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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 fulvestrant's 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 D'Cruz, 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

The RAS/MAPK pathway is a major driver in oncogenesis and is dysregulated in approximately 30% of human cancers, primarily by mutations in BRAF or RAS genes. The extracellular-signal-regulated kinases (ERK1 and ERK2) serve as key central nodes within this pathway. The feasibility of targeting the RAS/MAPK pathway has been demonstrated by the initial clinical responses observed to BRAF and MEK inhibitors in BRAF V600E/K metastatic melanoma, however resistance frequently develops by reactivation of the pathway. Direct targeting of ERK1/2, may provide another therapeutic option in tumours with mutations in BRAF or RAS genes. Importantly, ERK1/2 inhibition may have clinical utility in overcoming acquired resistance to RAF and MEK inhibitors where RAS/MAPK pathway reactivation has occurred, such as relapsed BRAF V600E/K melanoma. Starting from our published work,1 we will describe for the first time, a scaffold hopping approach leading to the identification of AZD0364, a pre-clinical ERK1/2 inhibitor candidate drug. Driven by conformational modelling and structure-based design, and by utilising novel sulfamidate ring opening chemistry, a high lipophilicity efficiency core was identified. Structure based, multi-parameter based optimisation of this improved core ultimately led to AZD0364. AZD0364 exhibits high cellular potency against a direct downstream substrate on the MAPK pathway (e.g. inhibition of phospho-p90RSK1 in BRAFV600E mutant A375 cells, IC50 = 6 nM). The molecule is a highly selective kinase inhibitor (10/329 kinases tested are inhibited at >50% at a 1 µM) and has long residence time on the protein (as determined by SPR on human unphosphorylated-ERK2: pKd = 10; t1/2 = 277 mins). The good in vitro potency and selectivity is complemented by excellent physico-chemical properties (maximum absorbable dose estimated to be >4 g) and good oral pharmacokinetics across species, leading to a low predicted dose to man. In xenograft models, AZD0364 inhibits phospho-p90RSK1 in tumors in a dose-dependent manner. AZD0364 induces regressions in the KRAS mutant NSCLC Calu 6 xenograft model. AZD0364 can also be combined safely and effectively with the MEK1/2 inhibitor selumetinib in KRAS mutant NSCLC xenograft models. 1Richard A. Ward et. al. Structure-Guided Discovery of Potent and Selective Inhibitors of ERK1/2 from a Modestly Active and Promiscuous Chemical Start Point, J. Med. Chem. 2017, 60, 3438−3450. Citation Format: Iain Simpson, Mark J. Anderton, David M. Andrews, Jason Breed, Emma Davies, Judit E. Debreczeni, Vikki Flemington, Francis D. Gibbons, Mark A. Graham, Philip Hopcroft, Tina Howard, Julian Hudson, Clifford D. Jones, Christopher Jones, Nicola Lindsay, J Elizabeth Pease, Philip Rawlins, Karen Roberts, Steve Swallow, Steve St-Gallay, Michael E. Tonge, Richard A. Ward. Discovery of AZD0364, a potent and selective oral inhibitor of ERK1/2 that is efficacious in both monotherapy and combination therapy in models of NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1647.

