, and lack of significant bone marrow toxicity in animal studies, EO9 has been selected for clinical evaluation within the framework of the EORTC.

Piritrexim is a lipid-soluble antifolate which, like methotrexate, has a potent capacity to inhibit dihydrofolate reductase. We performed a multicentre phase 11 study with piritrexim in patients with locally advanced or metastatic breast cancer. Twenty-four patients of which sixteen had received prior chemotherapy, were initially treated with 25 mg piritrexim orally administered trice daily for four days, repeated weekly, with provision for dose escalation or reduction according to observed toxicity. Of twenty-one patients evaluable for tumour response, one patient achieved a partial response which lasted for 24 weeks. Three patients had stable disease during 12 weeks of treatment, seventeen had progressive disease. Piritrexim was generally well tolerated, in eighteen patients the dose could be escalated. Myelotoxicity was the most frequent observed toxicity of this piritrexim regimen. Leucopenia and thrombocytopenia grade 3/4 occurred in 38% of the patients sometime during treatment. Pharmacokinetic analysis of piritrexim in three patients during the first treatment cycle, revelaed peak levels 1 to 2 h after an oral dose, with a trend towards a higher peak plasma levels and AUCs on the fourth dosing day compared with the first dosing day. In conclusion, orally administered piritrexim appears to be a regimen with little activity in patients with locally advanced or metastatic breast carcinoma.

Tyrosine kinases are associated with the cytoplasmic domains of growth factor receptors as well as oncoproteins and many have the potential to cause transformation if mutated or hyperexpressed. Tyrosine kinases therefore represent an excellent target for the development of cancer drugs. A large number of inhibitors have now been identified and many show promising cytostatic activity, particularly using in vitro models. Some in vivo activity has been reported. Progress with various structural classes is reviewed. It is not clear whether specific or broad spectrum tyrosine kinase inhibitors should be developed as potential anticancer drugs. It does seem likely, however, that tyrosine inhibitors will enter clinical trial in cancer patients.

Anthracyclines possessing either a 9-alkyl modification in the A-ring of the tetracyclic aglycone and/or specific changes to the amino sugar moiety retain effective cytotoxic activity against multidrug resistant (MDR) cell lines. To obtain a better understanding of the structural features responsible for this potentially valuable behaviour, we used the MTT tetrazolium dye reduction assay to calculate resistance factors (RF = the ratio of ID50 for the drug-resistant line to that for the parental line) for the EMT6/P mouse mammary tumour and its MDR variant EMT6/AR1.0, and the H69/P human small cell lung cancer line and its MDR counterpart H69/LX4. Both MDR lines exhibit marked resistance to doxorubicin, MDR 1 gene amplification, hyperexpression of the membrane P-glycoprotein and reduced drug accumulation. RF values for doxorubicin were 34 and 131 in the EMT6 and H69 cell line pairs, respectively. The 9-alkyl-substituted anthracyclines were confirmed as having RF values 9- to 15-fold lower than those for doxorubicin. The 9-ethyl analogues Ro 31-1966 (RF for EMT6 2.2, RF for H69 4.7) and Ro 31-1749 (RF for EMT6 3.9, RF for H69 9.5) were superior to the previously studied 9-methyl analogue Ro 31-1215 (RF for EMT6 8.1 RF for H69 12.4). A clear trend for RF values to decrease with increasing 9-alkyl chain length was also noted in the structurally more complex aclacinomycin series. For example, 13-methyl-aclacinomycin (RF for EMT6 1.0, RF for H69 2.2) featuring a 9-isopropyl moiety was superior to the 9-alkyl-containing aclacinomycin A (RF for EMT6 4.7, RF for H69 5.8), and this was in turn more effective than the 9-methyl analogue sulfurmycin A (RF for EMT6 6.4, RF for H69 14.2). The trisaccharide moiety was not an essential feature for activity against MDR lines in the aclacinomycins, as shown by the low RF value with aklavine (RF for EMT6 2.1, RF for H69 2.5). However, a small change in one of the sugar moieties of aclacinomycin A, as in marcellomycin, resulted in a considerable increase in RF values (RF for EMT6 18.5, RF for H69 25.3). The complex anthracyclines AD 32 (RF for EMT6 6.5, RF for H69 11.7) and particularly tetrahydropyranyl-doxorubicin (RF for EMT6 1.4, RF for H69 3.2) were effective against MDR lines.(ABSTRACT TRUNCATED AT 400 WORDS)

New drug discovery continues to follow the time-honored paths of screening and novel target identification combined with analogue development and serendipity. The agents selected here reflect these various approaches. They include drugs already showing significant antitumor activity, eg, anthrapyrazoles, temozolomide, camptothecin analogues, and taxotere, and updated information is provided on their development. Other drugs just entering or currently in phase I trials include rhizoxin, which seems capable of overcoming multidrug resistance; the novel bioreductive agent EO9 [corrected]; and bryostatin, a highly potent protein kinase C agonist. Sequence specificity could well prove to be an important factor in the development of DNA-interactive agents, and information is provided on the progress with distamycin mustard and the new cyclopropylpyrroloindole analogues carzelesin and adozelesin.

In the rational development of anticancer drugs it is important to employ all the available pharmacological information. Early clinical trials provide an opportunity for hypothesis testing. MRS techniques have the potential to provide valuable data on the preclinical and clinical pharmacokinetics and pharmacodynamics of drugs non-invasively. Here we illustrate advantages and pitfalls of MRS using studies of two fluorine-containing cancer drugs: a beta,beta-difluoro analogue of the alkylating agent chlorambucil and a fluorinated derivative of the nitroimidazole misonidazole, Ro 07-0741. Limitations include signal quenching via protein binding and inadequate sensitivity for more potent drugs like beta,beta-difluorochlorambucil; but fluoromisonidazole was shown to accumulate in tumours and shows promise as a chemical probe for tumour hypoxia, detectable by 19F MRS.

SRI 62-834 ([tetrahydro-2-(octadecycloxy)methylfuran- 2-yl]methoxylphosphocholine; CRC 86-05; NSC 614383) is a cyclic antitumour ether lipid (AEL) with a novel, but ill-defined, mechanism of action. AELs are believed to act on membranes and cell signals, but the precise mechanisms of selectivity are unclear. Receptor-mediated mechanisms can often be identified by the differential activity of the individual stereoisomers of a drug. We have therefore compared the R- and S-enantiomers of SRI 62-834 for: (1) cytotoxicity against the human HT29 colon carcinoma cell line using a tetrazolium dye reduction assay and (2) membrane-damaging effects monitored by 51Cr radiolabel release. The tetrazolium assay revealed near-identical mean ID50 values around of 2-3 microM for the R- and S-isomers as well as for the racemic mixture. Moreover, pre- and co-incubation of the cells with the potent platelet-activating factor (1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine;PAF) receptor antagonist WEB 2086BS (3-[4-(chorophenyl)-9-methyl-6H-thieno[3,2- f][1,2,4]triazolo-[4,3-a][1,4]-diazepin-2-yl]-1-(4- morpholinyl)-1-propanone) had no effect on the cytotoxicity of either isomer or the racemate. Short-term membrane damage was not evident at low micromolar concentrations and between 139 and 163 microM either lipid was required to release 50% of the incorporated 51Cr label. Again, there was no difference in potency between the enantiomers and the racemate. Coincubation with WEB 2086BS also failed to modulate the membrane-lytic potency of the AELs. These results indicate that the site(s) of cytotoxic action of SRI 62-834 is (are) not stereospecific and also appear to rule out the involvement of a conventional PAF receptor in the mechanism of action of SRI 62-834.

