Kevin Michael Foote

AZD1152, a Selective Inhibitor of Aurora B Kinase, Inhibits Human Tumor Xenograft Growth by Inducing Apoptosis

Purpose: In the current study, we examined the in vivo effects of AZD1152, a novel and specific inhibitor of Aurora kinase activity (with selectivity for Aurora B). Experimental Design: The pharmacodynamic effects and efficacy of AZD1152 were determined in a panel of human tumor xenograft models. AZD1152 was dosed via several parenteral (s.c. osmotic mini-pump, i.p., and i.v.) routes. Results: AZD1152 potently inhibited the growth of human colon, lung, and hematologic tumor xenografts (mean tumor growth inhibition range, 55% to ≥100%; P < 0.05) in immunodeficient mice. Detailed pharmacodynamic analysis in colorectal SW620 tumor-bearing athymic rats treated i.v. with AZD1152 revealed a temporal sequence of phenotypic events in tumors: transient suppression of histone H3 phosphorylation followed by accumulation of 4N DNA in cells (2.4-fold higher compared with controls) and then an increased proportion of polyploid cells (>4N DNA, 2.3-fold higher compared with controls). Histologic analysis showed aberrant cell division that was concurrent with an increase in apoptosis in AZD1152-treated tumors. Bone marrow analyses revealed transient myelosuppression with the drug that was fully reversible following cessation of AZD1152 treatment. Conclusions: These data suggest that selective targeting of Aurora B kinase may be a promising therapeutic approach for the treatment of a range of malignancies. In addition to the suppression of histone H3 phosphorylation, determination of tumor cell polyploidy and apoptosis may be useful biomarkers for this class of therapeutic agent. AZD1152 is currently in phase I trials.

Validating Aurora B as an anti-cancer drug target

The Aurora kinases, a family of mitotic regulators, have received much attention as potential targets for novel anti-cancer therapeutics. Several Aurora kinase inhibitors have been described including ZM447439, which prevents chromosome alignment, spindle checkpoint function and cytokinesis. Subsequently, ZM447439-treated cells exit mitosis without dividing and lose viability. Because ZM447439 inhibits both Aurora A and B, we set out to determine which phenotypes are due to inhibition of which kinase. Using molecular genetic approaches, we show that inhibition of Aurora B kinase activity phenocopies ZM447439. Furthermore, a novel ZM compound, which is 100 times more selective for Aurora B over Aurora A in vitro, induces identical phenotypes. Importantly, inhibition of Aurora B kinase activity induces a penetrant anti-proliferative phenotype, indicating that Aurora B is an attractive anti-cancer drug target. Using molecular genetic and chemical-genetic approaches, we also probe the role of Aurora A kinase activity. We show that simultaneous repression of Aurora A plus induction of a catalytic mutant induces a monopolar phenotype. Consistently, another novel ZM-related inhibitor, which is 20 times as potent against Aurora A compared with ZM447439, induces a monopolar phenotype. Expression of a drug-resistant Aurora A mutant reverts this phenotype, demonstrating that Aurora A kinase activity is required for spindle bipolarity in human cells. Because small molecule-mediated inhibition of Aurora A and Aurora B yields distinct phenotypes, our observations indicate that the Auroras may present two avenues for anti-cancer drug discovery.

Discovery of 4-{4-[(3R)-3-Methylmorpholin-4-yl]-6-[1-(methylsulfonyl)cyclopropyl]pyrimidin-2-yl}-1H-indole (AZ20): a potent and selective inhibitor of ATR protein kinase with monotherapy in vivo antitumor activity.

ATR is an attractive new anticancer drug target whose inhibitors have potential as chemo- or radiation sensitizers or as monotherapy in tumors addicted to particular DNA-repair pathways. We describe the discovery and synthesis of a series of sulfonylmorpholinopyrimidines that show potent and selective ATR inhibition. Optimization from a high quality screening hit within tight SAR space led to compound 6 (AZ20) which inhibits ATR immunoprecipitated from HeLa nuclear extracts with an IC50 of 5 nM and ATR mediated phosphorylation of Chk1 in HT29 colorectal adenocarcinoma tumor cells with an IC50 of 50 nM. Compound 6 potently inhibits the growth of LoVo colorectal adenocarcinoma tumor cells in vitro and has high free exposure in mouse following moderate oral doses. At well tolerated doses 6 leads to significant growth inhibition of LoVo xenografts grown in nude mice. Compound 6 is a useful compound to explore ATR pharmacology in vivo.

