Fearon, D., Westwood, I.M., van Montfort, R.L., Bayliss, R., Jones, K. & Bavetsias, V.
(2018). Synthesis and profiling of a 3-aminopyridin-2-one-based kinase targeted fragment library: Identification of 3-amino-5-(pyridin-4-yl)pyridin-2(1H)-one scaffold for monopolar spindle 1 (MPS1) and Aurora kinases inhibition. Bioorg med chem,
Screening a 3-aminopyridin-2-one based fragment library against a 26-kinase panel representative of the human kinome identified 3-amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-2(1H)-one (2) and 3-amino-5-(pyridin-4-yl)pyridin-2(1H)-one (3) as ligand efficient inhibitors of the mitotic kinase Monopolar Spindle 1 (MPS1) and the Aurora kinase family. These kinases are well recognised as attractive targets for therapeutic intervention for treating cancer. Elucidation of the binding mode of these fragments and their analogues has been carried out by X-ray crystallography. Structural studies have identified key interactions with a conserved lysine residue and have highlighted potential regions of MPS1 which could be targeted to improve activity and selectivity..
Cheeseman, M.D., Chessum, N.E., Rye, C.S., Pasqua, A.E., Tucker, M.J., Wilding, B., Evans, L.E., Lepri, S., Richards, M., Sharp, S.Y., et al.
(2017). Discovery of a Chemical Probe Bisamide (CCT251236): An Orally Bioavailable Efficacious Pirin Ligand from a Heat Shock Transcription Factor 1 (HSF1) Phenotypic Screen. J med chem,
Phenotypic screens, which focus on measuring and quantifying discrete cellular changes rather than affinity for individual recombinant proteins, have recently attracted renewed interest as an efficient strategy for drug discovery. In this article, we describe the discovery of a new chemical probe, bisamide (CCT251236), identified using an unbiased phenotypic screen to detect inhibitors of the HSF1 stress pathway. The chemical probe is orally bioavailable and displays efficacy in a human ovarian carcinoma xenograft model. By developing cell-based SAR and using chemical proteomics, we identified pirin as a high affinity molecular target, which was confirmed by SPR and crystallography..
McGrath, S., Tortorici, M., Drouin, L., Solanki, S., Vidler, L., Westwood, I., Gimeson, P., Van Montfort, R. & Hoelder, S.
(2017). Structure-Enabled Discovery of a Stapled Peptide Inhibitor to Target the Oncogenic Transcriptional Repressor TLE1. Chemistry,
TLE1 is an oncogenic transcriptional co-repressor that exerts its repressive effects through binding of transcription factors. Inhibition of this protein-protein interaction represents a putative cancer target, but no small-molecule inhibitors have been published for this challenging interface. Herein, the structure-enabled design and synthesis of a constrained peptide inhibitor of TLE1 is reported. The design features the introduction of a four-carbon-atom linker into the peptide epitope found in many TLE1 binding partners. A concise synthetic route to a proof-of-concept peptide, cycFWRPW, has been developed. Biophysical testing by isothermal titration calorimetry and thermal shift assays showed that, although the constrained peptide bound potently, it had an approximately five-fold higher Kd than that of the unconstrained peptide. The co-crystal structure suggested that the reduced affinity was likely to be due to a small shift of one side chain, relative to the otherwise well-conserved conformation of the acyclic peptide. This work describes a constrained peptide inhibitor that may serve as the basis for improved inhibitors..
Pettinger, J., Le Bihan, Y.-., Widya, M., van Montfort, R.L., Jones, K. & Cheeseman, M.D.
(2017). An Irreversible Inhibitor of HSP72 that Unexpectedly Targets Lysine-56. Angew chem int ed engl,
The stress-inducible molecular chaperone, HSP72, is an important therapeutic target in oncology, but inhibiting this protein with small molecules has proven particularly challenging. Validating HSP72 inhibitors in cells is difficult owing to competition with the high affinity and abundance of its endogenous nucleotide substrates. We hypothesized this could be overcome using a cysteine-targeted irreversible inhibitor. Using rational design, we adapted a validated 8-N-benzyladenosine ligand for covalent bond formation and confirmed targeted irreversible inhibition. However, no cysteine in the protein was modified; instead, we demonstrate that lysine-56 is the key nucleophilic residue. Targeting this lysine could lead to a new design paradigm for HSP72 chemical probes and drugs..
Pettinger, J., Le Bihan, Y.-., Widya, M., van Montfort, R.L., Jones, K. & Cheeseman, M.D.
(2017). An Irreversible Inhibitor of HSP72 that Unexpectedly Targets Lysine-56. Angewandte chemie-international edition,
Faisal, A., Mak, G.W., Gurden, M.D., Xavier, C.P., Anderhub, S.J., Innocenti, P., Westwood, I.M., Naud, S., Hayes, A., Box, G., et al.
(2017). Characterisation of CCT271850, a selective, oral and potent MPS1 inhibitor, used to directly measure in vivo MPS1 inhibition vs therapeutic efficacy. Br j cancer,
BACKGROUND: The main role of the cell cycle is to enable error-free DNA replication, chromosome segregation and cytokinesis. One of the best characterised checkpoint pathways is the spindle assembly checkpoint, which prevents anaphase onset until the appropriate attachment and tension across kinetochores is achieved. MPS1 kinase activity is essential for the activation of the spindle assembly checkpoint and has been shown to be deregulated in human tumours with chromosomal instability and aneuploidy. Therefore, MPS1 inhibition represents an attractive strategy to target cancers. METHODS: To evaluate CCT271850 cellular potency, two specific antibodies that recognise the activation sites of MPS1 were used and its antiproliferative activity was determined in 91 human cancer cell lines. DLD1 cells with induced GFP-MPS1 and HCT116 cells were used in in vivo studies to directly measure MPS1 inhibition and efficacy of CCT271850 treatment. RESULTS: CCT271850 selectively and potently inhibits MPS1 kinase activity in biochemical and cellular assays and in in vivo models. Mechanistically, tumour cells treated with CCT271850 acquire aberrant numbers of chromosomes and the majority of cells divide their chromosomes without proper alignment because of abrogation of the mitotic checkpoint, leading to cell death. We demonstrated a moderate level of efficacy of CCT271850 as a single agent in a human colorectal carcinoma xenograft model. CONCLUSIONS: CCT271850 is a potent, selective and orally bioavailable MPS1 kinase inhibitor. On the basis of in vivo pharmacodynamic vs efficacy relationships, we predict that more than 80% inhibition of MPS1 activity for at least 24 h is required to achieve tumour stasis or regression by CCT271850..
