Discovery and Characterization of a Cryptic Secondary Binding Site in the Molecular Chaperone HSP70.

Heat Shock Protein 70s (HSP70s) are key molecular chaperones that are overexpressed in many cancers and often associated with metastasis and poor prognosis. It has proven difficult to develop ATP-competitive, drug-like small molecule inhibitors of HSP70s due to the flexible and hydrophilic nature of the HSP70 ATP-binding site and its high affinity for endogenous nucleotides. The aim of this study was to explore the potential for the inhibition of HSP70 through alternative binding sites using fragment-based approaches. A surface plasmon resonance (SPR) fragment screen designed to detect secondary binding sites in HSP70 led to the identification by X-ray crystallography of a cryptic binding site in the nucleotide-binding domain (NBD) of HSP70 adjacent to the ATP-binding site. Fragment binding was confirmed and characterized as ATP-competitive using SPR and ligand-observed NMR methods. Molecular dynamics simulations were applied to understand the interactions with the protein upon ligand binding, and local secondary structure changes consistent with interconversion between the observed crystal structures with and without the cryptic pocket were detected. A virtual high-throughput screen (vHTS) against the cryptic pocket was conducted, and five compounds with diverse chemical scaffolds were confirmed to bind to HSP70 with micromolar affinity by SPR. These results identified and characterized a new targetable site on HSP70. While targeting HSP70 remains challenging, the new site may provide opportunities to develop allosteric ATP-competitive inhibitors with differentiated physicochemical properties from current series.

High Proliferation Rate and a Compromised Spindle Assembly Checkpoint Confers Sensitivity to the MPS1 Inhibitor BOS172722 in Triple-Negative Breast Cancers.

BOS172722 (CCT289346) is a highly potent, selective, and orally bioavailable inhibitor of spindle assembly checkpoint kinase MPS1. BOS172722 treatment alone induces significant sensitization to death, particularly in highly proliferative triple-negative breast cancer (TNBC) cell lines with compromised spindle assembly checkpoint activity. BOS172722 synergizes with paclitaxel to induce gross chromosomal segregation defects caused by MPS1 inhibitor-mediated abrogation of the mitotic delay induced by paclitaxel treatment. In in vivo pharmacodynamic experiments, BOS172722 potently inhibits the spindle assembly checkpoint induced by paclitaxel in human tumor xenograft models of TNBC, as measured by inhibition of the phosphorylation of histone H3 and the phosphorylation of the MPS1 substrate, KNL1. This mechanistic synergy results in significant in vivo efficacy, with robust tumor regressions observed for the combination of BOS172722 and paclitaxel versus either agent alone in long-term efficacy studies in multiple human tumor xenograft TNBC models, including a patient-derived xenograft and a systemic metastasis model. The current target indication for BOS172722 is TNBC, based on their high sensitivity to MPS1 inhibition, the well-defined clinical patient population with high unmet need, and the synergy observed with paclitaxel.

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).

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.

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.

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.

Characterisation of CCT271850, a selective, oral and potent MPS1 inhibitor, used to directly measure in vivo MPS1 inhibition vs therapeutic efficacy.

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.

A fragment-based approach applied to a highly flexible target: Insights and challenges towards the inhibition of HSP70 isoforms.

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.

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).

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.

Rapid Discovery of Pyrido[3,4-d]pyrimidine Inhibitors of Monopolar Spindle Kinase 1 (MPS1) Using a Structure-Based Hybridization Approach.

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.

Exploiting Protein Conformational Change to Optimize Adenosine-Derived Inhibitors of HSP70.

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.

8-Substituted Pyrido[3,4-d]pyrimidin-4(3H)-one Derivatives As Potent, Cell Permeable, KDM4 (JMJD2) and KDM5 (JARID1) Histone Lysine Demethylase Inhibitors.

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.

Naturally Occurring Mutations in the MPS1 Gene Predispose Cells to Kinase Inhibitor Drug Resistance.

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.

Differences between murine arylamine N-acetyltransferase type 1 and human arylamine N-acetyltransferase type 2 defined by substrate specificity and inhibitor binding.

