Michael Tonge

AZD9496: An Oral Estrogen Receptor Inhibitor That Blocks the Growth of ER-Positive and ESR1-Mutant Breast Tumors in Preclinical Models.

Fulvestrant is an estrogen receptor (ER) antagonist administered to breast cancer patients by monthly intramuscular injection. Given its present limitations of dosing and route of administration, a more flexible orally available compound has been sought to pursue the potential benefits of this drug in patients with advanced metastatic disease. Here we report the identification and characterization of AZD9496, a nonsteroidal small-molecule inhibitor of ERα, which is a potent and selective antagonist and downregulator of ERα in vitro and in vivo in ER-positive models of breast cancer. Significant tumor growth inhibition was observed as low as 0.5 mg/kg dose in the estrogen-dependent MCF-7 xenograft model, where this effect was accompanied by a dose-dependent decrease in PR protein levels, demonstrating potent antagonist activity. Combining AZD9496 with PI3K pathway and CDK4/6 inhibitors led to further growth-inhibitory effects compared with monotherapy alone. Tumor regressions were also seen in a long-term estrogen-deprived breast model, where significant downregulation of ERα protein was observed. AZD9496 bound and downregulated clinically relevant ESR1 mutants in vitro and inhibited tumor growth in an ESR1-mutant patient-derived xenograft model that included a D538G mutation. Collectively, the pharmacologic evidence showed that AZD9496 is an oral, nonsteroidal, selective estrogen receptor antagonist and downregulator in ER(+) breast cells that could provide meaningful benefit to ER(+) breast cancer patients. AZD9496 is currently being evaluated in a phase I clinical trial. Cancer Res; 76(11); 3307-18. ©2016 AACR.

Structure-Guided Design of Highly Selective and Potent Covalent Inhibitors of Erk1/2.

The RAS/RAF/MEK/ERK signaling pathway has been targeted with a number of small molecule inhibitors in oncology clinical development across multiple disease indications. Importantly, cell lines with acquired resistance to B-RAF and MEK inhibitors have been shown to maintain sensitivity to ERK1/2 inhibition by small molecule inhibitors. There are a number of selective, noncovalent ERK1/2 inhibitors reported along with the promiscuous hypothemycin (and related analogues) that act via a covalent mechanism of action. This article reports the identification of multiple series of highly selective covalent ERK1/2 inhibitors informed by structure-based drug design (SBDD). As a starting point for these covalent inhibitors, reported ERK1/2 inhibitors and a chemical series identified via high-throughput screening were exploited. These approaches resulted in the identification of selective covalent tool compounds for potential in vitro and in vivo studies to assess the risks and or benefits of targeting this pathway through such a mechanism of action.

Optimization of a Novel Binding Motif to (E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-Fluoro-2-Methylpropyl)-3-Methyl-2, 3,4,9-Tetrahydro-1H-Pyrido[3,4-B]Indol-1-Yl)Phenyl)Acrylic Acid (Azd9496), a Potent and Orally Bioavailable Selective Estrogen Receptor Downregulator and Antagonist.

The discovery of an orally bioavailable selective estrogen receptor downregulator (SERD) with equivalent potency and preclinical pharmacology to the intramuscular SERD fulvestrant is described. A directed screen identified the 1-aryl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole motif as a novel, druglike ER ligand. Aided by crystal structures of novel ligands bound to an ER construct, medicinal chemistry iterations led to (E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (30b, AZD9496), a clinical candidate with high oral bioavailability across preclinical species that is currently being evaluated in phase I clinical trials for the treatment of advanced estrogen receptor (ER) positive breast cancer.

Development of a High-Throughput Fluorescence Polarization DNA Cleavage Assay for the Identification of FEN1 Inhibitors

Flap endonuclease-1 (FEN1) is a highly conserved metallonuclease and is the main human flap endonuclease involved in the recognition and cleavage of single-stranded 5′ overhangs from DNA flap structures. The involvement of FEN1 in multiple DNA metabolism pathways and the identification of FEN1 overexpression in a variety of cancers has led to interest in FEN1 as an oncology target. In this article, we describe the development of a 1536-well high-throughput screening assay based on the change in fluorescence polarization of a FEN1 DNA substrate labeled with Atto495 dye. The assay was subsequently used to screen 850 000 compounds from the AstraZeneca compound collection, with a Z′ factor of 0.66 ± 0.06. Hits were followed up by IC50 determination in both a concentration-response assay and a technology artifact assay.

A Screening Assay Cascade to Identify and Characterize Novel Selective Estrogen Receptor Downregulators (SERDs)

Here, we describe an approach to identify novel selective estrogen receptor downregulator (SERD) compounds with improved properties such as oral bioavailability and the potential of increased efficacy compared to currently marketed drug treatments. Previously, methodologies such as Western blotting and transient cell reporter assays have been used to identify and characterize SERD compounds, but such approaches can be limited due to low throughput and sensitivity, respectively. We have used an endogenous cell-imaging strategy that has both the throughput and sensitivity to support a large-scale hit-to-lead program to identify novel compounds. A screening cascade with a suite of assays has been developed to characterize compounds that modulate estrogen receptor α (ERα)-mediated signaling or downregulate ERα levels in cells. Initially, from a focused high-throughput screening, novel ERα binders were identified that could be modified chemically into ERα downregulators. Following this, cellular assays helped determine the mechanism of action of compounds to distinguish between on-target and off-target compounds and differentiate SERDs, selective estrogen receptor modulator (SERM) compounds, and agonist ERα ligands. Data are shown to exemplify the characterization of ERα-mediated signaling inhibitors using a selection of literature compounds and illustrate how this cascade has been used to drive the chemical design of novel SERD compounds.