Rankin, S.S., Caldwell, J.J., Cronin, N.B., van Montfort, R.L. & Collins, I.
(2016). Synthesis of a Ribose-Incorporating Medium Ring Scaffold via a Challenging Ring-Closing Metathesis Reaction. European j org chem,
A practical synthesis of a novel oxabicyclo[6.2.1]undecenetriol useful as a medicinal chemistry scaffold has been developed starting from l-ribose. The sequence involves an oxidation/Grignard addition sequence and a challenging ring-closing metathesis (RCM) reaction as the ring forming step. Exploration of the RCM substrate protecting groups revealed the key factor for successful nine-membered medium ring formation to be conformational bias of the reacting alkenes of the RCM substrate by very bulky silyl ether protecting groups. The synthesis also allowed access to an epimeric triol and saturated and unsaturated variants of the nine-membered ring. The medium ring conformation of the oxabicyclo[6.2.1]undecenetriol was determined by X-ray crystallography and correlated to the solution state conformation by NMR experiments..
Bavetsias, V., Lanigan, R.M., Ruda, G.F., Atrash, B., McLaughlin, M.G., Tumber, A., Mok, N.Y., Le Bihan, Y.-., Dempster, S., Boxall, K.J., et al.
(2016). 8-Substituted Pyrido[3,4-d]pyrimidin-4(3H)-one Derivatives As Potent, Cell Permeable, KDM4 (JMJD2) and KDM5 (JARID1) Histone Lysine Demethylase Inhibitors. J med chem,
We report the discovery of N-substituted 4-(pyridin-2-yl)thiazole-2-amine derivatives and their subsequent optimization, guided by structure-based design, to give 8-(1H-pyrazol-3-yl)pyrido[3,4-d]pyrimidin-4(3H)-ones, a series of potent JmjC histone N-methyl lysine demethylase (KDM) inhibitors which bind to Fe(II) in the active site. Substitution from C4 of the pyrazole moiety allows access to the histone peptide substrate binding site; incorporation of a conformationally constrained 4-phenylpiperidine linker gives derivatives such as 54j and 54k which demonstrate equipotent activity versus the KDM4 (JMJD2) and KDM5 (JARID1) subfamily demethylases, selectivity over representative exemplars of the KDM2, KDM3, and KDM6 subfamilies, cellular permeability in the Caco-2 assay, and, for 54k, inhibition of H3K9Me3 and H3K4Me3 demethylation in a cell-based assay. .
Loveday, C., Tatton-Brown, K., Clarke, M., Westwood, I., Renwick, A., Ramsay, E., Nemeth, A., Campbell, J., Joss, S., Gardner, M., et al.
(2015). Mutations in the PP2A regulatory subunit B family genes PPP2R5B, PPP2R5C and PPP2R5D cause human overgrowth. Hum mol genet,
Overgrowth syndromes comprise a group of heterogeneous disorders characterised by excessive growth parameters, often in association with intellectual disability. To identify new causes of human overgrowth, we have been undertaking trio-based exome sequencing studies in overgrowth patients and their unaffected parents. Prioritisation of functionally relevant genes with multiple unique de novo mutations revealed four mutations in protein phosphatase 2A (PP2A) regulatory subunit B family genes protein phosphatase 2, regulatory Subunit B', beta (PPP2R5B); protein phosphatase 2, regulatory Subunit B', gamma (PPP2R5C); and protein phosphatase 2, regulatory Subunit B', delta (PPP2R5D). This observation in 3 related genes in 111 individuals with a similar phenotype is greatly in excess of the expected number, as determined from gene-specific de novo mutation rates (P = 1.43 × 10(-10)). Analysis of exome-sequencing data from a follow-up series of overgrowth probands identified a further pathogenic mutation, bringing the total number of affected individuals to 5. Heterozygotes shared similar phenotypic features including increased height, increased head circumference and intellectual disability. The mutations clustered within a region of nine amino acid residues in the aligned protein sequences (P = 1.6 × 10(-5)). We mapped the mutations onto the crystal structure of the PP2A holoenzyme complex to predict their molecular and functional consequences. These studies suggest that the mutations may affect substrate binding, thus perturbing the ability of PP2A to dephosphorylate particular protein substrates. PP2A is a major negative regulator of v-akt murine thymoma viral oncogene homolog 1 (AKT). Thus, our data further expand the list of genes encoding components of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT signalling cascade that are disrupted in human overgrowth conditions. .
Gurden, M.D., Westwood, I.M., Faisal, A., Naud, S., Cheung, K.-., McAndrew, C., Wood, A., Schmitt, J., Boxall, K., Mak, G., et al.
(2015). Naturally Occurring Mutations in the MPS1 Gene Predispose Cells to Kinase Inhibitor Drug Resistance. Cancer res,
Acquired resistance to therapy is perhaps the greatest challenge to effective clinical management of cancer. With several inhibitors of the mitotic checkpoint kinase MPS1 in preclinical development, we sought to investigate how resistance against these inhibitors may arise so that mitigation or bypass strategies could be addressed as early as possible. Toward this end, we modeled acquired resistance to the MPS1 inhibitors AZ3146, NMS-P715, and CCT251455, identifying five point mutations in the kinase domain of MPS1 that confer resistance against multiple inhibitors. Structural studies showed how the MPS1 mutants conferred resistance by causing steric hindrance to inhibitor binding. Notably, we show that these mutations occur in nontreated cancer cell lines and primary tumor specimens, and that they also preexist in normal lymphoblast and breast tissues. In a parallel piece of work, we also show that the EGFR p.T790M mutation, the most common mutation conferring resistance to the EGFR inhibitor gefitinib, also preexists in cancer cells and normal tissue. Our results therefore suggest that mutations conferring resistance to targeted therapy occur naturally in normal and malignant cells and these mutations do not arise as a result of the increased mutagenic plasticity of cancer cells..
Cheeseman, M.D., Faisal, A., Rayter, S., Barbeau, O.R., Kalusa, A., Westlake, M., Burke, R., Swan, M., van Montfort, R., Linardopoulos, S., et al.
(2014). Targeting the PPM1D phenotype; 2,4-bisarylthiazoles cause highly selective apoptosis in PPM1D amplified cell-lines. Bioorg med chem lett,
The metal-dependent phosphatase PPM1D (WIP1) is an important oncogene in cancer, with over-expression of the protein being associated with significantly worse clinical outcomes. In this communication we describe the discovery and optimization of novel 2,4-bisarylthiazoles that phenocopy the knockdown of PPM1D, without inhibiting its phosphatase activity. These compounds cause growth inhibition at nanomolar concentrations, induce apoptosis, activate p53 and display impressive cell-line selectivity. The results demonstrate the potential for targeting phenotypes in drug discovery when tackling challenging targets or unknown mechanisms. .