The design, development, and application of bioreductive antitumor agents in a rational way requires a detailed understanding of the mechanisms involved in their action. In addition to measuring and manipulating tumor hypoxia, we need to elucidate the particulars of the activation versus bioprotection pathways and the nature and properties of the participating enzymes. These areas are reviewed with particular reference to the development of novel quinone, nitro and N-oxide bioreductives.

DT-diaphorase is a unique two electron (2e) donating reductase catalyzing either bioactivation or bioprotection reactions. Using human and rodent DT-diaphorase preparations (cell extracts and purified enzyme) we have characterized the reductive metabolism of the hypoxic cell cytotoxins EO9, mitomycin C (MMC), CB 1954, and SR 4233 in vitro. Drug metabolism was assayed spectrophotometrically or by HPLC, with dicoumarol as a selective inhibitor. DNA damage was measured using an agarose gel mobility technique with plasmid pBR322 DNA. The developmental indoloquinone, EO9, was metabolized by both rat Walker and human HT29 tumor DT-diaphorases. Reduction proceeded 5-fold more efficiently with the rat than the human tumor enzyme and resulted in single-strand breaks in plasmid DNA. The structurally related MMC was metabolized much more slowly than EO9 by the rat Walker tumor enzyme and there was no detectable reaction with the human HT29 tumor DT-diaphorase. No DNA damage was seen with MMC for either enzyme. The dinitrophenylaziridine CB 1954 was reduced by both human and rat enzymes forming, preferentially, the highly toxic 4-hydroxylamine as a 4e reduction product. Rates were 3-fold lower than for the human tumor enzyme. SR 4233 was also reduced by the rat tumor enzyme predominantly via 4e reduction to the benzotriazine SR 4330, in a novel reaction mechanism. This appears to be a bioprotection pathway that bypasses the toxic 1e radical formed by other reductases. Such information may be valuable in the selection of hypoxic cell cytoxins to treat human tumors high or low in DT-diaphorase and should facilitate 'enzyme-directed' analogue development.

The flavoenzyme DT-diaphorase has the potential either to bioactivate or to detoxify different bioreductive cytotoxins. Elucidation of structural features governing the ability to act as a substrate for DT-diaphorase should facilitate rational optimization or elimination of this reductive pathway for a particular class of bioreductive drug. We have examined structure-activity relationships governing both the cytotoxicity and the DT-diaphorase mediated reduction of two groups of bioreductive alkylating agents: (1) Indoloquinones related to EO9 [3-hydroxy-methyl-5-aziridinyl-1-methyl-2-(1H-indole-4,7-dione)prop-beta - en-alpha-ol]; and (2) derivatives of diaziridinyl benzoquinone or diaziquone [2,5-bis(carboethoxyamino)-3,6-diaziridinyl-1,4-benzoquinone]. The rat U.K. 256 Walker tumor cell line and the human HT29 colon carcinoma line were studied because of their high DT-diaphorase content. Enzyme activity was measured spectrophotometrically by dicoumarol inhibitable cytochrome c reduction in the presence of drug, and aerobic cytotoxicity was assessed by the MTT assay. EO9 acted as a good substrate for both enzyme preparations and was highly potent in each cell line, especially in Walker tumor cells (ID50 0.039 nM). AZQ was also reduced efficiently and gave an ID50 of 6 nM in the Walker tumor line. Slight modifications in structure resulted in large variations in both DT-diaphorase metabolism and toxicity for both types of agent. There was a clear tendency for the most efficiently reduced analogues to exhibit greater cytotoxic potency. Inclusion of an aziridine moiety in the structure appears to be desirable, but not essential, for both rapid reduction and cytotoxicity. There was no evidence of active site-directed enzyme inhibition.

The indoloquinone EO9 is a novel and potent bioreductive agent related in structure to mitomycin C but differing in many aspects of its antitumor activity, toxicity, and enzymatic activation. Because it is about to undergo clinical trial, we have investigated the pharmacokinetics of EO9 in mice and rats. At the highest tolerated dose in male C3H/He mice (12 mg/kg iv) the initial plasma concentration (Co) was 1.8 micrograms/ml. The drug was cleared rapidly with a t1/2 of 1.9 min. The volume of distribution (Vd) was large (7.5 ml g-1) and the plasma clearance (Clp) correspondingly high (2.6 ml g-1 min-1). The AUCo-infinity was 4.8 micrograms ml-1 min. Equally rapid elimination was noted at the lower dose of 6 mg/kg iv. For comparison, the t1/2 for the same dose of mitomycin C was much longer at 16 min and the peak plasma level 4-fold higher. In male Sprague-Dawley rats receiving 3 mg kg-1 the Co was 1.5 micrograms/ml and the t1/2 was again short at 3.0 min. Vd was 2.2 ml g-1, Clp was 0.5 ml g-1 min-1, and AUCo-infinity was 6.2 micrograms ml-1 min. No parent drug was detected in urine, but extensive biotransformation was confirmed by the detection of around 20% of the dose as metabolites, including the aziridine ring-opened hydrolysis product EO5A. No drug or metabolite was detected in tumor or tissues. The results show that cytotoxic drug levels can be achieved for a short period in rodent plasma. The extremely fast excretion is consistent with the rapid rates of bioreductive metabolism in vitro. These data should be useful in the forthcoming clinical trials of EO9, where a pharmacokinetically guided dose escalation may be used, and also in the design and development of second generation analogues.

Phase I studies requiring multiple dose escalation steps have led to the development of pharmacokinetically guided dose escalation (PGDE) strategies to expedite the conduct of early clinical trials. This article critically reviews PGDE strategies for a number of new anticancer agents including amphethinile, brequinar sodium, iodo-doxorubicin, the anthrapyrazoles (DuP 941, DuP 942 and DuP 937), rhizoxin, and aphidicolin glycinate. The benefits and problems associated with PGDE are examined. Recommendations are made for the optimal deployment of pharmacological information in future phase I studies.

SR 4233 or WIN 59075 (3-amino-1,2,4-benzotriazine-1,4-dioxide) is a novel and highly selective hypoxic cell cytotoxin requiring reductive bioactivation for its impressive antitumour effects. Expression of appropriate reductases will contribute to therapeutic selectivity. Here we provide more detailed information on the role of cytochrome P450 and cytochrome P450 reductase in SR 4233 reduction by mouse liver microsomes. Reduction of SR 4233 to the mono-N-oxide SR 4317 (3-amino-1,2,4-benzotriazine-1-oxide) is NADPH, enzyme and hypoxia dependent. An inhibitory antibody to cytochrome P450 reductase decreased the microsomal SR 4233 reduction rate by around 20%. Moreover, studies with purified rat cytochrome P450 reductase showed unequivocally that this enzyme was able to catalyse SR 4233 reduction at a rate of 20-30% of that for microsomes with equivalent P450 reductase activity. Exposure to the specific cytochrome P450 inhibitor carbon monoxide (CO) inhibited microsomal reduction by around 70% and CO plus reductase antibody blocked essentially all activity. Additional confirmation of cytochrome P450 involvement was provided by the use of other P450 ligands: beta-diethylaminoethyl diphenylpropylacetate hydrochloride gave a slight stimulation while aminopyrine, n-octylamine and 2,4-dichloro-6-phenylphenoxyethylamine were inhibitory. Induction of SR 4233 reduction was seen with phenobarbitone, pregnenalone-16-alpha-carbonitrile and beta-napthoflavone, suggesting that cytochrome P450 subfamilies IIB, IIC and IIIA may be involved. Since cytochrome P450 and P450 reductase catalyse roughly 70 and 30%, of mouse liver microsomal SR 4233 reduction respectively, we propose that expression of these and other reductases in normal and tumour tissue is likely to be a major factor governing the toxicity and antitumour activity of the drug.

