Felisberto-Rodrigues, C., Thomas, J.C., McAndrew, C., Le Bihan, Y.-., Burke, R., Workman, P. & van Montfort, R.L.
(2019). Structural and functional characterisation of human RNA helicase DHX8 provides insights into the mechanism of RNA-stimulated ADP release. Biochemical journal,
DHX8 is a crucial DEAH-box RNA helicase involved in splicing and required for the release of mature mRNA from the spliceosome. Here, we report the biochemical characterisation of full-length human DHX8 and the catalytically active helicase core DHX8Δ547, alongside crystal structures of DHX8Δ547 bound to ADP and a structure of DHX8Δ547 bound to poly(A)6 single-strand RNA. Our results reveal that DHX8 has an in vitro binding preference for adenine-rich RNA and that RNA binding triggers the release of ADP through significant conformational flexibility in the conserved DEAH-, P-loop and hook-turn motifs. We demonstrate the importance of R620 and both the hook-turn and hook-loop regions for DHX8 helicase activity and propose that the hook-turn acts as a gatekeeper to regulate the directional movement of the 3′ end of RNA through the RNA-binding channel. This study provides an in-depth understanding of the activity of DHX8 and contributes insights into the RNA-unwinding mechanisms of the DEAH-box helicase family..
Woodward, H.L., Innocenti, P., Cheung, K.-., Hayes, A., Roberts, J., Henley, A.T., Faisal, A., Mak, G.W., Box, G., Westwood, I.M., et al.
(2018). Introduction of a Methyl Group Curbs Metabolism of Pyrido[3,4- d]pyrimidine Monopolar Spindle 1 (MPS1) Inhibitors and Enables the Discovery of the Phase 1 Clinical Candidate N2-(2-Ethoxy-4-(4-methyl-4 H-1,2,4-triazol-3-yl)phenyl)-6-methyl- N8-neopentylpyrido[3,4- d]pyrimidine-2,8-diamine (BOS172722). J med chem,
Monopolar spindle 1 (MPS1) occupies a central role in mitosis and is one of the main components of the spindle assembly checkpoint. The MPS1 kinase is an attractive cancer target, and herein, we report the discovery of the clinical candidate BOS172722. The starting point for our work was a series of pyrido[3,4- d]pyrimidine inhibitors that demonstrated excellent potency and kinase selectivity but suffered from rapid turnover in human liver microsomes (HLM). Optimizing HLM stability proved challenging since it was not possible to identify a consistent site of metabolism and lowering lipophilicity proved unsuccessful. Key to overcoming this problem was the finding that introduction of a methyl group at the 6-position of the pyrido[3,4- d]pyrimidine core significantly improved HLM stability. Met ID studies suggested that the methyl group suppressed metabolism at the distant aniline portion of the molecule, likely by blocking the preferred pharmacophore through which P450 recognized the compound. This work ultimately led to the discovery of BOS172722 as a Phase 1 clinical candidate..
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. British journal of cancer,
van Montfort, R.L. & Workman, P.
(2017). Structure-based drug design: aiming for a perfect fit. Essays in biochemistry,
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..
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 - a european journal,
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.
(2016). A fragment-based approach applied to a highly flexible target: Insights and challenges towards the inhibition of HSP70 isoforms. Scientific reports,
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. Journal of medicinal chemistry,
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.
(2016). 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..
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.
(2016). 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..
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 research,
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. .
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..
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..
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,
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..
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..
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..
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., 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..
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,