Tatton-Brown, K., Seal, S., Ruark, E., Harmer, J., Ramsay, E., Del Vecchio Duarte, S., Zachariou, A., Hanks, S., O'Brien, E., Aksglaede, L., et al.
(2014). Mutations in the DNA methyltransferase gene DNMT3A cause an overgrowth syndrome with intellectual disability. Nat genet,
Overgrowth disorders are a heterogeneous group of conditions characterized by increased growth parameters and other variable clinical features such as intellectual disability and facial dysmorphism. To identify new causes of human overgrowth, we performed exome sequencing in ten proband-parent trios and detected two de novo DNMT3A mutations. We identified 11 additional de novo mutations by sequencing DNMT3A in a further 142 individuals with overgrowth. The mutations alter residues in functional DNMT3A domains, and protein modeling suggests that they interfere with domain-domain interactions and histone binding. Similar mutations were not present in 1,000 UK population controls (13/152 cases versus 0/1,000 controls; P < 0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and greater height. DNMT3A encodes a DNA methyltransferase essential for establishing methylation during embryogenesis and is commonly somatically mutated in acute myeloid leukemia. Thus, DNMT3A joins an emerging group of epigenetic DNA- and histone-modifying genes associated with both developmental growth disorders and hematological malignancies. .
Tatton-Brown, K., Seal, S., Ruark, E., Harmer, J., Ramsay, E., Duarte, S.D., Zachariou, A., Hanks, S., O'Brien, E., Aksglaede, L., et al.
(2014). Mutations in the DNA methyltransferase gene DNMT3A cause an overgrowth syndrome with intellectual disability (vol 46, pg 385, 2014). Nature genetics,
Workman, P. & van Montfort, R.
(2014). EML4-ALK fusions: propelling cancer but creating exploitable chaperone dependence. Cancer discov,
The crystal structure of a conserved tubulin-binding region of the EML1 protein reveals a highly atypical fold in one of its β-propeller domains. Disruption of the EML1 core region domain in many of the oncogenic EML4-ALK fusion protein variants that drive non-small cell lung cancer explains their dependence on the HSP90 molecular chaperone, provides a basis to allow more precise patient stratification for therapy, and suggests a more general model for other oncogenic fusion proteins..
Tatton-Brown, K., Seal, S., Ruark, E., Harmer, J., Ramsay, E., Del Vecchio Duarte, S., Zachariou, A., Hanks, S., O'Brien, E., Aksglaede, L., et al.
(2014). Corrigendum: Mutations in the DNA methyltransferase gene DNMT3A cause an overgrowth syndrome with intellectual disability. Nat genet,
Newbatt, Y., Hardcastle, A., McAndrew, P.C., Strover, J.A., Mirza, A., Morgan, G.J., Burke, R., Davies, F.E., Collins, I. & van Montfort, R.L., et al.
(2013). Identification of autophosphorylation inhibitors of the inositol-requiring enzyme 1 alpha (IRE1α) by high-throughput screening using a DELFIA assay. J biomol screen,
Inositol-requiring enzyme 1 alpha (IRE1α) is a transmembrane sensor protein with both kinase and ribonuclease activity, which plays a crucial role in the unfolded protein response (UPR). Protein misfolding in the endoplasmic reticulum (ER) lumen triggers dimerization and subsequent trans-autophosphorylation of IRE1α. This leads to the activation of its endoribonuclease (RNase) domain and splicing of the mRNA of the transcriptional activator XBP1, ultimately generating an active XBP1 (XBP1s) implicated in multiple myeloma survival. Previously, we have identified human IRE1α as a target for the development of kinase inhibitors that could modulate the UPR in human cells, which has particular relevance for multiple myeloma and other secretory malignancies. Here we describe the development and validation of a 384-well high-throughput screening assay using DELFIA technology that is specific for IRE1α autophosphorylation. Using this format, a focused library of 2312 potential kinase inhibitors was screened, and several novel IRE1α kinase inhibitor scaffolds were identified that could potentially be developed toward new therapies to treat multiple myeloma..
Allen, C.E., Chow, C.L., Caldwell, J.J., Westwood, I.M., van Montfort, R.L. & Collins, I.
(2013). Synthesis and evaluation of heteroaryl substituted diazaspirocycles as scaffolds to probe the ATP-binding site of protein kinases. Bioorg med chem,
With the success of protein kinase inhibitors as drugs to target cancer, there is a continued need for new kinase inhibitor scaffolds. We have investigated the synthesis and kinase inhibition of new heteroaryl-substituted diazaspirocyclic compounds that mimic ATP. Versatile syntheses of substituted diazaspirocycles through ring-closing metathesis were demonstrated. Diazaspirocycles directly linked to heteroaromatic hinge binder groups provided ligand efficient inhibitors of multiple kinases, suitable as starting points for further optimization. The binding modes of representative diazaspirocyclic motifs were confirmed by protein crystallography. Selectivity profiles were influenced by the hinge binder group and the interactions of basic nitrogen atoms in the scaffold with acidic side-chains of residues in the ATP pocket. The introduction of more complex substitution to the diazaspirocycles increased potency and varied the selectivity profiles of these initial hits through engagement of the P-loop and changes to the spirocycle conformation, demonstrating the potential of these core scaffolds for future application to kinase inhibitor discovery..
Langdon, S.R., Westwood, I.M., van Montfort, R.L., Brown, N. & Blagg, J.
(2013). Scaffold-focused virtual screening: prospective application to the discovery of TTK inhibitors. J chem inf model,
We describe and apply a scaffold-focused virtual screen based upon scaffold trees to the mitotic kinase TTK (MPS1). Using level 1 of the scaffold tree, we perform both 2D and 3D similarity searches between a query scaffold and a level 1 scaffold library derived from a 2 million compound library; 98 compounds from 27 unique top-ranked level 1 scaffolds are selected for biochemical screening. We show that this scaffold-focused virtual screen prospectively identifies eight confirmed active compounds that are structurally differentiated from the query compound. In comparison, 100 compounds were selected for biochemical screening using a virtual screen based upon whole molecule similarity resulting in 12 confirmed active compounds that are structurally similar to the query compound. We elucidated the binding mode for four of the eight confirmed scaffold hops to TTK by determining their protein-ligand crystal structures; each represents a ligand-efficient scaffold for inhibitor design..
Couty, S., Westwood, I.M., Kalusa, A., Cano, C., Travers, J., Boxall, K., Chow, C.L., Burns, S., Schmitt, J., Pickard, L., et al.