3-Amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233; WIN 59075) is a highly selective hypoxic cell cytotoxin soon to enter phase I clinical trial. The compound is thought to exert its action through a toxic one-electron reduced free radical intermediate. Preliminary data have suggested that SR 4233 may be metabolized by DT-diaphorase [NAD(P)H: (quinone acceptor) oxidoreductase (EC 1.6.99.2)] to both two- and four-electron reduced products and that this route of biotransformation may represent a bioprotection pathway. In this study, a highly purified enzyme preparation was employed in order to investigate further the metabolism of SR 4233 by DT-diaphorase and to examine the mechanism of reduction in more detail. Spectrophotometric analysis showed that SR 4233 underwent reduction by DT-diaphorase with an apparent Km of 1.23 +/- 0.27 mM and Vmax of 8.55 +/- 1.67 nmol/min/microgram protein. This reaction was inhibited completely by dicoumarol (100 microM) and partially by an antiserum raised against the purified enzyme. Characterization of the products of SR 4233 reduction by reverse-phase HPLC confirmed that both two- (SR 4317) and four- (SR 4330) electron reduction products were generated, the latter being the predominant metabolite, particularly in prolonged incubations. Further experiments showed that the four-electron reduction product, but not the two-electron reduction product, was also a substrate for DT-diaphorase with an apparent Km of 1.14 mM and a Vmax of 57.12 nmol/min/micrograms protein. The results presented confirm that SR 4233 is indeed a substrate for DT-diaphorase and that a mixture of two-, four- and six-electron reduced products may be formed. The possible toxicological and pharmacodynamic significance of this metabolism is discussed.

Flavone acetic acid (FAA) showed impressive effects against murine solid tumours but no activity in clinical studies. The mechanism of action in mice may involve damage to tumour vasculature or immunomodulation, and these effects may be species-specific. Alternatively, concentrations of FAA achieved in mouse tumours may be higher than in human tumours. It is important to resolve this issue since it raises important questions about the relevance of in vitro versus in vivo tumour screens and the development of FAA analogues. As part of a Cancer Research Campaign Phase II study of metastatic melanoma in which 8.4 g m-2 FAA was given as a 6 h infusion, six tumour biopsies were obtained from four patients. FAA tumour concentrations were determined by HPLC and compared with subcutaneous murine solid tumours within the same analytical laboratory. Tumour/plasma percentages (range 26-61%; mean +/- SD, 43.9 +/- 11.4%) were similar to those in mice, as was the area under the curve (AUC) extrapolated to infinity and the AUC above the putative activity threshold of 100-mu-g ml-1. We conclude that the exposure of drug-refractory human melanoma tissue to FAA was comparable to that of sensitive mouse tumours. This suggests that reduced penetration of FAA into human tumours is unlikely to explain the lack of antitumour activity observed in clinical studies and that differences in mechanism of action are predominant.

The indoloquinone EO9 exhibits promising in vitro and in vivo antitumour activity. EO9 is metabolised to DNA damaging species by DT-diaphorase in vitro. In the present study DT-diaphorase specific activity was 16 fold higher in the mouse adenocarcinoma MAC 16, a tumour which is quite responsive to EO9 in vivo, compared with levels in the more resistant mouse adenocarcinoma MAC 26. This order of responsiveness is the reverse of that seen with the most active of the clinically used agents in these tumours [chloroethylnitrosoureas and 5-fluorouracil (5-FU)]. In addition, when the in vitro sensitivity of two human colon carcinoma cell lines was compared, EO9 was 15-30 fold more active in the DT-diaphorase rich HT29 line than in the enzyme-deficient BE cell line counterpart. These results are consistent with the hypothesis that DT-diaphorase expression may be a major determinant of the sensitivity of tumours to EO9. This should be considered in the clinical development of the drug.

Both electron spectroscopic imaging (ESI) and electron energy-loss spectroscopy (EELS) have great potential for use in several areas of cancer research. In biologically targeted radiotherapy, cytotoxic drug therapy and boron neutron capture therapy the effectiveness of many drugs is often critically dependent upon the intracellular localization of the agent employed. We describe the use of parallel EEL spectral imaging to assess the penetration and location of the iodine-containing drug meta-iodobenzyl guanidine, of potential value in targeted radiotherapy, and for the rapid detection of boron within borate-adsorbed polystyrene beads, of potential value in boron neutron capture therapy. We also describe elemental mapping of boron following low-temperature embedding. These results show how the techniques could be applied to many forms of cancer research by discussing the validity and limitations of the techniques experimentally. We also provide an outline of other areas in this field which could benefit from the future application of ESI and EELS.

A subline (COR-L23/R) of the human large cell lung line [corrected] COR-L23, derived by in vivo exposure to doxorubicin, exhibits an unusual multidrug resistant (MDR) phenotype. This subline shows cross-resistance to daunorubicin, vincristine, colchicine and etoposide but does not express P-glycoprotein. Interestingly, COR-L23/R [corrected] shows little or no resistance to a range of structurally-modified analogues of doxorubicin comprising 9-alkyl and/or sugar modified anthracyclines. We have previously identified these same compounds as effective agents against P-glycoprotein-positive MDR cell lines. In contrast to typical MDR cell lines, COR-L23/R [corrected] shows only minimal chemosensitisation by verapamil and no collateral sensitivity to verapamil. Compared to the parental cell line, COR-L23/R [corrected] displays reduced accumulation of doxorubicin and daunorubicin. Accumulation defects were apparent only after 0.5-1 h of incubation of cells with these agents. The rate of daunorubicin efflux was shown to be enhanced by COR-L23/R [corrected] and this efflux was demonstrated to be energy-dependent. The use of anthracyclines which retain activity in MDR cells thus appears to be a valid approach for the circumvention of MDR, not only in cells which express P-glycoprotein, but also where defective drug accumulation is due to other mechanisms possibly involving an alternative multidrug transporter.

EO9 [3-hydroxymethyl-5-aziridinyl-1-methyl-2-(H-indole-4, 7-indione)-propenol] is a novel indoloquinone structurally related to mitomycin C, a quinone anticancer drug that requires reductive bioactivation. NAD(P)H: (quinone-acceptor) oxidoreductase (quinone reductase, DT-diaphorase, EC 1.6.99.2) is an obligate 2-electron donating enzyme that can reduce a variety of quinones resulting either in bioactivation or bioprotection. Using quinone reductase (QR) preparations from rat Walker 256 mammary tumor cells and human HT29 colon carcinoma cells, we have characterized the role of this enzyme in EO9 reductive metabolism. QR activity was assayed under optimal conditions by following cytochrome c reduction at 550 nm in the presence of enzyme, quinone substrate, NADH, and bovine albumin, and confirmed by loss of EO9 absorbance at 550 nm. Both the rat and human tumor cell enzymes catalyzed reduction of the benchmark quinone menadione with a similar Km of 1.4-3.1 microM, although the Vmax was 7 to 8-fold lower for the human preparation. EO9 was readily reduced by the rat Walker QR. The mean Km was about 5-fold higher than for menadione at around 15 microM and the Vmax was 6-fold lower at around 2.5 mumol of cytochrome c reduced mg-1 of protein. EO9 was also metabolized by QR from HT29 human colon carcinoma cells but rather less efficiently than by the rat tumor enzyme. For example, the rate was 6-fold lower than that for the Walker tumor enzyme at 100 microM substrate concentration after correcting for the 7- to 8-fold difference in specific activity for the two preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