Progress in the Development of Selective Inhibitors of Aurora Kinases

Errors in the mitotic process are thought to be one of the principal sources of the genetic instability that hallmarks cancer. Unsurprisingly, many of the proteins that regulate mitosis are aberrantly expressed in tumour cells when compared to their normal counterparts. These may represent a good source of targets for the development of novel anti-cancer agents. The Aurora kinases represent one such family of mitotic regulators. In recent years there has been intense interest in both understanding the role of the Aurora kinases in cell cycle regulation and also in developing small molecule inhibitors as potential novel anti-cancer drugs. With several companies now starting to take Aurora kinase inhibitors into clinical development, the time is right to review the medicinal chemistry contribution to developing the field, in particular to review the increasingly broad range of small molecule inhibitors with activity against this kinase family.

Protein-Ligand Crystal Structures Can Guide the Design of Selective Inhibitors of the FGFR Tyrosine Kinase.

The design of compounds that selectively inhibit a single kinase is a significant challenge, particularly for compounds that bind to the ATP site. We describe here how protein-ligand crystal structure information was able both to rationalize observed selectivity and to guide the design of more selective compounds. Inhibition data from enzyme and cellular screens and the crystal structures of a range of ligands tested during the process of identifying selective inhibitors of FGFR provide a step-by-step illustration of the process. Steric effects were exploited by increasing the size of ligands in specific regions in such a way as to be tolerated in the primary target and not in other related kinases. Kinases are an excellent target class to exploit such approaches because of the conserved fold and small side chain mobility of the active form.

Drugging ATR: progress in the development of specific inhibitors for the treatment of cancer

In this article, we review the ATR inhibitor field from initial pharmacological tools to first-generation clinical candidates with the potential to bring benefit to cancer patients. ATR is a critical part of the cell DNA-damage response. Over the past decade or more, compounds with weak ATR potency and low specificity have been used as tools in early studies to elucidate ATR pharmacology. More recently highly potent, selective and in vivo active ATR inhibitors have been developed enabling detailed preclinical in vitro and in vivo target assessment to be made. The published studies reveal the potential of ATR inhibitors for use as monotherapy or in combination with DNA-damaging agents. To date, VX-970 and AZD6738, have entered clinical assessment.

Abstract 2348: Discovery of AZD6738, a potent and selective inhibitor with the potential to test the clinical efficacy of ATR kinase inhibition in cancer patients.

AZD6738 is a potent and selective inhibitor of ataxia telangiectasia and rad3 related (ATR) kinase with the potential to be used in the clinic. We report the discovery of a morpholino-pyrimidine series and its subsequent optimisation to the pre-clinical candidate AZD6738 which has a good overall balance of potency, selectivity, pharmacokinetic and biopharmaceutical properties suitable for oral dosing. ATR is a serine/threonine protein kinase involved in DNA damage response signalling caused by DNA replication associated stress. Activation of ATR at stalled replication forks leads to suppression of replication fork origin firing, promotes repair and S/G2-cell cycle checkpoints to prevent premature mitosis and maintain genomic integrity. Failure to resolve damage leads to genomic instability and if sufficiently high, cell death. Stalled replication forks may collapse leading to formation of DNA double stranded breaks and activation of the ataxia telangiectasia mutated (ATM) kinase. ATM works in conjunction with ATR to efficiently resolve replication associated DNA damage creating a co-dependency with loss of one leading to a greater reliance on the other to maintain genomic stability. ATM is frequently inactivated across B-cell malignancies, head and neck, breast and lung cancers through chromosomal deletion, promoter hypermethylation or mutation. ATM-deficient tumours are hypothesised to be more reliant on ATR for survival and specific inhibition of ATR may lead enhanced anti-tumour activity while minimizing normal tissue toxicity. AZD6378 has the potential to test the clinical efficacy of ATR inhibition in cancer patients as monotherapy against ATM-deficient tumours or in combination with chemo or radiotherapy. Citation Format: Clifford D. Jones, Kevin Blades, Kevin M. Foote, Sylvie M. Guichard, Philip J. Jewsbury, Thomas McGuire, Johannes W. Nissink, Rajesh Odedra, Kin Tam, Pia Thommes, Paul Turner, Gary Wilkinson, Christine Wood, James W. Yates. Discovery of AZD6738, a potent and selective inhibitor with the potential to test the clinical efficacy of ATR kinase inhibition in cancer patients. [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 2348. doi:10.1158/1538-7445.AM2013-2348

