Mark J. Anderton

Discovery of a Potent and Selective EGFR Inhibitor (AZD9291) of Both Sensitizing and T790M Resistance Mutations That Spares the Wild Type Form of the Receptor

Epidermal growth factor receptor (EGFR) inhibitors have been used clinically in the treatment of non-small-cell lung cancer (NSCLC) patients harboring sensitizing (or activating) mutations for a number of years. Despite encouraging clinical efficacy with these agents, in many patients resistance develops leading to disease progression. In most cases, this resistance is in the form of the T790M mutation. In addition, EGFR wild type receptor inhibition inherent with these agents can lead to dose limiting toxicities of rash and diarrhea. We describe herein the evolution of an early, mutant selective lead to the clinical candidate AZD9291, an irreversible inhibitor of both EGFR sensitizing (EGFRm+) and T790M resistance mutations with selectivity over the wild type form of the receptor. Following observations of significant tumor inhibition in preclinical models, the clinical candidate was administered clinically to patients with T790M positive EGFR-TKI resistant NSCLC and early efficacy has been observed, accompanied by an encouraging safety profile.

Induction of Heart Valve Lesions by Small-Molecule ALK5 Inhibitors

Aberrant signaling by transforming growth factor-β (TGF-β) and its type I (ALK5) receptor has been implicated in a number of human diseases and this pathway is considered a potential target for therapeutic intervention. Transforming growth factor-β signaling via ALK5 plays a critical role during heart development, but the role of ALK5 in the adult heart is poorly understood. In the current study, the preclinical toxicology of ALK5 inhibitors from two different chemistry scaffolds was explored. Ten-week-old female Han Wistar rats received test compounds by the oral route for three to seven days. Both compounds induced histopathologic heart valve lesions characterized by hemorrhage, inflammation, degeneration, and proliferation of valvular interstitial cells. The pathology was observed in all animals, at all doses tested, and occurred in all four heart valves. Immunohistochemical analysis of ALK5 in rat hearts revealed expression in the valves, but not in the myocardium. Compared to control animals, protein levels of ALK5 were unchanged in the heart valves of treated animals. We also observed a physeal dysplasia in the femoro-tibial joint of rats treated with ALK5 inhibitors, a finding consistent with a pharmacological effect described previously with ALK5 inhibitors. Overall, these findings suggest that TGF-β signaling via ALK5 plays a critical role in maintaining heart valve integrity.

Structure- and Reactivity-Based Development of Covalent Inhibitors of the Activating and Gatekeeper Mutant Forms of the Epidermal Growth Factor Receptor (EGFR)

A novel series of small-molecule inhibitors has been developed to target the double mutant form of the epidermal growth factor receptor (EGFR) tyrosine kinase, which is resistant to treatment with gefitinib and erlotinib. Our reported compounds also show selectivity over wild-type EGFR. Guided by molecular modeling, this series was evolved to target a cysteine residue in the ATP binding site via covalent bond formation and demonstrates high levels of activity in cellular models of the double mutant form of EGFR. In addition, these compounds show significant activity against the activating mutations, which gefitinib and erlotinib target and inhibition of which gives rise to their observed clinical efficacy. A glutathione (GSH)-based assay was used to measure thiol reactivity toward the electrophilic functionality of the inhibitor series, enabling both the identification of a suitable reactivity window for their potency and the development of a reactivity quantitative structure-property relationship (QSPR) to support design.

Reprogramming of the HepG2 genome by long interspersed nuclear element-1

Long Interspersed Nuclear Element‐1 (LINE‐1 or L1) is an autonomous, mobile element within the human genome that transposes via a “copy and paste” mechanism and relies upon L1‐encoded endonuclease and reverse transcriptase (RT) activities to compromise genome integrity. L1 has been implicated in various forms of cancer, but its role in the regulation of the oncogenic phenotype is not understood. The present studies were conducted to evaluate mechanisms of genetic regulatory control in HepG2 cells by human L1, or a D702Y mutant deficient in RT activity, and their influence on cellular phenotype. Forced expression of synthetic L1 ORF1p and ORF2p was associated with formation of cytoplasmic foci and minor association with the nuclear compartment. While de novo L1 mobilizations were only identified in cells expressing wild type L1, and were absent in the D702Y mutant, changes in gene expression profiles involved RT dependent as well as RT independent mechanisms. Synthetic L1 altered the expression of 24 in silico predicted genetic targets; ten of which showed RT‐dependence, ten RT‐independence, and four reciprocal regulatory control by both wild type and RT mutant. Of five targets examined, only VCAM1 and PTPRB colocalized with newly retrotransposed wild type L1. Biological discretization to partition patterns of gene expression into unique frequencies identified adhesion, inflammation, and cellular metabolism as key processes targeted for molecular interference with disruption of epithelial‐to‐mesenchymal programming seen irrespective of the RT phenotype. These findings establish L1 as a key regulator of genome plasticity and EMT via mechanisms independent of RT activity.