RB 6145 is a novel hypoxic cell sensitizer and cytotoxin containing both an essential bioreductive nitro group and a bromoethylamino substituent designed to form an alkylating aziridine moiety under physiological conditions. In mice, RB 6145 is 2.5 times less toxic but only slightly less active than the aziridine analogue RSU 1069, giving rise to an improved therapeutic index. However, the mechanism for the enhanced selectivity is not clear. Reasoning that this may lie in a more beneficial pharmacokinetic profile, we investigated the plasma pharmacokinetics, tissue distribution and metabolism of RB 6145 in mice using a specially developed reversed-phase HPLC technique. An i.p. dose of 190 mg kg-1 (0.5 mmol kg-1) RB 6145 produced peak plasma concentrations of about 50 micrograms ml-1 of the pharmacologically active target molecule RSU 1069 as compared with levels of around twice this value that were obtained using an equimolar i.p. dose of RSU 1069 itself. The plasma AUC0-infinity value for administered RSU 1069 was ca. 47 micrograms ml-1 h and that for the analogue RSU 1069 was ca. 84 micrograms ml-1 h. No prodrug was detectable. Another major RB 6145 metabolite in plasma was the corresponding oxazolidinone, apparently formed on interaction of the drug with hydrogen carbonate. The oxazolidinone initially occurred at higher concentrations than did RSU 1069, with the levels becoming very similar from 30 min onwards. Post-peak plasma concentrations of both RB 6145 metabolites declined exponentially, displaying an elimination t1/2 of ca. 25 min, very similar to the 30-min value observed for injected RSU 1069. The plasma AUC0-infinity value for the metabolite RSU 1069 was about 1.3 and 1.6 times higher following i.p. injection of 95 mg kg-1 (0.25 mmol kg-1) of the prodrug as compared with administration via the oral and i.v. routes, respectively. After i.v. injection, peak levels of the oxazolidinone metabolite were twice those observed following both i.p. and oral dosing and possibly contributed to the acute toxicity. After an i.p. dose of 190 mg kg-1 RB 6145, concentrations of RSU 1069 and the oxazolidinone metabolites rose to 40% and 33%, respectively, of the ambient plasma level in i.d. KHT tumours. The peak level of metabolite RSU 1069 was ca. 6 micrograms g-1 as compared with 10 micrograms g-1 following an equimolar dose of RSU 1069 itself; the tumour AUC0-infinity value for the metabolite RSU 1069 was some 35% lower.(ABSTRACT TRUNCATED AT 400 WORDS)

During a clinical toxicity study it was possible to obtain urine samples from six patients receiving either the R-(-)- or S-(+)-stereoenantiomeric forms of the developmental 2-nitroimidazole radiosensitizer Ro 03-8799 (pimonidazole). Paired plasma samples were also obtained from four patients. The pharmacokinetic data were compared with those for the racemic mixture in the same individuals. The results revealed no major differences in the plasma pharmacokinetics, urinary clearance or N-oxidation of the individual enantiomers as compared with the racemic mixture. A similar lack of stereoselectivity with respect to the acute dose-limiting CNS toxicity syndrome suggests that this may not involve a specific CNS receptor interaction.

(+/-)-2-{Hydroxy(tetrahydro-2(octadecyloxy)methylfuran-2-yl]methoxyl}phosphinyloxy-N,N,N-trimethylethaniminium hydroxide, inner salt (SRI 62-834) is a tetrahydrofuran analogue of platelet activating factor (PAF) that is currently entering clinical trial. Like other ether lipids it is of interest as a membrane-active antitumor agent. Here, we have used two-color multiparameter flow cytometry to study simultaneously its effects on cell membrane permeability, intracellular pH, and cell size/structure of EMT6 mouse mammary tumor cells and HL-60 human promyelocytic leukemia cells in vitro. Concentrations as low as 1-mu-M up to 100-mu-M SRI 62-834 caused a rapid, dose-dependent increase in membrane permeability, initially towards outward efflux of the preloaded flurescein probe bis(carboxyethyl)carboxyfluorescein (green fluorescence) and then towards influx of extracellular propidium (red fluorescence). At the same time, median cell size from light scatter was reduced with an increased coefficient of variation, and the proportion of cell debris was elevated. In vitro antitumor activity was seen over the same concentration range, as measured by tetrazolium dye reduction and cell growth curves. Neither low concentrations of PAF (50-mu-M) nor the potent PAF antagonist3-[4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-f][1,2,4]triazola[4,3a][1,4]diazepin-2-yl]-1(4-morpholinyl)-1-propanone (0.5-100 mu-M) had any influence on the membrane effects of SRI 62-834, and at higher concentrations (1-200 mu-M) PAF mimicked the behavior of SRI 62-834. In addition, the PAF antagonist did not modulate the cytotoxicity of SRI 62-834 or PAF. HL-60 cells were more sensitive to SRI 62-834 than were EMT6 cells in terms of both cytotoxicity and membrane permeability. However, PAF was more potent than SRI 62-834 in causing membrane permeabilization with both cell lines, whereas PAF was less active than SRI 62-834 in cytotoxicity assays. The results support a membrane-damaging role in the cytotoxicity of SRI 62-834 but suggest that additional factors are also involved. Membrane permeabilization may be related to its reported effects on protein kinase C-dependent intracellular calcium signaling but apparently does not involve a conventional PAF receptor in HL-60 or EMT6 cells.

In the concluding Discussion session, emphasis focussed on the potential for interfering selectively with cell membranes and cell signalling in tumour as against normal tissues. There could be no doubt that tremendous advances are being made in our understanding of the molecular changes associated with malignancy and that the information available for the rational design of inhibitors of particular signalling pathways is increasingly sophisticated. There was a consensus that we need more information on the qualitative and quantitative differences in the structure and function of membranes and the signalling machinery in various normal tissues as compared to their cancerous counterparts. Ideally we will develop drug against, for example, specific forms of, let us say, protein kinase C or tyrosine kinase which are found to be predominantly active in neoplastic cells. This may well prove possible, at least in some instances, in which case a safe therapeutic margin will be assured. But differences may in other situations turn out to be in the level of expression rather than purely qualitative in nature, and the scale of the disparate expression may not always be great. Even in such situations, adequate therapeutic selectivity may still be achieved. This may derive from a "damping down" of signalling in the hyperactive tumour. Although there are legitimate concerns regarding the possible toxic effects of administering signal-wrecking molecules in man, we should not be pessimistic as there are clear precedents elsewhere in medicine for drugs acting on membrane signals proving to be safe and effective against expectation informed by hindsight. There may also be concerns about new forms of drug resistance. But this will be so for any new agent or novel target. And with mechanism of action clearly to the fore we should be able to predict resistance pathways in advance and devise appropriate circumvention strategies or targeted second line therapies. There was a palpable buzz at the meeting that this is a valid, different and above all rational approach. Not only that, but the new therapeutic molecules which we discover will themselves prove to be valuable tools with which to probe further into the mechanisms of malignancy and signal transduction. We had expected to see a bewildering amount of new information from the basic sciences of molecularbiology and cell physiology, and we got it. But it was also impressive to witness the number of new compounds coming through which look like real drugs or at least exciting lead compounds. The membrane-active ether lipids are in clinical trial. Bryostatin 1 will shortly join them.(ABSTRACT TRUNCATED AT 400 WORDS)

In summary doxorubicin-3'-NH-oestrone-17-oximethyl-carbonyl (Dox-Oes) is a covalent adduct of the anthracycline antitumor agent doxorubicin and oestrogen. Dox-Oes does not generate free radicals in rat liver microsomes as detected by electron spin resonance spectroscopy or redox cycle as shown by lack of superoxide anion formation and NADPH oxidation. Furthermore Dox-Oes actually inhibits free radical formation by doxorubicin used in equimolar amounts. The lack of free radical formation by doxorubicin when covalently linked to oestrone supports the development of Dox-Oes as a non-cardiotoxic derivative whilst potentially improving its targeting to oestrogen positive breast tumour cells.

