Jessica Schmitt

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.

Aurora Isoform Selectivity: Design and Synthesis of Imidazo[4,5-B]Pyridine Derivatives as Highly Selective Inhibitors of Aurora-A Kinase in Cells.

Aurora-A differs from Aurora-B/C at three positions in the ATP-binding pocket (L215, T217, and R220). Exploiting these differences, crystal structures of ligand–Aurora protein interactions formed the basis of a design principle for imidazo[4,5-b]pyridine-derived Aurora-A-selective inhibitors. Guided by a computational modeling approach, appropriate C7-imidazo[4,5-b]pyridine derivatization led to the discovery of highly selective inhibitors, such as compound 28c, of Aurora-A over Aurora-B. In HCT116 human colon carcinoma cells, 28c and 40f inhibited the Aurora-A L215R and R220K mutants with IC50 values similar to those seen for the Aurora-A wild type. However, the Aurora-A T217E mutant was significantly less sensitive to inhibition by 28c and 40f compared to the Aurora-A wild type, suggesting that the T217 residue plays a critical role in governing the observed isoform selectivity for Aurora-A inhibition. These compounds are useful small-molecule chemical tools to further explore the function of Aurora-A in cells.

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.

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.

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.

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-4H-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.

Abstract 3642: Structure enabled design of inhibitors of the mitotic kinase MPS1

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA MPS1 (also known as TTK), is a dual-specificity protein kinase that is essential for the proper attachment of chromosomes to the mitotic spindle. MPS1 expression is elevated in a variety of human cancers and is correlated with higher histological grade, aggressiveness and poor patient survival. It has been also shown that basal, PTEN-deficient and triple negative breast cancers are sensitive to MPS1 inhibition. These data together provide strong support for selective antitumour action of MPS1 inhibitors in human cancers. Here we report the discovery of pyridopyrimidines as a new class of inhibitors of MPS1 exploring a hybridization approach. Rapid structure based optimisation of the initial hits led to highly potent, selective and ligand efficient compounds. We will discuss our initial design approach and the structural features that are critical for potent biochemical and cellular inhibition as derived from SAR and co-crystal structures. Furthermore, we will report pharmacokinetic and in vivo properties of selected compounds and comment on our strategy to optimise this series towards preclinical candidates. Citation Format: Paolo Innocenti, Hannah Woodward, Kwai_Ming J. Cheung, Sebastien Naud, Savade Solanki, Isaac M. Westwood, Amir Faisal, Angela Hayes, Jessica Schmitt, Ross Baker, Berry Matijssen, Rosemary Burke, Suzanne A. Eccles, Florence I. Raynaud, Spiros Linardopoulos, Julian Blagg, Rob L M van Montfort, Swen Hoelder. Structure enabled design of inhibitors of the mitotic kinase MPS1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3642. doi:10.1158/1538-7445.AM2015-3642

Abstract 5450: Naturally occurring mutations in the MPS1 gene predispose cells to kinase inhibitor drug-resistance

Acquired resistance is the greatest challenge to the effectiveness of targeted anti-cancer therapies in the clinic. With several MPS1 inhibitors under pre-clinical development, we aimed to investigate how cancer cells will develop resistance against these inhibitors; therefore we modeled acquired resistance using a range of MPS1 inhibitors. We identified and characterized five point mutations in the kinase domain of MPS1 that confer resistance against multiple inhibitors. Structural studies showed that several MPS1 mutants conferred resistance by causing steric hindrance to inhibitor binding. One mutation in particular, p.C604W, which is close to the gatekeeper residue, rendered MPS1 resistant to all the inhibitors we tested. However, we were able to design new compounds to specifically overcome this mutation, which in fact targeted the mutant with more potency than the wild-type MPS1 protein. Importantly, we show that these mutations are present in untreated cancer cell lines and primary tumour samples, and also pre-exist in normal lymphoblast and breast tissues. Furthermore, to confirm this is not specific to MPS1, we show that the EGFR p.T790M mutation is also pre-existing in cancer cell lines and normal tissue. Our data therefore suggest that mutations conferring resistance to targeted therapy are naturally occurring mutations in normal and cancer cells that are not introduced due to cancer cells being more mutagenic. Citation Format: Mark D. Gurden, Isaac Westwood, Amir Faisal, Sebastien Naud, Jack Cheung, Craig McAndrew, Amy Wood, Jessica Schmitt, Kathy Boxall, Grace Mak, Paul Workman, Rosemary Burke, Swen Hoelder, Julian Blagg, Rob Van Montfort, Spiros Linardopoulos. Naturally occurring mutations in the MPS1 gene predispose cells to kinase inhibitor drug-resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5450. doi:10.1158/1538-7445.AM2015-5450