(2013). The discovery of potent ribosomal S6 kinase inhibitors by high-throughput screening and structure-guided drug design. Oncotarget,
The ribosomal P70 S6 kinases play a crucial role in PI3K/mTOR regulated signalling pathways and are therefore potential targets for the treatment of a variety of diseases including diabetes and cancer. In this study we describe the identification of three series of chemically distinct S6K1 inhibitors. In addition, we report a novel PKA-S6K1 chimeric protein with five mutations in or near its ATP-binding site, which was used to determine the binding mode of two of the three inhibitor series, and provided a robust system to aid the optimisation of the oxadiazole-substituted benzimidazole inhibitor series. We show that the resulting oxadiazole-substituted aza-benzimidazole is a potent and ligand efficient S6 kinase inhibitor, which blocks the phosphorylation of RPS6 at Ser235/236 in TSC negative HCV29 human bladder cancer cells by inhibiting S6 kinase activity and thus provides a useful tool compound to investigate the function of S6 kinases..
Naud, S., Westwood, I.M., Faisal, A., Sheldrake, P., Bavetsias, V., Atrash, B., Cheung, K.M., Liu, M., Hayes, A., Schmitt, J., et al.
(2013). Structure-based design of orally bioavailable 1H-pyrrolo[3,2-c]pyridine inhibitors of the mitotic kinase monopolar spindle 1 (MPS1). J med chem,
Newbatt, Y., Hardcastle, A., McAndrew, P.C., Strover, J.A., Mirza, A., Morgan, G.J., Burke, R., Davies, F.E., Collins, I. & van Montfort, R.L., et al.
(2013). Identification of Autophosphorylation Inhibitors of the Inositol-Requiring Enzyme 1 Alpha (IRE1 alpha) by High-Throughput Screening Using a DELFIA Assay. Journal of biomolecular screening,
Silva-Santisteban, M.C., Westwood, I.M., Boxall, K., Brown, N., Peacock, S., McAndrew, C., Barrie, E., Richards, M., Mirza, A., Oliver, A.W., et al.
(2013). Fragment-based screening maps inhibitor interactions in the ATP-binding site of checkpoint kinase 2. Plos one,
Checkpoint kinase 2 (CHK2) is an important serine/threonine kinase in the cellular response to DNA damage. A fragment-based screening campaign using a combination of a high-concentration AlphaScreen™ kinase assay and a biophysical thermal shift assay, followed by X-ray crystallography, identified a number of chemically different ligand-efficient CHK2 hinge-binding scaffolds that have not been exploited in known CHK2 inhibitors. In addition, it showed that the use of these orthogonal techniques allowed efficient discrimination between genuine hit matter and false positives from each individual assay technology. Furthermore, the CHK2 crystal structures with a quinoxaline-based fragment and its follow-up compound highlight a hydrophobic area above the hinge region not previously explored in rational CHK2 inhibitor design, but which might be exploited to enhance both potency and selectivity of CHK2 inhibitors..
Faisal, A., Naud, S., Schmitt, J., Westwood, I., Hayes, A., Gurden, M., Bavetsias, V., Berry, T., Mak, G., Innocenti, P., et al.
(2012). Characterisation of CCT251455, a novel, selective and highly potent Mps1 kinase inhibitor. Cancer research,
Matijssen, C., Silva-Santisteban, M.C., Westwood, I.M., Siddique, S., Choi, V., Sheldrake, P., van Montfort, R.L. & Blagg, J.
(2012). Benzimidazole inhibitors of the protein kinase CHK2: clarification of the binding mode by flexible side chain docking and protein-ligand crystallography. Bioorg med chem,
Two closely related binding modes have previously been proposed for the ATP-competitive benzimidazole class of checkpoint kinase 2 (CHK2) inhibitors; however, neither binding mode is entirely consistent with the reported SAR. Unconstrained rigid docking of benzimidazole ligands into representative CHK2 protein crystal structures reveals an alternative binding mode involving a water-mediated interaction with the hinge region; docking which incorporates protein side chain flexibility for selected residues in the ATP binding site resulted in a refinement of the water-mediated hinge binding mode that is consistent with observed SAR. The flexible docking results are in good agreement with the crystal structures of four exemplar benzimidazole ligands bound to CHK2 which unambiguously confirmed the binding mode of these inhibitors, including the water-mediated interaction with the hinge region, and which is significantly different from binding modes previously postulated in the literature..
Barillari, C., Duncan, A.L., Westwood, I.M., Blagg, J. & van Montfort, R.L.
(2011). Analysis of water patterns in protein kinase binding sites. Proteins,
Deregulation of protein kinases is associated with numerous diseases, making them important targets for drug discovery. The majority of drugs target the catalytic site of these proteins, but due to the high level of similarity within the ATP binding sites of protein kinases, it is often difficult to achieve the required pharmacological selectivity. In this study, we describe the identification and subsequent analysis of water patterns in the ATP binding sites of 171 protein kinase structures, comprising 19 different kinases from various branches of the kinome, and demonstrate that structurally similar binding sites often have significantly different water patterns. We show that the observed variations in water patterns of different, but structurally similar kinases can be exploited in the structure-based design of potent and selective kinase inhibitors..
Reader, J.C., Matthews, T.P., Klair, S., Cheung, K.-., Scanlon, J., Proisy, N., Addison, G., Ellard, J., Piton, N., Taylor, S., et al.
(2011). Structure-guided evolution of potent and selective CHK1 inhibitors through scaffold morphing. J med chem,
Pyrazolopyridine inhibitors with low micromolar potency for CHK1 and good selectivity against CHK2 were previously identified by fragment-based screening. The optimization of the pyrazolopyridines to a series of potent and CHK1-selective isoquinolines demonstrates how fragment-growing and scaffold morphing strategies arising from a structure-based understanding of CHK1 inhibitor binding can be combined to successfully progress fragment-derived hit matter to compounds with activity in vivo. The challenges of improving CHK1 potency and selectivity, addressing synthetic tractability, and achieving novelty in the crowded kinase inhibitor chemical space were tackled by multiple scaffold morphing steps, which progressed through tricyclic pyrimido[2,3-b]azaindoles to N-(pyrazin-2-yl)pyrimidin-4-amines and ultimately to imidazo[4,5-c]pyridines and isoquinolines. A potent and highly selective isoquinoline CHK1 inhibitor (SAR-020106) was identified, which potentiated the efficacies of irinotecan and gemcitabine in SW620 human colon carcinoma xenografts in nude mice..
Solanki, S., Innocenti, P., Mas-Droux, C., Boxall, K., Barillari, C., van Montfort, R.L., Aherne, G.W., Bayliss, R. & Hoelder, S.
