Hannah Woodward

Discovery of Novel Small-Molecule Inhibitors of BRD4 Using Structure-Based Virtual Screening.

Bromodomains (BRDs) are epigenetic readers that recognize acetylated-lysine (KAc) on proteins and are implicated in a number of diseases. We describe a virtual screening approach to identify BRD inhibitors. Key elements of this approach are the extensive design and use of substructure queries to compile a set of commercially available compounds featuring novel putative KAc mimetics and docking this set for final compound selection. We describe the validation of this approach by applying it to the first BRD of BRD4. The selection and testing of 143 compounds lead to the discovery of six novel hits, including four unprecedented KAc mimetics. We solved the crystal structure of four hits, determined their binding mode, and improved their potency through synthesis and the purchase of derivatives. This work provides a validated virtual screening approach that is applicable to other BRDs and describes novel KAc mimetics that can be further explored to design more potent inhibitors.

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.

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 193: Inhibitors of MPS1: Discovery of CCT289346, a highly potent, selective and orally available preclinical candidate

MPS1 (also known as TTK), is a dual-specificity protein kinase and one of the main components of the spindle assembly checkpoint. Cancer cells heavily rely on MPS1 to cope with aneuploidy resulting from aberrant numbers of chromosomes and MPS1 has been found to be upregulated in a large number of tumor types. Extensive work by us and other groups has shown that MPS1 inhibitors are effective against a variety of cancers, particularly when used in combination with other drugs, for example, tubulin-targeting agents. We recently reported the structure-based design and discovery of a series of pyrido[3,4-d]pyrimidines inhibitors of MPS1 (1). Advanced compounds showed very potent inhibition of MPS1 in biochemical and cellular assays. However, these compounds suffered from high lipophilicity and pronounced metabolism in human liver microsomes preventing progression into preclinical development. Here we report the optimisation of this series ultimately yielding CCT289346, our preclinical candidate. CCT289346 shows excellent potency, kinase selectivity, and ADME properties including stability in human liver microsomes. The compound has been produced on a kilogram scale and is currently undergoing preclinical development. We will discuss our design approach and hypotheses leading to the discovery of CCT289346 and disclose in vivo efficacy data. References 1. Innocenti P et al. Rapid Discovery of Pyrido[3,4-d]pyrimidine Inhibitors of Monopolar Spindle Kinase 1 (MPS1) Using a Structure-Based Hybridization Approach. Journal of Medicinal Chemistry. 2016; 59(8):3671-88. Citation Format: Hannah L. Woodward, Paolo Innocenti, Kwai-Ming J. Cheung, Sebastien Naud, Angela Hayes, Alan T. Henley, Amir Faisal, Grace Mak, Gary Box, Isaac M. Westwood, Michael Carter, Melanie Valenti, Alexis De Haven Brandon, Lisa O’Fee, Harry Saville, Rosemary Burke, Rob van Montfort, Florence Raynaud, Suzanne A. Eccles, Spiros Linardopoulos, Julian Blagg, Swen Hoelder. Inhibitors of MPS1: Discovery of CCT289346, a highly potent, selective and orally available preclinical candidate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 193. doi:10.1158/1538-7445.AM2017-193

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 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