We describe an improved twin-probe multiparameter flow cytometric technique to examine cell membrane permeability. Ability to retain preloaded intracellular bis-carboxyethyl carboxy fluorescein (BCECF, green fluorescence) and to exclude extracellular propidium (red fluorescence) is measured, simultaneously with forward and right-angle scatter. This has significant advantages over an earlier method using fluorescein together with ethidium. In addition to the two expected cell populations which were stained green positive, red negative (by convention membrane "intact" and "viable," Region 1) and green negative, red positive ("membrane-damaged" and "non-viable," Region 3), a third population was seen which fluoresced neither green nor red and displayed intermediate light scatter characteristics (Region 2). This was true for each of 9 cell types in vitro. For EMT6 mouse mammary tumour cells held under sub-optimal conditions or treated with membrane-active drugs, progression from Region 1 to Region 2 was observed, followed by further progression from Region 2 to Region 3. Cells eventually accumulated in Region 3. These results suggest that sequential changes in membrane structure lead to increased permeability, first with respect to intracellular BCECF and in turn to extracellular propidium.

SR 4233 (3-amino-1,2,4-benzotriazine-1,4-dioxide) is a novel benzotriazine di-N-oxide which shows unusually high selective toxicity towards hypoxic cells, probably as a result of reductive bioactivation. Using an HPLC assay for the parent drug and its 2- and 4-electron reduction products (SR 4317 and SR 4330, respectively), we have examined the enzymology of SR 4233 reductive metabolism in vitro using a variety of different enzyme preparations. SR 4233 was converted extremely rapidly to SR 4317 under N2 by mouse liver microsomes, and showed a marked preference for NADPH over NADH as a reduced cofactor. The reaction was inhibited completely in air and boiled preparations. It was also inhibited by 78-86% in carbon monoxide (CO), implicating cytochrome P-450 as the major microsomal SR 4233 reductase. The kinetics of reductive metabolism of SR 4233 to SR 4317 by mouse liver microsomes conformed to Michaelis-Menten kinetics, with a Km of 1.4 mM and a Vmax of 950 nmol/min/mg protein. SR 4233 reduction was also catalysed by mouse liver cytosol under N2. However, rates were markedly slower than for microsomes and showed an equal dependency on NADH and NADPH. The cytosolic enzymes aldehyde oxidase and xanthine oxidase both catalysed SR 4233 reduction to SR 4317 under N2. Purified buttermilk xanthine oxidase also catalysed this reaction. In contrast to other enzyme preparations, DT-diaphorase from Walker 256 tumour cells reduced SR 4233 predominantly to SR 4330, and this reaction occurred under aerobic conditions. These data illustrate that SR 4233 is reduced rapidly by a wide variety of reductases. We propose that the therapeutic selectivity of SR 4233 will be controlled by the relative expression of reductases in tumour versus normal tissues, and in particular by the differential participation of putative activating versus detoxifying enzymes.

The pimonidazole-etanidazole combination shows promise for improved radiosensitization, although pimonidazole may increase the dose-limiting peripheral neurotoxicity of etanidazole in man. Induction of liver microsomal drug metabolizing enzymes by phenobarbitone resulted in reduced exposure to pimonidazole in plasma, brain, and tumor in mice. Peak tumor concentrations were lowered, but to a lesser extent. Phenobarbitone induction caused no change in the urinary ratio of pimonidazole to its N-oxide metabolite, and in fact, markedly reduced the circulating metabolite concentrations in plasma. There was no effect of phenobarbitone on plasma or tissue pharmacokinetics of etanidazole, which is eliminated by renal clearance. The results suggest that hepatic microsomal enzyme induction may be a possible approach to reducing the toxicity of the pimonidazole-etanidazole combination, and may also provide valuable information on the enzymology of pimonidazole metabolism.

The hydrophilic 2-nitroimidazole radiosensitizer etanidazole is currently undergoing clinical evaluation. Although considerably less neurotoxic than misonidazole because of its rapid renal clearance and partial exclusion from the nervous system, total dose is limited by peripheral neuropathy. Monitoring plasma etanidazole concentration in patients to determine the area under the curve (AUC0-infinity) has been proposed as a method of predicting patients at risk, and of providing a quantitative basis for dose reduction in such patients. Successful application of this policy requires accurate assessment of AUC0-infinity. We have analyzed plasma data for 18 patients receiving 2 g/m2 etanidazole to determine the errors introduced in the estimation of AUC0-infinity caused by omitting selected time points from the analysis. A 'baseline' AUC0-infinity value was calculated by integration of the rate equation for the 2-compartment model using data points at 0, 15, and 30 min and 1, 2, 4, 8, 12, and 24 hr after the end of infusion. The mean +/- SD area for AUC0-infinity was 502 +/- 152 micrograms ml-1 h (2.35 +/- 0.71 mM.h). Omitting the zero or the 24 hr time point, the average errors were quite small (2.5% in both cases), but errors of up to 16.4 and 7.3%, respectively, were seen for individual patients. Leaving out both the 8 hr and 12 hr points at the same time gave a similar low average error of 2.9%, with a highest error of 7.3%. Omitting all data points after 4 hr, the mean error was 24.7% and 15 of 18 patients had errors in excess of 10%. In addition, failure to correct for infusion time results in an underestimation of AUC0-infinity averaging 4.5% (range 1.9-8.7%). The choice of sampling times for toxicological monitoring will depend upon the accuracy with which the AUC0-infinity must be known. Including all data points between 0 and 24 hr will minimize errors. Considering the general similarity in the errors introduced by omitting the 8 hr and 12 hr points together compared to those seen with exclusion of the single 24 hr point, the choice between these truncated sampling options would be expected to lie in the relative inconvenience caused to patients and medical staff for the particular dose schedule used. The short sampling schedule (0-4 hr) should not be used.

A range of anthracyclines and related compounds were evaluated for activity against murine and human cell lines exhibiting multidrug resistance (MDR). Cell lines used were the NCI-H69 human small-cell lung cancer line and the EMT6 murine mammary tumour line, together with their multidrug-resistant counterparts produced by in vitro exposure to Adriamycin (ADM). Chemosensitivity testing was carried out using the tetrazolium (MTT) dye assay. Results were expressed as the ratio of the ID50 for the resistant line to that obtained in the parent, i.e. the resistance factor (RF). Compounds exhibiting much lower RF values than ADM in both resistant cell lines were identified as those anthracyclines with 9-alkyl substituents and those with certain changes to the amino sugar residue at position 3' and 4', together with the anthracenedione mitoxantrone (MIT). In a further attempt to overcome resistance, we used four of these compounds, Ro 31-1215, 4'-deoxy-4'-iodo-ADM (iodo-ADM), aclacinomycin A (ACL) and MIT (all yielding low RF values), in combination with the resistance modifiers verapamil (VRP) and cyclosporin A (CYA). Additional enhancement of chemosensitivity was achieved in the ADM-resistant sublines, as shown by the further decrease in RF values. At the concentrations used, the largest effects were generally seen with CYA, and the combination of this modifier with ACL and MIT was particularly effective. For the H69/LX4 resistant line, the latter combinations gave RF values approaching unity. These findings point to the use of analogues with the 9-alkyl substituent and/or specific changes to the sugar residue in combination with resistance modifiers as a therapeutic strategy for circumvention of the MDR phenotype.