Synthesis and SAR of 1-acetanilide-4-aminopyrazole-substituted quinazolines: Selective inhibitors of Aurora B kinase with potent anti-tumor activity

A new class of 1-acetanilide-4-aminopyrazole-substituted quinazoline Aurora kinase inhibitors has been discovered possessing highly potent cellular activity. Continuous infusion into athymic mice bearing SW620 tumors of the soluble phosphate derivative 2 led to dose-proportional exposure of the des-phosphate compound 8 with a high-unbound fraction. The combination of potent cell activity and high free-drug exposure led to pharmacodynamic changes in the tumor at low doses, indicative of Aurora B-kinase inhibition and a reduction in tumor volume.

An Enantiospecific Polyene Cyclization Initiated by an Enantiomerically Pure Bromonium Ion: Enantiospecific Bromonium Ion Polyene Cycl.

Dimethylaluminum triflate-mediated activation of tetrafluorobenzoates of enantiomerically pure bromohydrins results in enantiospecific polyene cyclizations. The initiation of cyclization by enantiomerically pure bromonium ions and subsequent propagation is not subject to catastrophic erosion of enantiomeric purity by any intramolecular or intermolecular bromonium ion-to-alkene transfer.

Abstract 3343: The pre-clinical in vitro and in vivo activity of AZD6738: A potent and selective inhibitor of ATR kinase.

AZD6738 is a potent and selective orally bioavailable kinase inhibitor of ataxia telangiectasia and rad3 related (ATR). Here we report the pre-clinical in vitro and in vivo and biological profile of AZD6738. ATR is a serine/threonine protein kinase involved in DNA damage response signalling caused by DNA replication associated stress. Activation of ATR at stalled replication forks leads to suppression of replication fork origin firing, promotes repair and S/G2-cell cycle checkpoints to prevent premature mitosis and maintain genomic integrity. Failure to resolve damage leads to genomic instability and if sufficiently high, cell death. Stalled replication forks may collapse leading to formation of DNA double stranded breaks and activation of the ataxia telangiectasia mutated (ATM) kinase. ATM works in conjunction with ATR to efficiently resolve replication associated DNA damage creating a co-dependency with loss of one leading to a greater reliance on the other to maintain genomic stability. ATM is frequently inactivated across B-cell malignancies, head and neck, breast and lung cancers through chromosomal deletion, promoter hypermethylation or mutation. ATM-deficient tumours are hypothesised to be more reliant on ATR for survival and specific inhibition of ATR may lead enhanced anti-tumour activity while minimizing normal tissue toxicity. AZD6738 demonstrates this preclinical profile. AZD6738 inhibits the phosphorylation of direct downstream substrate CHK1 while increasing the phosphorylation of ATM-dependent substrate CHK2 and DNA damage marker γH2AX. This is associated with impaired S-phase cell cycle progression with prolonged inhibition causing cell death, indicative of replication fork stalling, collapse and irreversible damage. AZD6738 is active as a single agent across cancer cell line panels but shows enhanced sensitivity in cell lines with ATM-pathway defects. AZD6738 when used in combination with DNA damaging inducing agents’ gemcitabine, cisplatin or ionising radiation (IR) shows enhanced synergistic cell killing activity. In vivo, AZD6738 monotherapy treatment leads to significant anti-tumour activity in ATM-deficient but not ATM-proficient xenograft models at equivalent, tolerated doses. When AZD6738 is used in combination with carboplatin or IR anti-tumour growth inhibitory activity or regression is observed. AZD6738 in vivo activity is also associated with a persistent increased γH2AX staining in tumour tissue but only a transient increase in normal bone marrow or gut tissue suggesting a favourable therapeutic index can be achieved. Early pre-clinical safety studies support these findings with target related bone marrow suppression, neutropenia and GI tract impact only at high-doses. These data merit further investigation of AZD6738 as a monotherapy or in combination with chemo or radiotherapy. Citation Format: Sylvie M. Guichard, Elaine Brown, Rajesh Odedra, Adina Hughes, Dan Heathcote, Jen Barnes, Alan Lau, Steve Powell, Clifford D. Jones, Willem Nissink, Kevin M. Foote, Philip J. Jewsbury, Martin Pass. The pre-clinical in vitro and in vivo activity of AZD6738: A potent and selective inhibitor of ATR kinase. [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 3343. doi:10.1158/1538-7445.AM2013-3343