Discovery and Optimization of Allosteric Inhibitors of Mutant Isocitrate Dehydrogenase 1 (R132H IDH1) Displaying Activity in Human Acute Myeloid Leukemia Cells

A collaborative high throughput screen of 1.35 million compounds against mutant (R132H) isocitrate dehydrogenase IDH1 led to the identification of a novel series of inhibitors. Elucidation of the bound ligand crystal structure showed that the inhibitors exhibited a novel binding mode in a previously identified allosteric site of IDH1 (R132H). This information guided the optimization of the series yielding submicromolar enzyme inhibitors with promising cellular activity. Encouragingly, one compound from this series was found to induce myeloid differentiation in primary human IDH1 R132H AML cells in vitro.

Structure-Guided Discovery of Potent and Selective Inhibitors of ERK1/2 from a Modestly Active and Promiscuous Chemical Start Point.

There are a number of small-molecule inhibitors targeting the RAS/RAF/MEK/ERK signaling pathway that have either been approved or are in clinical development for oncology across a range of disease indications. The inhibition of ERK1/2 is of significant current interest, as cell lines with acquired resistance to BRAF and MEK inhibitors have been shown to maintain sensitivity to ERK1/2 inhibition in preclinical models. This article reports on our recent work to identify novel, potent, and selective reversible ERK1/2 inhibitors from a low-molecular-weight, modestly active, and highly promiscuous chemical start point, compound 4. To guide and inform the evolution of this series, inhibitor binding mode information from X-ray crystal structures was critical in the rapid exploration of this template to compound 35, which was active when tested in in vivo antitumor efficacy experiments.

Abstract DDT01-03: Discovery and pre-clinical pharmacology of AZD9496: An oral, selective estrogen receptor down-regulator (SERD)

With over 70% of breast cancers expressing estrogen receptor alpha protein (ERα), treatment with either anti-hormonal therapies that directly block ERα function (e.g. tamoxifen) or therapies that block the production of estrogen itself (e.g. anastrozole) have proven to be effective treatments for the disease. Following the discovery of the ERα antagonist tamoxifen in the 1960s, identification of the selective estrogen receptor down-regulator (SERD) fulvestrant represented a further step forward in the treatment of advanced ER+ breast cancer, especially in the endocrine resistance setting where ERα appears to be activated by a ligand independent route through other growth factor signaling pathways. In addition, fulvestrant has also shown significant overall survival (OS) results in the FIRST trial comparing 500 mg fulvestrant with anastrozole in first line advanced ER+ve patients where the majority of patients had not received prior endocrine therapy. Given fulvestrant9s low bioavailability following intramuscular injection and the levels of ERα protein in clinical samples after treatment, the question remains as to whether an agent that could achieve higher steady state levels of drug more rapidly and drive further decreases in ERα levels would give enhanced clinical benefit. We have identified a novel, potent, non-steroidal SERD that can be administered orally and could yield improved exposure and clinical benefit. This presentation will describe the discovery and pre-clinical pharmacology of AZD9496, a small molecule that can antagonise ERα and induce receptor degradation in breast cancer cell lines at picomolar concentrations. The good oral pharmacokinetic properties of the compound in pre-clinical species led to significant tumor growth inhibition in an endocrine sensitive MCF-7 xenograft model at a dose of 5 mg/kg and >90% reduction in ER-regulated, progesterone receptor (PR) levels. Tumor regressions were seen in a long term estrogen deprived (LTED) in vivo model, representing the aromatase resistant setting, and corresponded with significant reductions in ERα protein levels, >90% at 5 mg/kg dose. AZD9496 also showed antagonist and down-regulation activity against ERα mutant protein both in vitro and in vivo. These findings strongly supported selection of AZD9496 as a clinical candidate for the treatment of ER+ve breast cancer and the drug is now under evaluation in a Phase 1 clinical trial. Citation Format: Hazel Weir, Mandy Lawson, Rowena Callis, Michael Hulse, Michael Tonge, Gareth Davies, Graeme Walker, Rachel Rowlinson, Jon Curwen, Zena Wilson, Steve Powell, Robert Bradbury, Alfred Rabow, Craig Donald, David Buttar, Richard Norman, Camila de Almeida, Peter Ballard, Gordon Currie, David Andrews, Graham Richmond, Anne Marie Mazzola, Ermira Pazolli, Brendon Ladd, Celina D9Cruz, Chris De Savi. Discovery and pre-clinical pharmacology of AZD9496: An oral, selective estrogen receptor down-regulator (SERD). [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 DDT01-03. doi:10.1158/1538-7445.AM2015-DDT01-03