Using flow cytometry and conventional spectrofluorimetry we have previously shown that chloroethylnitrosoureas (CNUs) can exhibit marked inhibition of cellular enzymes catalysing hydrolysis of fluorescein diacetate (FDA). More potent inhibition was seen for the carbamoylating CNUs, whereas alkylating agents were largely inactive. We now report results obtained with the developmental imidazotetrazines mitozolomide and temozolomide in comparison with BCNU, the novel alkylating agents clomesome and cyclodisone, and the active mitozolomide metabonate MCTIC. Inhibition of EMT6 mouse mammary-tumour esterases was seen for mitozolomide and temozolomide, and activity against purified porcine carboxylesterase was demonstrated. Flow cytometric analysis showed that inhibition occurred across the entire EMT6 cell population, with no evidence of a subpopulation resistant to enzyme inhibition. Inhibitory potency for the imidazotetrazines was much weaker than for BCNU. With EMT6 cells, I50 values from flow cytometry were 9.7 x 10(-3) M and 1.5 x 10(-3) M for mitozolomide and temozolomide compared with 3.7 x 10(-4) M for BCNU. These were higher than the ID50 values for in vitro antitumour activity (MTT assay), 8.5 x 10(-6) M in the case of mitozolomide and 1.2 x 10(-5) M for BCNU, but similar to that of 5.6 x 10(-4) M for the less toxic temozolomide. MCTIC and cyclodisone showed very low activity, but significant inhibition was seen for clomesome. The results are consistent with the view that the imidazotetrazines do not exhibit major carbamoylating ability, although significant effects are seen at cytotoxic concentrations of temozolomide. In addition, the potential for the generation of carbamoylating species at the enzyme active site cannot be ruled out.

We have examined the cellular accumulation of anthracycline compounds, alone or in conjunction with resistance modifiers, in an attempt to identify mechanisms by which multidrug resistance (MDR) can be circumvented. This was facilitated by using the EMT6 mouse mammary tumour cell line EMT6/P and its MDR subline EMT6/AR1.0 with 30-fold resistance to Adriamycin (ADM), and the human small cell lung cancer line H69/P together with its MDR subline H69/LX4 with 100-fold resistance to ADM. Both MDR lines hyperexpress membrane P-170 glycoprotein. The accumulation of ADM was compared to that seen for the anthracycline analogues aclacinomycin A (ACL), Ro 31-1215 and 4'-deoxy-4'-iodo-Adriamycin (iodo-ADM). These analogues were selected because of their high activity against MDR sublines, including H69/LX4 and EMT6/AR1.0. Both MDR cell lines exhibited a deficiency in ADM accumulation compared to the parent lines. Smaller differentials were seen using Ro 31-1215 or iodo-ADM. Both resistant sublines were able to accumulate ACL in identical amounts to their respective parental sublines. Improved drug accumulation is likely to contribute to the improved activity of the analogues against MDR cell lines. However, the relative accumulation defects in the resistant lines did not correlate exactly with the degree of resistance to a particular compound. Cyclosporin A (5 micrograms/ml) or verapamil (3.3 micrograms/ml) caused a preferential increase in uptake in both MDR sublines, with a small or negligible effect for the parental line. A smaller effect was observed with iodo-ADM and Ro 31-1215, and levels of ACL were unchanged in the MDR lines in the presence of either resistance modifier. These results indicate two mechanisms for circumventing drug resistance due to reduced drug accumulation. Structurally modified derivatives can partially or completely eliminate uptake differentials between parent and drug resistant cell lines. Any residual uptake can be eliminated using resistance modifiers. The two mechanisms may both operate via inhibition or circumvention of P-170 mediated efflux. The situation is complex, however, and this study indicates the possible involvement of additional resistance mechanisms.

Classical pharmacokinetic analysis of plasma, urine and tissue specimens continues to be of major value to the rational development of chemical modifiers of cancer treatment. However, in addition, increasingly sophisticated analytical techniques are becoming available which allow the pathways of microdistribution and micrometabolism of drugs to be traced down to the cellular and molecular level. New developments described here include flow cytometry, magnetic resonance spectroscopy, and molecular enzymology. These are predicted to have a major impact on the optimization of chemical modification.

The hypoxic cell cytotoxins SR 4233, benznidazole (Benzo), and CB 1954 were readily reduced by anaerobic mouse liver microsomes in vitro to their respective amino or single N-oxide derivatives. The reactions were inhibited in air and required reduced cofactors, particularly NADPH. The rates of reductive bioactivation were markedly different for each drug, with SR 4233 much greater than CB 1954 greater than Benzo. Using purified cytochrome P-450 reductase (P-450 reductase) and an inhibitory antibody to this enzyme, we demonstrated that P-450 reductase was involved in the reductive bioactivation of all 3 compounds. It had a minor role in SR 4233 reduction, but a more important involvement in CB 1954 metabolism to its 4-amino metabolite. Using carbon monoxide, a specific inhibitor of cytochrome P-450 (P-450), we demonstrated that P-450 was involved in both SR 4233 and Benzo reduction. P-450 had a major role both in SR 4233 conversion to SR 4317 and in the latter steps of Benzo amine formation. Purified xanthine oxidase was shown to reduce SR 4233 and Benzo in vitro, but cytosolic aldehyde oxidase activity was only detectable with Benzo as substrate. Characterizing the relative participation of the various reductases in tumor versus normal tissues may allow a more rational selection and application of hypoxic cell cytotoxins in cancer therapy.

We have investigated the effects of localized tumor hyperthermia (LTH; 43.5 degrees C x 30 min) on the reductive bioactivation of the 2-nitroimidazole benznidazole in C3H mouse normal tissues and KHT tumors. Mice were allocated to one of three treatment groups: (a) unrestrained controls, (b) sham tumor treatment, and (c) LTH. Concentrations of benznidazole and its amine metabolite were determined by high-performance liquid chromatography. Conscious mice were given LTH or sham treatment 2.5 h after 2.5 mmol/kg benznidazole i.p. This gave steady-state plasma benznidazole concentrations of 120-170 micrograms/ml at 2-5 h in all three groups. Plasma amine concentrations were very low at 0.1-1 micrograms/ml in all cases. Liver benznidazole concentrations were similar to plasma but amine concentrations were 30-40-fold greater at 20-40 micrograms/g in all three groups, implicating the liver as a major site of reductive metabolism. Benznidazole concentrations in tumors from unrestrained mice were comparable to those in plasma and liver, with tumor/plasma ratios of 85-113%. Tumor amine concentrations were intermediate at about 2-3 micrograms/g, indicating reductive bioactivation had occurred. Sham treatment decreased tumor benznidazole concentrations by 25-50%, particularly at later times, and amine concentrations were correspondingly increased. This may be a result of sham tumor treatment at 37 degrees C, a temperature 3-4 degrees C higher than in unrestrained controls. More importantly, LTH further decreased tumor benznidazole concentrations over sham treatment, e.g., by 59% from 114 to 47 micrograms/g (P less than 0.01) immediately after heating. Amine concentrations were correspondingly elevated, e.g., by 40% from 5.1 to 8.4 micrograms/g (P less than 0.01). These results clearly show that LTH can selectively enhance the reductive bioactivation of benznidazole in KHT tumors in mice, and support a particular role for the use of bioreductive agents with heat.