(2011). Benzimidazole inhibitors induce a DFG-out conformation of never in mitosis gene A-related kinase 2 (Nek2) without binding to the back pocket and reveal a nonlinear structure-activity relationship. J med chem,
We describe herein the structure-activity relationship (SAR) and cocrystal structures of a series of Nek2 inhibitors derived from the published polo-like kinase 1 (Plk1) inhibitor (R)-1. Our studies reveal a nonlinear SAR for Nek2 and our cocrystal structures show that compounds in this series bind to a DFG-out conformation of Nek2 without extending into the enlarged back pocket commonly found in this conformation. These observations were further investigated, and structure-based design led to Nek2 inhibitors derived from (R)-1 with more than a hundred-fold selectivity against Plk1..
Workman, P. & van Montfort, R.L.
(2010). PI(3) KINASES Revealing the delta lady. Nat chem biol,
Workman, P. & van Montfort, R.L.
(2010). Revealing the delta lady (vol 6, pg 82, 2010). Nat chem biol,
Whelligan, D.K., Solanki, S., Taylor, D., Thomson, D.W., Cheung, K.-., Boxall, K., Mas-Droux, C., Barillari, C., Burns, S., Grummitt, C.G., et al.
(2010). Aminopyrazine inhibitors binding to an unusual inactive conformation of the mitotic kinase Nek2: SAR and structural characterization. J med chem,
We report herein the first systematic exploration of inhibitors of the mitotic kinase Nek2. Starting from HTS hit aminopyrazine 2, compounds with improved activity were identified using structure-based design. Our structural biology investigations reveal two notable observations. First, 2 and related compounds bind to an unusual, inactive conformation of the kinase which to the best of our knowledge has not been reported for other types of kinase inhibitors. Second, a phenylalanine residue at the center of the ATP pocket strongly affects the ability of the inhibitor to bind to the protein. The implications of these observations are discussed, and the work described here defines key features for potent and selective Nek2 inhibition, which will aid the identification of more advanced inhibitors of Nek2..
Powers, M.V., Jones, K., Barillari, C., Westwood, I., van Montfort, R.L. & Workman, P.
(2010). Targeting HSP70: The second potentially druggable heat shock protein and molecular chaperone?. Cell cycle,
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 drugs including HSP90 inhibitors. Here we provide a perspective on the progress made so far in discovering small molecules which target the HSP70 family, in the context of the available structural data and potential issues in drugging this key chaperone..
Workman, P., Clarke, P.A., Raynaud, F.I. & van Montfort, R.L.
(2010). Drugging the PI3 kinome: from chemical tools to drugs in the clinic. Cancer res,
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..
Matthews, T.P., McHardy, T., Klair, S., Boxall, K., Fisher, M., Cherry, M., Allen, C.E., Addison, G.J., Ellard, J., Aherne, G.W., et al.
(2010). Design and evaluation of 3,6-di(hetero)aryl imidazo[1,2-a]pyrazines as inhibitors of checkpoint and other kinases. Bioorg med chem lett,
A range of 3,6-di(hetero)arylimidazo[1,2-a]pyrazine ATP-competitive inhibitors of CHK1 were developed by scaffold hopping from a weakly active screening hit. Efficient synthetic routes for parallel synthesis were developed to prepare analogues with improved potency and ligand efficiency against CHK1. Kinase profiling showed that the imidazo[1,2-a]pyrazines could inhibit other kinases, including CHK2 and ABL, with equivalent or better potency depending on the pendant substitution. These 3,6-di(hetero)aryl imidazo[1,2-a]pyrazines appear to represent a general kinase inhibitor scaffold..
Workman, P. & van Montfort, R.L.
(2010). Unveiling the secrets of the ancestral PI3 kinase Vps34. Cancer cell,
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..
van Montfort, R.L. & Workman, P.
(2009). Structure-based design of molecular cancer therapeutics. Trends biotechnol,
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..
van Montfort, R.L. & Collins, I.
(2009). Fragment-Based Methods in Drug Discovery: It's the Small Things that Matter. Curr top med chem,
Matthews, T.P., Klair, S., Burns, S., Boxall, K., Cherry, M., Fisher, M., Westwood, I.M., Walton, M.I., McHardy, T., Cheung, K.-., et al.
(2009). Identification of inhibitors of checkpoint kinase 1 through template screening. J med chem,
Checkpoint kinase 1 (CHK1) is an oncology target of significant current interest. Inhibition of CHK1 abrogates DNA damage-induced cell cycle checkpoints and sensitizes p53 deficient cancer cells to genotoxic therapies. Using template screening, a fragment-based approach to small molecule hit generation, we have identified multiple CHK1 inhibitor scaffolds suitable for further optimization. The sequential combination of in silico low molecular weight template selection, a high concentration biochemical assay and hit validation through protein-ligand X-ray crystallography provided 13 template hits from an initial in silico screening library of ca. 15000 compounds. The use of appropriate counter-screening to rule out nonspecific aggregation by test compounds was essential for optimum performance of the high concentration bioassay. One low molecular weight, weakly active purine template hit was progressed by iterative structure-based design to give submicromolar pyrazolopyridines with good ligand efficiency and appropriate CHK1-mediated cellular activity in HT29 colon cancer cells..
Westwood, I., Cheary, D.-., Baxter, J.E., Richards, M.W., van Montfort, R.L., Fry, A.M. & Bayliss, R.
(2009). Insights into the conformational variability and regulation of human Nek2 kinase. J mol biol,
The Nek family of serine/threonine kinases regulates centrosome and cilia function; in addition, several of its members are potential targets for drug discovery. Nek2 is dimeric, is cell cycle regulated and functions in the separation of centrosomes at G2/M. Here, we report the crystal structures of wild-type human Nek2 kinase domain bound to ADP at 1.55-A resolution and T175A mutant in apo form as well as that bound to a non-hydrolyzable ATP analog. These show that regions of the Nek2 structure around the nucleotide-binding site can adopt several different but well-defined conformations. None of the conformations was the same as that observed for the previously reported inhibitor-bound structure, and the two nucleotides stabilized two conformations. The structures suggest mechanisms for the auto-inhibition of Nek2 that we have tested by mutagenesis. Comparison of the structures with Aurora-A and Cdk2 gives insight into the structural mechanism of Nek2 activation. The production of specific inhibitors that target individual kinases of the human genome is an urgent challenge in drug discovery, and Nek2 is especially promising as a cancer target. We not only identify potential challenges to the task of producing Nek2 inhibitors but also propose that the conformational variability provides an opportunity for the design of Nek2 selective inhibitors because one of the conformations may provide a unique target..