Epigenetics – relevance to drug safety science

Epigenetics describes the study of heritable changes in gene expression that occur in the absence of a change to the DNA sequence. Specific patterns of epigenetic signatures can be stably transmitted through mitosis and cell division and form the molecular basis for developmental stage- and cell type-specific gene expression. Associations have been observed that endogenous and exogenous stimuli can change the epigenetic control of both somatic and stem cell differentiation and thus influence phenotypic behaviours and/or disease progression. In relation to drug safety, DNA methylation changes have been identified in many stages of tumour development following exposure to non-genotoxic carcinogens. However, it is not clear whether DNA methylation changes cause cancer, or arise as a consequence of the transformed state. Toxic agents could act at different levels, by directly modifying the epigenome or indirectly by altering signalling pathways. These alterations in chromatin structure may or may not be heritable but are probably reversible. That said, there is currently insufficient data to support inclusion of epigenetic profiling into pre-clinical evaluation studies. Several international collaborations aim to generate data to determine whether epigenetic modifications are causal links in disease and/or tumour progression. It will only be when an understanding of chemical mode-of-action is required that evaluation of epigenetic changes might be considered. The current toxicological testing battery is expected to identify any potential adverse effects regardless of the mechanism, epigenetic or otherwise. It is recommended that toxicologists keep a close watch of new developments in this field, in particular identification of early epigenetic markers for non-genotoxic carcinogenicity. Scientific collaborations between academia and industry will help to understand inter-individual variations in response to drug and toxin exposure to be able to distinguish between adverse and non-adverse epigenetic changes.

Abstract B94: Discovery of and first disclosure of the clinical candidate AZD9291, a potent and selective third-generation EGFR inhibitor of both activating and T790M resistant mutations that spares the wild type form of the receptor.

Abstracts: AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics--Oct 19-23, 2013; Boston, MA Small molecule inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase such as gefitinib and erlotinib have been employed successfully in the treatment of non-small cell lung cancer (NSCLC) patients harboring an activating mutation (EGFRm+). However, resistance to these inhibitors in the form of additional mutations in the kinase domain such as T790M is emerging as a growing clinical issue. This presentation will describe the discovery of AZD9291, an orally bioavailable, irreversible EGFR inhibitor of both the resistance (NCI-H1975, cell phosphorylation IC50 0.5 μM). Wild type EGFR inhibition is believed to drive the observed dose limiting toxicities (such as skin rash and diarrhea) for these first generation therapies in the clinic. New data will be discussed for the first time including the medicinal chemistry program that led to the identification of AZD9291, details of significant in vivo oral activity in pre-clinical xenograft models (including tumor regression in the L858R/T790M double mutant setting at a dose of 5 mpk) and the first disclosure of the candidate drug structure. The pre-clinical findings from this work strongly supported selection of AZD9291 as a clinical candidate, and first dose in man was achieved with AZD9291 in March 2013. View this table: Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B94. Citation Format: M. Raymond V. Finlay, Mark Anderton, Susan Ashton, Peter G. Ballard, Rob H. Bradbury, Sam Butterworth, Nicola Colclough, Darren A. E. Cross, Heather L. McFarland, Martine J. Mellor, Richard A. Ward, Mike J. Waring. Discovery of and first disclosure of the clinical candidate AZD9291, a potent and selective third-generation EGFR inhibitor of both activating and T790M resistant mutations that spares the wild type form of the receptor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B94.

De novo LINE-1 retrotransposition in HepG2 cells preferentially targets gene poor regions of chromosome 13.