We have investigated the effects of localised tumour hyperthermia (LTH; 43.5 degrees C x 30 min) on the acute toxicity and pharmacokinetics of the hypoxic cell sensitizer pimonidazole (Ro 03-8799) in mice. There were three treatment groups: unrestrained controls, sham-treated and LTH treated mice. LTH had minimal effects on the acute toxicity (LD50/7d) of pimonidazole with no significant difference between the three treatment groups. Pharmacokinetic studies were carried out at the maximum tolerated dose (MTD; approximately 60% LD50) of 437 micrograms g-1 i.v. in plasma, brain and tumour. Sham tumour treatment consistently increased plasma drug concentrations compared to unrestrained controls but had minimal effects on the elimination t1/2. The AUC0-infinitive was increased by 35% and the plasma clearance decreased by 26%. By contrast, LTH had minimal effects on these parameters compared to sham treatment. Brain pimonidazole concentrations were increased in restrained mice (sham and LTH treatments) compared to unrestrained controls, but average brain/plasma ratios were similar in all three groups at between 400 and 500%. Sham tumour treatment markedly reduced peak tumour pimonidazole concentrations compared to unrestrained controls giving a 29% lower AUC0-180min. Average tumour/plasma ratios were reduced from 236 to 129%. The most important finding was that LTH further reduced pimonidazole tumour concentrations, giving a 31% lower AUC0-180 min compared to sham treated tumours. Tumour/plasma ratios for pimonidazole were reduced by 41%. Plasma exposure to the pimonidazole N-oxide metabolite, Ro 31-0313, was unaltered by LTH. The markedly reduced drug concentrations in heated tumours may be a result of hyperthermia-stimulated bioreductive drug activation.

We investigated the effects of a range of temperatures (33 degrees-44 degrees C) on the stability and kinetics of C3H mouse liver microsomal misonidazole (MISO) O-demethylase in vitro. Microsomal O-demethylase activity was stable for 60 min at 37 degrees C and for 30 min at 41 degrees C but was steadily inactivated with longer incubation times. Inactivation at 44 degrees and 47 degrees C was exponential, with half-lives of 41 and 11 min, respectively. MISO O-demethylation followed Michaelis-Menten kinetics from 33 degrees to 44 degrees C. The apparent Vmax for desmethylmisonidazole (Ro 05-9963) formation was decreased by 32% (from 2.14 to 1.47 nmol min-1 mg-1 protein) with a 4 degrees decrease from 37 degrees to 33 degrees C. An increase of 4 degrees from 37 degrees to 41 degrees C enhanced the Vmax by 47%, but there was only an additional 9% increase for a further 3 degrees rise to 44 degrees C. Apparent Km values were unaltered at about 1.6 mM. These results show that elevated temperatures in the clinically relevant hyperthermia range (41 degrees-44 degrees C) can enhance a model mixed-function oxidase reaction in vitro. Such effects may be important for the metabolism, activity and toxicity of anticancer drugs combined with hyperthermia in vivo.

In a series of 22 patients, high dose BCNU (800-1,000mg m-2) with autologous bone marrow transplantation was given as the first post-surgical treatment for grade IV astrocytoma and followed by full dose radiotherapy. When compared to historical experience and matched to control patients in national studies, there appeared to be a small prolongation of survival but no increase in the proportion of long survivors. Acute myelosuppression was mild but toxicity to lung and liver was substantial and limited further dose escalation. Late bone marrow failure was seen in 4 patients. Pharmacokinetic studies were performed and suggested that the late marrow failure was due to persistence of BCNU at the time of marrow return. Despite the suggestion of a prolongation of survival this approach is not routinely recommended and a randomised trial is probably not justified.

The hypoxic cell radiosensitizers Ro 03-8799 (pimonidazole) and SR 2508 (etanidazole) have been evaluated for their simultaneous penetration into human brain tumors and surrounding normal tissue. Thirteen patients received a dose of 1 g of each agent, infused over a 10 minute period during neurosurgery. Samples of glioma (20), brain (10) and cerebrospinal fluid (1) were obtained at a mean time (+/- SD) of 31 +/- 18 min from the end of infusion. A 24 hr plasma time course was measured in six patients. Nitroimidazole concentrations were determined by HPLC. For a mean dose of 0.55 g/m2 of each agent, the mean tumor concentrations (+/- SD) were 17.0 +/- 12.0 micrograms/g for Ro 03-8799 and 13.5 +/- 10.9 micrograms/g for SR 2508. The tumor/plasma ratios were 279 +/- 230% and 47 +/- 34% respectively. For adjacent 'normal' brain tissue, the radiosensitizer concentrations were 29.9 +/- 13.1 micrograms/g for Ro 03-8799, and 4.0 +/- 1.7 micrograms/g for SR 2508, and the brain/plasma ratios were 430 +/- 29% and 14 +/- 8% respectively. There was a significant trend towards increasing accumulation of both agents with time, in both tumor and normal brain. Concentrations in cerebrospinal fluid were very low. Plasma pharmacokinetics for Ro 03-8799 were similar to previous experience, but for SR 2508 the terminal half-life was greater in this series by a factor of 1.3. The results confirm that Ro 03-8799 is distributed widely in the central nervous system, and demonstrate that SR 2508 can achieve high tumor concentrations when the blood-brain barrier is compromised. The concentrations achieved with the combination are indicative of a significant advantage over metronidazole, misonidazole, or either agent alone, and normalized to the therapeutic dose of 0.75 g/m2 plus 2.0 g/m2 SR 2508 are consistent with those giving additive sensitization in an in vivo mouse tumor model.

We describe a simple and rapid method to determine the amount by which the area under the curve (AUC) is underestimated when a drug is given by i.v. infusion, but the infusion period is ignored and the post-infusion data are analysed as if they derive from a bolus injection. Charts are provided that allow the investigator to determine the approximate underestimation for drugs following the one- and two-compartment models, and hence to decide whether it is acceptable to ignore the infusion period in a given case (e.g., underestimation less than 5% or 10%). Equations are also provided that allow the exact underestimation to be calculated, together with the true value of the AUC.

The hypoxic cell radiosensitizers Ro 03-8799 (pimonidazole) and SR 2508 (etanidazole) have differing physico-chemical properties and clinical toxicities. The former is basic, lipophilic and produces an acute but transient central nervous system syndrome; the latter is neutral, hydrophilic and causes cumulative peripheral neuropathy. We therefore investigated the possibility of combining these agents to achieve additive radiosensitization with no enhancement of toxicity, as demonstrated in a rodent tumor model. Following a single dose study which showed a lack of interaction with respect to both toxicity and pharmacokinetics, twenty-one patients have now completed simultaneous drug administration on an escalating, multiple dose schedule. There has been no adverse acute interaction up to 0.75 g/m2 Ro 03-8799 with 2 g/m2 SR 2508 for 15 doses. At this dose-level, however, all patients experienced peripheral neuropathy. There was no adverse pharmacokinetic interaction, or perturbation of plasma pharmacokinetics between initial and final infusions. Tumor concentrations were determined in 48 biopsy samples 0-60 min after administration. Mean values normalized to a dose of 0.75 g/m2 Ro 03-8799 plus 2 g/m2 SR 2508 were 33 micrograms/g Ro 03-8799 and 74 micrograms/g SR 2508. These would be expected to produce a single-dose sensitizer enhancement ratio of around 1.5. The combination is predicted to be around 6.8 times more active than misonidazole, and superior to any single agent tested to date. The current schedules are reaching the limits of clinical tolerance, and an attempt is now being made to define the optimal regimen for use in a randomized clinical trial of the combination.