Howard, N., Abell, C., Blakemore, W., Chessari, G., Congreve, M., Howard, S., Jhoti, H., Murray, C.W., Seavers, L.C. & van Montfort, R.L., et al.
(2006). Application of fragment screening and fragment linking to the discovery of novel thrombin inhibitors. J med chem,
The screening of fragments is an alternative approach to high-throughput screening for the identification of leads for therapeutic targets. Fragment hits have been discovered using X-ray crystallographic screening of protein crystals of the serine protease enzyme thrombin. The fragment library was designed to avoid any well-precedented, strongly basic functionality. Screening hits included a novel ligand (3), which binds exclusively to the S2-S4 pocket, in addition to smaller fragments which bind to the S1 pocket. The structure of these protein-ligand complexes are presented. A chemistry strategy to link two such fragments together and to synthesize larger drug-sized compounds resulted in the efficient identification of hybrid inhibitors with nanomolar potency (e.g., 7, IC50 = 3.7 nM). These potent ligands occupy the same area of the active site as previously described peptidic inhibitors, while having very different chemical architecture..
Van Montfort, R.L., Bateman, O.A., Lubsen, N.H. & Slingsby, C.
(2003). Crystal structure of truncated human beta B1-crystallin. Protein sci,
Crystallins are long-lived proteins packed inside eye lens fiber cells that are essential in maintaining the transparency and refractive power of the eye lens. Members of the two-domain betagamma-crystallin family assemble into an array of oligomer sizes, forming intricate higher-order networks in the lens cell. Here we describe the 1.4 Angstrom resolution crystal structure of a truncated version of human betaB1 that resembles an in vivo age-related truncation. The structure shows that unlike its close homolog, betaB2-crystallin, the homodimer is not domain swapped, but its domains are paired intramolecularly, as in more distantly related monomeric gamma-crystallins. However, the four-domain dimer resembles one half of the crystallographic bovine betaB2 tetramer and is similar to the engineered circular permuted rat betaB2. The crystal structure shows that the truncated betaB1 dimer is extremely well suited to form higher-order lattice interactions using its hydrophobic surface patches, linker regions, and sequence extensions..
van Montfort, R.L., Congreve, M., Tisi, D., Carr, R. & Jhoti, H.
(2003). Oxidation state of the active-site cysteine in protein tyrosine phosphatase 1B. Nature,
Protein tyrosine phosphatases regulate signal transduction pathways involving tyrosine phosphorylation(1) and have been implicated in the development of cancer, diabetes, rheumatoid arthritis and hypertension(2). Increasing evidence suggests that the cellular redox state is involved in regulating tyrosine phosphatase activity through the reversible oxidization of the catalytic cysteine to sulphenic acid (Cys-SOH)(3-6). But how further oxidation to the irreversible sulphinic (Cys-SO2H) and sulphonic (Cys-SO3H) forms is prevented remains unclear. Here we report the crystal structures of the regulatory sulphenic and irreversible sulphinic and sulphonic acids of protein tyrosine phosphatase 1B (PTP1B), an important enzyme in the negative regulation of the insulin receptor(7,8) and a therapeutic target in type II diabetes and obesity(9). We also identify a sulphenyl-amide species that is formed through oxidation of its catalytic cysteine. Formation of the sulphenyl-amide causes large changes in the PTP1B active site, which are reversible by reduction with the cellular reducing agent glutathione. The sulphenyl-amide is a protective intermediate in the oxidative inhibition of PTP1B. In addition, it may facilitate reactivation of PTP1B by biological thiols and signal a unique state of the protein..
Bateman, O.A., Purkiss, A.G., van Montfort, R., Slingsby, C., Graham, C. & Wistow, G.
(2003). Crystal structure of eta-crystallin: adaptation of a class 1 aldehyde dehydrogenase for a new role in the eye lens. Biochemistry,
Eta-crystallin is a retinal dehydrogenase that has acquired a role as a structural protein in the eye lens of elephant shrews, members of an ancient order of mammals. While it retains some activity toward retinal, which is oxidized to retinoic acid, the protein has acquired a number of specific sequence changes that have presumably been selected to enhance the lens role. The crystal structure of eta-crystallin, in common with class 1 and 2 ALDHs, is a dimer of dimers. It has a better-defined NAD binding site than those of related mammalian ALDH1 enzymes with the cofactor bound in the "hydride transfer" position in all four monomers with small differences about the dimer dyads. Although the active site is well conserved, the substrate-binding site is larger in eta-crystallin, and there are some mutations to the substrate access tunnel that might affect binding or release of substrate and product. It is possible that eta-crystallin has lost flexibility to improve its role in the lens. Enhanced binding of cofactor could enable it to act as a UV/blue light filter in the lens, improving visual acuity. The structure not only gives a view of a "natural mutant" of ALDH1 illustrating the adaptive conflict that can arise in multifunctional proteins, but also provides a well-ordered NAD binding site structure for this class of enzymes with important roles in development and health..
van Montfort, R.L., Basha, E., Friedrich, K.L., Slingsby, C. & Vierling, E.
(2001). Crystal structure and assembly of a eukaryotic small heat shock protein. Nat struct biol,
Van Montfort, R., Slingsby, C. & Vierling, E.
(2001). Structure and function of the small heat shock protein/alpha-crystallin family of molecular chaperones. Adv protein chem,
Van Montfort, R.L. & Dijkstra, B.W.
(1998). The functional importance of structural differences between the mannitol-specific IIA(mannitol) and the regulatory IIA(nitrogen). Protein sci,
The three-dimensional structures of the IIA domain of the mannitol-specific phosphoenol-pyruvate dependent phosphotransferase system (PTS) of Escherichia coli and the regulatory IIA(ntr) enzyme have been compared. The enzymes are 20% identical in sequence and contain the sequence motif specific for IIA proteins belonging to the mannitol-fructose family of the PTS. The fold of the enzymes is nearly identical, which confirms their evolution from a common ancestor. Moreover, the phosphorylation site of IIA(mtl) (His65) and one of the observed conformations of the active site Arg49 are extremely similar to their equivalents (His73 and Arg57) in IIA(ntr). In contrast, His120, the equivalent of the second active site His111 of IIA(mtl), is not located in the active site of IIA(ntr) but points into the solvent on the other side of the molecule. The different position of His120 makes it unlikely that this residue assists in phosphoryl transfer in the regulatory IIA(ntr)s. As His120 is conserved in all IIA(ntr) enzymes, it could have an essential role in the recognition of the target protein of IIA(ntr)..
van Montfort, R.L., Pijning, T., Kalk, K.H., Hangyi, I., Kouwijzer, M.L., Robillard, G.T. & Dijkstra, B.W.