BACKGROUND: The mouse has three arylamine N-acetyltransferase genes, (MOUSE)Nat1, (MOUSE)Nat2 and (MOUSE)Nat3. These are believed to correspond to (HUMAN)NAT1, (HUMAN)NAT2 and NATP in humans. (MOUSE)Nat3 encodes an enzyme with poor activity and human NATP is a pseudogene. (MOUSE)Nat2 is orthologous to (HUMAN)NAT1 and their corresponding proteins are functionally similar, but the relationship between (MOUSE)Nat1 and (HUMAN)NAT2 is less clear-cut. METHODS: To determine whether the (MOUSE)NAT1 and (HUMAN)NAT2 enzymes are functionally equivalent, we expressed and purified (MOUSE)NAT1*1 and analysed its substrate specificity using a panel of arylamines and hydrazines. To understand how specific residues contribute to substrate selectivity, three site-directed mutants of (MOUSE)NAT2*1 were prepared: these were (MOUSE)NAT2_F125S, (MOUSE)NAT2_R127G and (MOUSE)NAT2_R127L. All three exhibited diminished activity towards "(MOUSE)NAT2-specific" arylamines but were more active against hydrazines than (MOUSE)NAT1*1. The inhibitory and colorimetric properties of a selective naphthoquinone inhibitor of (HUMAN)NAT1 and (MOUSE)NAT2 were investigated. RESULTS: Comparing (MOUSE)NAT1*1 with other mammalian NAT enzymes demonstrated that the substrate profiles of (MOUSE)NAT1 and (HUMAN)NAT2 are less similar than previously believed. Three key residues (F125, R127 and Y129) in (HUMAN)NAT1*4 and (MOUSE)NAT2*1 were required for enzyme inhibition and the associated colour change on naphthoquinone binding. In silico modelling of selective ligands into the appropriate NAT active sites further implicated these residues in substrate and inhibitor specificity in mouse and human NAT isoenzymes. CONCLUSIONS: Three non-catalytic residues within (HUMAN)NAT1*4 (F125, R127 and Y129) contribute both to substrate recognition and inhibitor binding by participating in distinctive intermolecular interactions and maintaining the steric conformation of the catalytic pocket. These active site residues contribute to the definition of substrate and inhibitor selectivity, an understanding of which is essential for facilitating the design of second generation (HUMAN)NAT1-selective inhibitors for diagnostic, prognostic and therapeutic purposes. In particular, since the expression of (HUMAN)NAT1 is related to the development and progression of oestrogen-receptor-positive breast cancer, these structure-based tools will facilitate the ongoing design of candidate compounds for use in (HUMAN)NAT1-positive breast tumours.

Structure-based design of orally bioavailable 1H-pyrrolo[3,2-c]pyridine inhibitors of mitotic kinase monopolar spindle 1 (MPS1).

The protein kinase MPS1 is a crucial component of the spindle assembly checkpoint signal and is aberrantly overexpressed in many human cancers. MPS1 is one of the top 25 genes overexpressed in tumors with chromosomal instability and aneuploidy. PTEN-deficient breast tumor cells are particularly dependent upon MPS1 for their survival, making it a target of significant interest in oncology. We report the discovery and optimization of potent and selective MPS1 inhibitors based on the 1H-pyrrolo[3,2-c]pyridine scaffold, guided by structure-based design and cellular characterization of MPS1 inhibition, leading to 65 (CCT251455). This potent and selective chemical tool stabilizes an inactive conformation of MPS1 with the activation loop ordered in a manner incompatible with ATP and substrate-peptide binding; it displays a favorable oral pharmacokinetic profile, shows dose-dependent inhibition of MPS1 in an HCT116 human tumor xenograft model, and is an attractive tool compound to elucidate further the therapeutic potential of MPS1 inhibition.

The discovery of potent ribosomal S6 kinase inhibitors by high-throughput screening and structure-guided drug design.

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.

Synthesis and evaluation of heteroaryl substituted diazaspirocycles as scaffolds to probe the ATP-binding site of protein kinases.

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.

Fragment-based screening maps inhibitor interactions in the ATP-binding site of checkpoint kinase 2.

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.

Scaffold-focused virtual screening: prospective application to the discovery of TTK inhibitors.

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.

Benzimidazole inhibitors of the protein kinase CHK2: clarification of the binding mode by flexible side chain docking and protein-ligand crystallography.

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.

Piperidinols that show anti-tubercular activity as inhibitors of arylamine N-acetyltransferase: an essential enzyme for mycobacterial survival inside macrophages.

Latent M. tuberculosis infection presents one of the major obstacles in the global eradication of tuberculosis (TB). Cholesterol plays a critical role in the persistence of M. tuberculosis within the macrophage during latent infection. Catabolism of cholesterol contributes to the pool of propionyl-CoA, a precursor that is incorporated into cell-wall lipids. Arylamine N-acetyltransferase (NAT) is encoded within a gene cluster that is involved in the cholesterol sterol-ring degradation and is essential for intracellular survival. The ability of the NAT from M. tuberculosis (TBNAT) to utilise propionyl-CoA links it to the cholesterol-catabolism pathway. Deleting the nat gene or inhibiting the NAT enzyme prevents intracellular survival and results in depletion of cell-wall lipids. TBNAT has been investigated as a potential target for TB therapies. From a previous high-throughput screen, 3-benzoyl-4-phenyl-1-methylpiperidinol was identified as a selective inhibitor of prokaryotic NAT that exhibited antimycobacterial activity. The compound resulted in time-dependent irreversible inhibition of the NAT activity when tested against NAT from M. marinum (MMNAT). To further evaluate the antimycobacterial activity and the NAT inhibition of this compound, four piperidinol analogues were tested. All five compounds exert potent antimycobacterial activity against M. tuberculosis with MIC values of 2.3-16.9 µM. Treatment of the MMNAT enzyme with this set of inhibitors resulted in an irreversible time-dependent inhibition of NAT activity. Here we investigate the mechanism of NAT inhibition by studying protein-ligand interactions using mass spectrometry in combination with enzyme analysis and structure determination. We propose a covalent mechanism of NAT inhibition that involves the formation of a reactive intermediate and selective cysteine residue modification. These piperidinols present a unique class of antimycobacterial compounds that have a novel mode of action different from known anti-tubercular drugs.

Structure-guided evolution of potent and selective CHK1 inhibitors through scaffold morphing.

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.