Antitumour chloroethylnitrosoureas (Cnus) decompose in physiological conditions yielding alkylating species and organic isocyanates. While antitumour activity is mainly attributed to the alkylation of DNA, carbamoylation of intracellular proteins by isocyanates may also have pharmacological and toxicological relevance. We previously reported a novel dynamic flow cytoenzymological assay for esterase inhibition in intact murine cells by BCNU and related isocyanates, and proposed that this might form the basis of an assay for intracellular carbamoylation. We have now examined a wide range of Cnus, isocyanates, and alkylating agents for their ability to inhibit cellular esterases. BCNU, CCNU, their derived isocyanates, and the 4-OH metabolites of CCNU exhibited potent inhibitory activity (I50 values 5.5 x 10(-5)-7.3 x 10(-4) M). Chlorozotocin and GANU were relatively inactive (I50 much greater than 10(-2) M). ACNU, TCNU and the 2-OH metabolites of CCNU exhibited intermediate activity (I50 values 1.1 x 10(-3)-2.3 x 10(-2) M). Compounds not able to form isocyanates were essentially inactive. Poor membrane permeability was also implicated in the weak activity of chlorozotocin and GANU. There was overall a good correlation between esterase inhibition and chemical carbamoylating activity, but some particular differences were identified. We concluded that flow cytoenzymological assay appears to have the potential to provide useful measurement of intracellular protein carbamoylation by existing Cnus and novel derivatives, and also offers the advantage of cell subpopulation identification for in vivo evaluation of these agents.

RSU 1069 is a leading compound in the class of mixed-function hypoxic cell sensitizers. Possessing an alkylating aziridine function as well as a nitro group, it represents an important prototype molecule for new sensitizer development. Using a novel HPLC assay for RSU 1069 and its metabolites with a cyanopropyl column, we studied the detailed pharmacokinetics and metabolism of this drug in mice. An i.v. dose of 100 mg kg-1 produced peak plasma concentrations of about 100 micrograms ml-1. Absorption was rapid after i.p. injection but peak plasma concentrations were some three- to fourfold lower, giving an i.p. bioavailability of 55%. The elimination t1/2 was route-dependent; e.g. after 50 mg kg-1 the t1/2 was 37.2 and 22.4 min for the i.v. and i.p. routes respectively (P less than 0.001). There was also an indication of dose-dependent kinetics, with a 37% increase in elimination t1/2 when the i.p. dose was doubled from 50 to 100 mg kg-1. Oral bioavailability was low. The volume of distribution was 0.65-1.31 ml g-1 at 50 mg kg-1, but tissue penetration was limited. Brain/plasma ratios ranged from 9.3% to 66.8%, while the mean steady-state tumour/plasma ratio was 28.4%, a value considerably less than the 80%-100% ratios occurring with the neutral 2-nitroimidazole misonidazole. About 18% and 8% of a dose were excreted as the parent drug and the ring-opened hydrolysis product (RSU 1137) in the 8 h urine, indicating the likelihood of extensive metabolism via aziridine-ring removal and nitroreduction. RSU 1137 was also detected in mouse plasma and tissues and, in contrast to the aziridine ring-intact parent compound, gave tumour/plasma ratios of 100%. These studies should provide a pharmacokinetic basis for the evaluation and development of improved mixed-function sensitizers.

Fluorescein esters are employed in assays of cell viability, membrane permeability and esterase activity. The ester most widely used, fluorescein diacetate (FDA), has the disadvantage of rapid cellular efflux of its hydrolysis product fluorescein. This is particularly problematic for flow cytoenzymology (FCE), where fluorescence is measured in individual cells allowing identification of subpopulations differing in esterase activity and/or membrane characteristics. We present a comparison of FDA with two potentially improved substrate probes for FCE, carboxyfluorescein diacetate (CFDA) and bis(carboxyethyl)-carboxyfluorescein-tetra acetoxy methyl ester (BCECF-AM). Substrates were characterized in terms of reaction and product efflux kinetics in EMT6 mouse mammary tumour cells, together with inhibition kinetics for the carbamoylating agent BCNU. Intact viable cells were analysed by FCE and spectrofluorimetry, and the latter was also used for cell sonicates and purified esterase. CFDA and BCECF-AM enter cells and are hydrolysed more slowly than FDA. CFDA and FDA hydrolyses obey Michaelis-Menten kinetics with Km values of around 19 and 2 microM, respectively, whereas BCECF-AM hydrolysis deviates from this classical behaviour. BCNU (5 X 10(-4) M) inhibits FDA and BCECF-AM hydrolyses by approximately 50%, compared to 30% for CFDA. CFDA may be partly hydrolysed by membrane-bound esterases. Efflux half-lives were 16 min, 94 min and greater than 2 h for products of FDA, CFDA and BCECF-AM, respectively. We conclude that BCECF-AM is the optimal substrate probe for FCE. This study emphasizes the need to optimize various parameters when selecting a substrate for flow cytoenzymological assay or when loading other reporter fluorochromes into cells via lipophilic esters.

Flow cytoenzymology is the determination of enzyme activities or concentrations in single intact cells. Using the flow cytometer built and designed in our laboratory and recent modifications to hardware and software, we have developed an improved dynamic flow cytoenzymological procedure for the assay of cellular enzyme kinetics. The reaction mixture is sampled continuously, and the computer clock incorporates time as a parameter for kinetic determinations. Conditions for cellular esterase analysis were optimized and the rates of hydrolysis of two fluorogenic substrates, fluorescein diacetate (FDA) and 4-methylumbelliferone acetate (MUA), by esterases in EMT6 mouse mammary tumor cells were studied. Reaction kinetics were characterized, and Km values of 19 and 72 microM were obtained for the hydrolysis of FDA and MUA respectively. The kinetics of the cellular efflux of fluorescein were investigated, and a half-life of 7.5 min obtained. Enzyme inhibition kinetics were investigated using the competitive substrates p-nitrophenyl acetate and phenyl acetate, and the carbamoylating agents physostigmine and n-butyl isocyanate. The latter was particularly potent with an I50 of 4.8 X 10(-5) M for FDA hydrolysis compared with 6.5 X 10(-3) M for physostigmine. The I50 of 8.8 X 10(-5) M for n-butyl isocyanate inhibition of MUA hydrolysis was similar to that obtained with FDA as substrate. By monitoring FDA and MUA reactions separately and simultaneously, we showed them to act as competitive substrates. A comparison of flow cytoenzymological and conventional spectrofluorimetric analysis was also made, and differences identified in some cases.

We present a novel dynamic flow cytoenzymological demonstration of the potent inhibition by the antitumour chloroethylnitrosourea BCNU of the intracellular hydrolysis of fluorescein diacetate by esterases of viable, intact murine and human tumour cells in vitro. The BCNU metabonate chloroethyl isocyanate and the related compound n-butyl isocyanate were also potent inhibitors. I50 values were in the range 4.2 X 10(-5)-2.0 X 10(-4) M. Generally similar quantitative results were obtained for intact cells and sonicates by conventional spectrofluorimetry, and inhibition of purified porcine liver carboxyl esterase (EC 3.1.1.1) was demonstrated. Little or no inhibitory activity was seen with the alkylating agents methyl methane-sulphonate, melphalan and nitrogen mustard. The results are consistent with carbamoylation of the esterase molecules by isocyanates, and this may provide a basis for the flow cytometric determination of intracellular carbamoylation in discrete subpopulations of heterogeneous samples.

Phosphatidylinositol-3-kinase (PI3K) is an important target in cancer due to the deregulation of the PI3K/ Akt signaling pathway in a wide variety of tumors. A series of thieno[3,2-d]pyrimidine derivatives were prepared and evaluated as inhibitors of PI3 kinase p110alpha. The synthesis, biological activity, and further profiling of these compounds are described. This work resulted in the discovery of 17, GDC-0941, which is a potent, selective, orally bioavailable inhibitor of PI3K and is currently being evaluated in human clinical trials for the treatment of cancer.