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA With over 70% of breast cancers expressing the estrogen receptor alpha (ERα), treatment with either anti-hormonal therapies that directly block ER function or therapies that block the production of estrogen itself have proven to be effective treatments of the disease. Following the pioneering discovery of the ERα antagonist tamoxifen in the 1960s, identification of the selective estrogen receptor downregulator (SERD) fulvestrant represented a further step forward in the treatment of advanced ER+ breast cancer especially in the endocrine resistance setting. The pure estrogen downregulator-antagonist fulvestrant offers the advantage of not only antagonising ERα-driven tumor cell growth but also degrading ERα via ubiquitinylation and thus diminishing growth due to reduced ERα content. Given its low level of bioavailability and metabolic profile, fulvestrant has been formulated as an intramuscular monthly injection. We have sought to identify a novel, non-steroidal ERα antagonist and down-regulator that can be administered orally and could yield improved exposure and clinical benefit. This presentation will describe the discovery of AZD9496, a novel, orally bioavailable small molecule that can induce ERα degradation in breast cancer cell lines at picomolar concentrations. Data will be shared for the first time on the medicinal chemistry efforts that led to AZD9496, the good oral pharmacokinetic properties of the compound which led to significant tumor efficacy in both endocrine sensitive model (complete tumor growth inhibition (TGI) in a MCF-7 model at a dose of 10 mpk) and resistant models (tumor regressions in a HCC1428 long term estrogen deprived (LTED) model, with significant down-regulation of ER protein (81%) at 1 mpk QD and tumour growth inhibition in an ESR1 mutant PDX model). These findings strongly supported selection of AZD9496 as a clinical candidate for the treatment of ER+ breast cancer, and on October 2014 the first patient was dosed with AZD9496. | | ER alpha human binding IC50 (μM) | ER downregulation cell IC50 (μM) | MCF7 anti-proliferation EC50 (μM) | Aqueous solubility, (pH = 7.4, μM) | MCF7 tumor xenograft efficacy (% TGI at 10 mpk PO QD for 22 days) | |:------- | -------------------------------- | -------------------------------- | --------------------------------- | ---------------------------------- | ----------------------------------------------------------------- | | AZD9496 | 0.0007 | 0.00014 | 0.00004 | 110 | 84 | Citation Format: Chris De Savi, Robert H. Bradbury, Alfred A. Rabow, Richard A. Norman, David Buttar, Gordon S. Currie, Hazel Weir, Craig Donald, David Andrews, Phil MacFaul, Peter Ballard, Jon Curwen, Zena Wilson, Graham Richmond, Celina D'Cruz, Steve Powell, Graeme Walker, Michael Hulse, Michael Tonge. Discovery of the clinical candidate AZD9496: a potent and orally bioavailable selective estrogen receptor downregulator and antagonist. [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 3650. doi:10.1158/1538-7445.AM2015-3650

Phosphatidylinositol (PI) is a phospholipid that resides primarily on the cytosolic surface of cell membranes. PI can be phosphorylated to generate seven different phosphatidylinositol phosphates (PIPs), all of which have distinct biological activities.1 A major first step in the production of these PIPs is the conversion of PI into phosphatidylinositol-4-phosphate (PI4P) by PI4 kinase.2 Here we describe the identification of inhibitors of PI4 kinase via targeted screening of a kinase subset. This exercise has delivered inhibitors that are potent inhibitors of PI4 kinase (pIC50 >8), which are selective the α- or the β-subtypes and do not inhibit related enzymes on the PI pathway (PI3 kinase pIC50 The effect of these useful probe compounds on cellular phosphatidylinositol-4,5-diphosphate and subsequent downstream markers will be described along with the comparison of these effects with the corresponding siRNA. 1. G. Di Paolo, P. De Camilli, Phosphoinositides in cell regulation and membrane dynamics. Nature 2006, 443, 651-657.2. T. Sasaki, S. Takasuga, J. Sasaki, S. Kofuji, S. Eguchi, M. Yamazaki, A. Suzuki, Mammalian phosphoinositide kinases and phosphatases. Progress in lipid research, 2009, 48, 307-343. Citation Format: Michael J. Waring, Darren Cross, David Andrews, Vikki Flemington, Carol Lenaghan, Jennifer McKelvie, Sarita Maman, Piotr Raubo, Graeme Robb, James Smith, Martin Swarbrick. Phosphatidylinositol-4-kinase - Potent and selective inhibitors of PI4Kα and PI4Kβ. [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 2228. doi:10.1158/1538-7445.AM2013-2228

Deubiquitinating enzymes (DUBs) represent promising opportunities for therapeutic intervention in oncology, and this study reports key information about the catalytic activity, assaying and inhibition of this novel enzyme class. The DUBs are proteases responsible for the cleavage of Ubiquitin from substrate molecules, and the resulting effects on the levels or activity of the substrates has been shown to play key roles in a range of cellular processes. Here we have utilized a range of in vitro and cellular assays of DUB activity to characterize various DUB enzymes, generate high throughput screens and profile DUB inhibition by hit molecules, to further our understanding in this area. The characterization of different DUBs has enabled us to highlight both differences and similarities in their behavior and requirements, particularly with respect to protein subunits, reducing conditions, and activity against different substrates. Profiling of small-molecule hits targeting these enzymes, under various in vitro conditions, and also in a cellular environment, has not only yielded information on the compounds, but has also demonstrated key considerations about the inhibition of these cysteine proteases. Together these findings further our knowledge of this novel target class, as well as providing valuable information to aid the hunt for therapeutic agents modulating these enzymes in a range of oncology settings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4043. doi:10.1158/1538-7445.AM2011-4043