(1998). The structure of the Escherichia coli phosphotransferase IIA(mannitol) reveals a novel fold with two conformations of the active site. Structure,
Background: The bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) catalyses the cellular uptake and subsequent phosphorylation of carbohydrates. Moreover, the PTS plays a crucial role in the global regulation of various metabolic pathways, The PTS consists of two general proteins, enzyme I and the histidine-containing protein (HPr), and the carbohydrate-specific enzyme II [E-II]. E(II)s are usually composed of two cytoplasmic domains, IIA and IIB, and a transmembrane domain, IIC. The IIA domains catalyse the transfer of a phosphoryl group from HPr to IIB, which phosphorylates the transported carbohydrate. Knowledge of the structures of the IIA proteins may provide insight into the mechanisms by which the PTS couples phosphorylation reactions with carbohydrate specificity.Results: We have determined the crystal structure of the Escherichia coli mannitol-specific IIA domain, IIA(mtl) (M-r 16.3 kDa), by multiple anomalous dispersion analysis of a selenomethionine variant of IIA(mtl). The structure was refined at 1.8 Angstrom resolution to an R factor of 19.0% (R-free 24.2%). The enzyme consists of a single five-stranded mixed beta sheet, flanked by helices on both sides. The phosphorylation site (His65) is located at the end of the third beta strand, in a shallow crevice lined with hydrophobic residues. The sidechains of two conserved active-site residues, Arg49 and His111, adopt two different conformations in the four independent IIA(mtl) molecules. Using a solution structure of phosphorylated HPr, and a combination of molecular modelling and NMR binding experiments, structural models of the HPr-IIA(mtl) complex were generated.Conclusions: The fold of IIA(mtl) is completely different from the structures of other IIA proteins determined so far, The two conformations of Arg49 and His111 might represent different states of the active site, required for the different phosphoryl transfer reactions in which IIA(mtl) is involved. A comparison of the HPr-IIA(mtl) model with models of HPr in complex with other IIA enzymes shows that the overall interaction mode between the two proteins is similar. Differences in the stabilisation of the invariant residue Arg17 of HPr by the different IIA proteins might be part of a subtle mechanism to control the hierarchy of carbohydrate utilisation by the bacterium..
vanMontfort, R.L., Pijning, T., Kalk, K.H., Reizer, J., Saier, M.H., Thunnissen, M.M., Robillard, G.T. & Dijkstra, B.W.
(1997). The structure of an energy-coupling protein from bacteria, IIBcellobiose, reveals similarity to eukaryotic protein tyrosine phosphatases. Structure,
Background: The bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) mediates the energy-driven uptake of carbohydrates and their concomitant phosphorylation, In addition, the PTS is intimately involved in the regulation of a variety of metabolic and transcriptional processes in the bacterium. The multiprotein PTS consists of a membrane channel and at least four cytoplasmic proteins or protein domains that sequentially transfer a phosphoryl group from phosphoenolpyruvate to the transported carbohydrate, Determination of the three-dimensional structure of the IIB enzymes within the multiprotein complex would provide insights into the mechanisms by which they promote efficient transport by the membrane channel IIC protein and phosphorylate the transported carbohydrate on the inside of the cell,Results: The crystal structure of the IIB enzyme specific for cellobiose, IIBcellobiose (molecular weight 11.4 kDa), has been determined to a resolution of 1.8 Angstrom and refined to an R factor of 18.7% (R(free) of 24.1%). The enzyme consists of a single four-stranded parallel beta sheet flanked by helices on both sides. The phosphorylation site (Cys10) is located at the C-terminal end of the first beta strand, No positively charged residues, which could assist in phosphoryl-transfer, can be found in or near the active site. The fold of IIBcellobiose is remarkably similar to that of the mammalian low molecular weight protein tyrosine phosphatases.Conclusions: A comparison between IIBcellobiose and the structurally similar low molecular weight protein tyrosine phosphatases provides insight into the mechanism of the phosphoryltransfer reactions in which IIBcellobiose is involved, The differences in tertiary structure and active-site composition between IIBcellobiose and the glucose-specific IIBglucose give a structural explanation why the carbohydrate-specific components of different families cannot complement each other..
Dijkstra, B.W. & vanMontfort, R.L.
(1996). Structure and function of PEP-dependent sugar transport systems. Prog biophys mol bio,
VANMONTFORT, R., PIJNING, T., KALK, K., SCHUURMANWOLTERS, G., REIZER, J., SAIER, M., ROBILLARD, G. & DIJKSTRA, B.
(1994). CRYSTALLIZATION OF ENZYME IIB OF THE CELLOBIOSE-SPECIFIC PHOSPHOTRANSFERASE SYSTEM OF ESCHERICHIA-COLI. J mol biol,
Lawson, C.L., van Montfort, R., Strokopytov, B., Rozeboom, H.J., Kalk, K.H., de Vries, G.E., Penninga, D., Dijkhuizen, L. & Dijkstra, B.W.
(1994). Nucleotide sequence and X-ray structure of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 in a maltose-dependent crystal form. J mol biol,
The cyclodextrin glycosyltransferase (CGTase, EC 220.127.116.11) gene from Bacillus circulans strain 251 was cloned and sequenced. It was found to code for a mature protein of 686 amino acid residues, showing 75% identity to the CGTase from B. circulans strain 8. The X-ray structure of the CGTase was elucidated in a maltodextrin-dependent crystal form and refined against X-ray diffraction data to 2.0 A resolution. The structure of the enzyme is nearly identical to the CGTase from B. circulans strain 8. Three maltose binding sites are observed at the protein surface, two in domain E and one in domain C. The maltose-dependence of CGTase crystallization can be ascribed to the proximity of two of the maltose binding sites to intermolecular crystal contacts. The maltose molecules bound in the E domain interact with several residues implicated in a raw starch binding motif conserved among a diverse group of starch converting enzymes..
van Montfort, R.L. & Workman, P.
Structure-based drug design: aiming for a perfect fit. Essays biochem,
Knowledge of the three-dimensional structure of therapeutically relevant targets has informed drug discovery since the first protein structures were determined using X-ray crystallography in the 1950s and 1960s. In this editorial we provide a brief overview of the powerful impact of structure-based drug design (SBDD), which has its roots in computational and structural biology, with major contributions from both academia and industry. We describe advances in the application of SBDD for integral membrane protein targets that have traditionally proved very challenging. We emphasize the major progress made in fragment-based approaches for which success has been exemplified by over 30 clinical drug candidates and importantly three FDA-approved drugs in oncology. We summarize the articles in this issue that provide an excellent snapshot of the current state of the field of SBDD and fragment-based drug design and which offer key insights into exciting new developments, such as the X-ray free-electron laser technology, cryo-electron microscopy, open science approaches and targeted protein degradation. We stress the value of SBDD in the design of high-quality chemical tools that are used to interrogate biology and disease pathology, and to inform target validation. We emphasize the need to maintain the scientific rigour that has been traditionally associated with structural biology and extend this to other methods used in drug discovery. This is particularly important because the quality and robustness of any form of contributory data determines its usefulness in accelerating drug design, and therefore ultimately in providing patient benefit..