Long interspersed nuclear elements (Line-1 or L1s) account for ~17% of the human genome. While the majority of human L1s are inactive, ~80-100 elements remain retrotransposition competent and mobilize through RNA intermediates to different locations within the genome. De novo insertions of L1s account for polymorphic variation of the human genome and disruption of target loci at their new location. In the present study, fluorescence in situ hybridization and DNA sequencing were used to characterize retrotransposition profiles of L1(RP) in cultured human HepG2 cells. While expression of synthetic L1(RP) was associated with full-length and truncated insertions throughout the entire genome, a strong preference for gene-poor regions, such as those found in chromosome 13 was observed for full-length insertions. These findings shed light into L1 targeting mechanisms within the human genome and question the putative randomness of L1 retrotransposition.

Abstract 3676: Influence of early toxicology assessment on the selection of azd9291

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Discovery toxicology is the concept of evaluating the safety of molecules early in the drug development process when there is chemical scope to refine candidate drug quality. The aim is to identify and mitigate safety liabilities to maximize the chance of clinical success. This poster describes the impact of discovery toxicology on the selection of AZD9291, an irreversible inhibitor of both EGFR-TKI sensitising and resistance mutations. Although, maintaining a margin of selectivity to wild type EGFR was a key medicinal chemistry goal, early in vitro selectivity evaluation of the lead series compounds revealed significant off-target inhibition against the insulin and insulin-like growth factor receptors (IR/IGFR). Further exploration of this liability in a rat in vivo study revealed that our early lead compound (Cmpd 1) was associated with severe hyperglycemia, rapid body weight loss and evidence of reduced gastric emptying. The reduction in bodyweight and the inhibition of gastric emptying was dose limiting and we hypothesized this was caused by the hyperglycaemia. A broader selection of compounds based on the same scaffold was tested both in vitro and in vivo in the hope of identifying one or more compounds without this liability. Compounds were tested in a modified single dose rat study, exploring insulin and glucose homeostasis and effects on gastric emptying. Compounds within the shortlist caused increases in insulin of up to 80-fold, 3-fold increases in glucose and up to a 60% reduction in gastric emptying. There was a clear link between in vitro IR/IGF inhibition, hyperinsulinemia and inhibition of gastric emptying. One of the shortlisted candidates (now selected as AZD9291), had up to a 30 fold improvement in in vitro IGFR cellular potency compared to previous candidates and had no effect on insulin/glucose homeostasis in vivo. AZD9291 has demonstrated tumor shrinkage in patients at doses that have been very well tolerated and to date, no effect on glucose homeostasis has been reported. View this table: Citation Format: Mark J. Anderton, Richard A. Ward, Paula Daunt, Anne Galer, Darren A. E. Cross, Louise Marks, M. Raymond V. Finlay. Influence of early toxicology assessment on the selection of azd9291. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3676. doi:10.1158/1538-7445.AM2014-3676

Abstract 4744: Structure-based development of covalent inhibitors of the activating and T790M gatekeeper mutant forms of the epidermal growth factor receptor (EGFR) leading to the discovery of AZD9291

Small molecule inhibitors of the Epidermal Growth Factor Receptor (EGFR) tyrosine kinase such as gefitinib and erlotinib have been employed successfully in the treatment of non-small cell lung cancer (NSCLC) patients harboring an activating mutation (EGFRm+). However, resistance to these inhibitors in the form of additional mutations such as T790M, (mutation of the gatekeeper residue), is recognized as a clinical issue. This presentation will describe the discovery and evolution of one of our novel chemical series, leading ultimately to the identification of AZD9291, an orally bioavailable, covalent EGFR inhibitor of both the resistance (NCI-H1975, cell phosphorylation IC50 0.5 uM). Wild type EGFR inhibition is believed to drive the observed dose limiting toxicities (such as skin rash and diarrhea) for these first generation therapies in the clinic. New data will be presented for the first time including a broader description of the medicinal chemistry program that led to the identification of AZD9291. We shall also present previously undisclosed work on the identification of additional distinct chemical series and an update of recent data from ongoing AZD9291 Phase I clinical studies in NSCLC patients. Citation Format: Richard A. Ward, Susan Ashton, Mark Anderton, Pete G. Ballard, Rob H. Bradbury, Sam Butterworth, Nicola Colclough, Darren A E Cross, M Ray V. Finlay, Heather L. McFarland, Martine Mellor, Mike J. Waring. Structure-based development of covalent inhibitors of the activating and T790M gatekeeper mutant forms of the epidermal growth factor receptor (EGFR) leading to the discovery of AZD9291. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4744. doi:10.1158/1538-7445.AM2014-4744