Abstract 3242: CCT271850, a novel, selective, highly potent and orally bioavailable Mps1 kinase inhibitor.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The main role of the cell cycle is to enable error-free DNA replication, chromosome segregation and cytokinesis. Surveillance mechanisms, the so-called checkpoint pathways, monitor passage through mitosis at several stages. One of the best characterised is the spindle assembly checkpoint that prevents anaphase onset until the appropriate tension and attachment across kinetochores is achieved. One of the first components of the spindle assembly checkpoint signal, identified by a genetic screen in budding yeast, was MPS1 (monopolar spindle 1; also known as TTK). MPS1 gene was shown to encode an essential dual-specificity kinase conserved from yeast to humans. MPS1 activity peaks at the G2/M transition and is enhanced upon activation of the spindle assembly checkpoint with nocodazole. We and others, have identified the autophosphorylation of T676 in the activation loop of MPS1 and shown that this is essential for MPS1 function. MPS1 has been found aberrantly overexpressed in a wide range of human tumours including breast, lung, oesophagus, and prostate. MPS1 is required for the establishment and maintenance of the spindle assembly checkpoint during mitosis. Aneuploid tumour cells possess a compromised spindle checkpoint to allow onset of anaphase and cell division. We have shown that depletion of MPS1 by siRNA induces cell death selectively in PTEN-deficient breast cancer cell lines. We have developed biochemical and cellular assays for MPS1 activity and a high throughput screening of our compound library delivered multiple hit series. We have previously reported the discovery of CCT251455 as a selective and orally bioavailable MPS1 inhibitor that inhibits the growth of a panel of human tumour cell lines, abrogates nocodazole-induced mitotic arrest and reduces the time spent in mitosis. Medicinal chemistry in combination with X-ray crystallography led to the discovery of CCT271850, a novel inhibitor of MPS1 kinase activity. CCT271850 selectively inhibits MPS1 kinase activity with an IC50 of 0.004 μM, inhibits autophosphorylation of MPS1 in cells with an IC50 of 0.07 μM and reduces the growth of a panel of human tumour cell lines, particularly PTEN-deficient cell lines. Tumour cells treated with CCT271850 contain aberrant numbers of chromosomes and the majority of cells divide their chromosomes without proper alignment. CCT271850 is orally bioavailable (F = 68%) and shows modulation of biomarkers in vivo. Citation Format: Amir Faisal, Paolo Innocenti, Isaac Westwood, Sebastan Naud, Jessica Schmitt, Angela Hayes, Grace Mak, Mark Gurden, Vassilios Bavetsias, Jack Cheung, Hannah Woodward, Peter Sheldrake, Butrus Atrash, Rosemary Burke, Ross Baker, Craig McAndrew, Martin Rowlands, Melanie Valenti, Paul Workman, Suzanne Eccles, Florence Raynaud, Rob vanMontfort, Swen Hoelder, Julian Blagg, Spiros Linardopoulos. CCT271850, a novel, selective, highly potent and orally bioavailable Mps1 kinase inhibitor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3242. doi:10.1158/1538-7445.AM2013-3242

Abstract 1817: Characterisation of CCT251455, a novel, selective and highly potent Mps1 kinase inhibitor

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Monopolar spindle 1 (Mps1, also known as TTK) is a dual-specificity, cell cycle-regulated kinase required for the establishment and maintenance of the spindle assembly checkpoint during mitosis. Aneuploid tumour cells possess a weak spindle checkpoint to allow onset of anaphase and cell division. Our hypothesis is that a complete inhibition of an already weakened mitotic checkpoint of cancer cells will cause gross chromosomal abnormalities leading to aneuploid cell death. We have shown that depletion of Mps1 by siRNA induces cell death selectively in aneuploid and PTEN-deficient cancer cell lines. We have demonstrated that Mps1 depletion inhibits MAD2 localisation to the kinetochores. We have developed biochemical and cellular assays for Mps1 activity and a high throughput screening of our Institutes compound library delivered multiple hit series. Medicinal chemistry in combination with X-ray crystallography led to the development of CCT251455, a small molecule inhibitor of Mps1 kinase activity. CCT251455 selectively inhibits Mps1 kinase with an IC50 of 0.003 μM, inhibits growth of a panel of human tumour cell lines with GI50 between 0.06 - 1 μM and is particularly potent in PTEN-deficient cell lines. Cells treated with CCT251455 abrogate nocodazole-induced mitotic arrest and reduce the time spent in mitosis. Mps1-inhibited cells contain aberrant numbers of chromosomes and the majority of cells divide their chromosomes without proper alignment. CCT251455 is orally bioavailable (F = 82%) and shows modulation of biomarkers in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1817. doi:1538-7445.AM2012-1817