Tisi, D., Chiarparin, E., Tamanini, E., Pathuri, P., Coyle, J.E., Hold, A., Holding, F.P., Amin, N., Martin, A.C., Rich, S.J., et al.
Structure of the Epigenetic Oncogene MMSET and Inhibition by N-Alkyl Sinefungin Derivatives. Acs chem biol,
The members of the NSD subfamily of lysine methyl transferases are compelling oncology targets due to the recent characterization of gain-of-function mutations and translocations in several hematological cancers. To date, these proteins have proven intractable to small molecule inhibition. Here, we present initial efforts to identify inhibitors of MMSET (aka NSD2 or WHSC1) using solution phase and crystal structural methods. On the basis of 2D NMR experiments comparing NSD1 and MMSET structural mobility, we designed an MMSET construct with five point mutations in the N-terminal helix of its SET domain for crystallization experiments and elucidated the structure of the mutant MMSET SET domain at 2.1 Å resolution. Both NSD1 and MMSET crystal systems proved resistant to soaking or cocrystallography with inhibitors. However, use of the close homologue SETD2 as a structural surrogate supported the design and characterization of N-alkyl sinefungin derivatives, which showed low micromolar inhibition against both SETD2 and MMSET..
Jones, A.M., Westwood, I.M., Osborne, J.D., Matthews, T.P., Cheeseman, M.D., Rowlands, M.G., Jeganathan, F., Burke, R., Lee, D., Kadi, N., et al.
A fragment-based approach applied to a highly flexible target: Insights and challenges towards the inhibition of HSP70 isoforms. Sci rep,
The heat shock protein 70s (HSP70s) are molecular chaperones implicated in many cancers and of significant interest as targets for novel cancer therapies. Several HSP70 inhibitors have been reported, but because the majority have poor physicochemical properties and for many the exact mode of action is poorly understood, more detailed mechanistic and structural insight into ligand-binding to HSP70s is urgently needed. Here we describe the first comprehensive fragment-based inhibitor exploration of an HSP70 enzyme, which yielded an amino-quinazoline fragment that was elaborated to a novel ATP binding site ligand with different physicochemical properties to known adenosine-based HSP70 inhibitors. Crystal structures of amino-quinazoline ligands bound to the different conformational states of the HSP70 nucleotide binding domain highlighted the challenges of a fragment-based approach when applied to this particular flexible enzyme class with an ATP-binding site that changes shape and size during its catalytic cycle. In these studies we showed that Ser275 is a key residue in the selective binding of ATP. Additionally, the structural data revealed a potential functional role for the ATP ribose moiety in priming the protein for the formation of the ATP-bound pre-hydrolysis complex by influencing the conformation of one of the phosphate binding loops..
Osborne, J.D., Matthews, T.P., McHardy, T., Proisy, N., Cheung, K.-., Lainchbury, M., Brown, N., Walton, M.I., Eve, P.D., Boxall, K.J., et al.
Multiparameter Lead Optimization to Give an Oral Checkpoint Kinase 1 (CHK1) Inhibitor Clinical Candidate: (R)-5-((4-((Morpholin-2-ylmethyl)amino)-5-(trifluoromethyl)pyridin-2-yl)amino)pyrazine-2-carbonitrile (CCT245737). J med chem,
Multiparameter optimization of a series of 5-((4-aminopyridin-2-yl)amino)pyrazine-2-carbonitriles resulted in the identification of a potent and selective oral CHK1 preclinical development candidate with in vivo efficacy as a potentiator of deoxyribonucleic acid (DNA) damaging chemotherapy and as a single agent. Cellular mechanism of action assays were used to give an integrated assessment of compound selectivity during optimization resulting in a highly CHK1 selective adenosine triphosphate (ATP) competitive inhibitor. A single substituent vector directed away from the CHK1 kinase active site was unexpectedly found to drive the selective cellular efficacy of the compounds. Both CHK1 potency and off-target human ether-a-go-go-related gene (hERG) ion channel inhibition were dependent on lipophilicity and basicity in this series. Optimization of CHK1 cellular potency and in vivo pharmacokinetic-pharmacodynamic (PK-PD) properties gave a compound with low predicted doses and exposures in humans which mitigated the residual weak in vitro hERG inhibition..
Innocenti, P., Woodward, H.L., Solanki, S., Naud, S., Westwood, I.M., Cronin, N., Hayes, A., Roberts, J., Henley, A.T., Baker, R., et al.
Rapid Discovery of Pyrido[3,4-d]pyrimidine Inhibitors of Monopolar Spindle Kinase 1 (MPS1) Using a Structure-Based Hybridization Approach. J med chem,
Monopolar spindle 1 (MPS1) plays a central role in the transition of cells from metaphase to anaphase and is one of the main components of the spindle assembly checkpoint. Chromosomally unstable cancer cells rely heavily on MPS1 to cope with the stress arising from abnormal numbers of chromosomes and centrosomes and are thus more sensitive to MPS1 inhibition than normal cells. We report the discovery and optimization of a series of new pyrido[3,4-d]pyrimidine based inhibitors via a structure-based hybridization approach from our previously reported inhibitor CCT251455 and a modestly potent screening hit. Compounds in this novel series display excellent potency and selectivity for MPS1, which translates into biomarker modulation in an in vivo human tumor xenograft model..
Cheeseman, M.D., Westwood, I.M., Barbeau, O., Rowlands, M., Dobson, S., Jones, A.M., Jeganathan, F., Burke, R., Kadi, N., Workman, P., et al.
Exploiting Protein Conformational Change to Optimize Adenosine-Derived Inhibitors of HSP70. J med chem,
HSP70 is a molecular chaperone and a key component of the heat-shock response. Because of its proposed importance in oncology, this protein has become a popular target for drug discovery, efforts which have as yet brought little success. This study demonstrates that adenosine-derived HSP70 inhibitors potentially bind to the protein with a novel mechanism of action, the stabilization by desolvation of an intramolecular salt-bridge which induces a conformational change in the protein, leading to high affinity ligands. We also demonstrate that through the application of this mechanism, adenosine-derived HSP70 inhibitors can be optimized in a rational manner..