NOVEL SMALL MOLECULE DUAL ANTAGONISTS OF XIAP AND CIAP1 GENERATED BY FRAGMENT-BASED DRUG DISCOVERY (FBDD) ARE EFFECTIVE IN PRE-CLINICAL MODELS Sandra Muench on behalf of the IAP project team Astex Pharmaceuticals, 436 Cambridge Science Park, Cambridge, CB4 0QA, United Kingdom. INTRODUCTION The inhibitor of apoptosis proteins (IAPs) are widely de-regulated in many tumours and contribute to cancer drug resistance. The targeted inhibition of IAPs can switch TNF-alpha signaling in cancer cells from pro-survival to pro-apoptotic. Therefore, IAPs represent an attractive target for cancer therapy. The IAP family member cellular IAP1 (cIAP1) is involved in the regulation of TNF-alpha signaling and X-linked IAP (XIAP) directly interacts with and inhibits caspases. IAP family members are characterized by BIR (baculoviral IAP repeat) domains, to which the endogenous inhibitor of IAPs SMAC (second mitochondria derived activator of caspases) binds. Peptidomimetic compounds based on the SMAC sequence have been developed, but they show high selectivity for cIAP1. We used our fragment based-drug discovery approach to generate a non-alanine, non- peptidomimetic IAP antagonist, which has dual potency for XIAP and cIAP1. Here we describe the characterization of this compound in in vitro and in vivo models of melanoma and breast cancer. Regulation of death receptor signaling by Smac mimetic (). Fulda S Clin Cancer Res 2014;20:3915-3920 OPTIMISATION OF FRAGMENTS CELLULAR ACTIVITY OF AT-IAP Poster presented at “Cancer Pharmacogenomics and Targeted Therapies 2014” 17-19 September 2014, Cambridge, UK © Astex Pharmaceuticals Inc. MELANOMA AND BREAST CANCER XENOGRAFT EFFICACY MODEL • AT-IAP was well tolerated up to 50 mg/kg. p.o. q.d. and shows significant in vivo activity in xenograft models. MELANOMA AND BREAST CANCER XENOGRAFT PK/PD DATA • Degradation of cIAP1 and induction of apoptosis markers observed in both tumour types after single doses of AT-IAP SUMMARY AND CONCLUSIONS • AT-IAP represents a novel IAP antagonist with a potent dual cIAP1 and XIAP antagonist profile. • In vitro cell line testing suggests significant activity against a panel of melanoma and primary tumor cell lines, which is enhanced on addition of exogenous TNF-α (1ng/ml). • In vitro biomarker evaluation shows robust inhibition of cIAP1 and XIAP-Caspase-9 interaction and up-regulation of apoptosis marker. AT-IAP ACTIVITY IN MELANOMA CELL LINES AND PRIMARY TUMORS APOPTOSIS EVALUATION OF MELANOMA CELL LINES BY FLOW CYTOMETRY • Increased caspase-3-substrate staining after 48h treatment with AT-IAP + 1ng/ml TNF-α A375 melanoma cell line BIOMARKER EVALUATION IN VITRO BY WESTERN BLOTTING • cIAP1/2 degradation and apoptosis marker 24h after AT-IAP addition in sensitive cancer cell lines • Cell viability assay screen of cell lines for AT-IAP sensitivity +/- TNF-α 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Hs 294 T Malme-3M RPMI-7951 LB1319-MEL SK-MEL-28 C81 61 BB74-MEL UZG4-MEL A-375 SK-MEL-2 SK-MEL-5 KUL58-MEL SK-MEL-24 CMEL-5 Activity Area AT-IAP AT-IAP + 1ng/ml TNF-a Cell lines (proliferation) 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 PDX-2 PDX-15 PDX-1 PDX-3 PDX-12 PDX-5 PDX-4 PDX-6 PDX-7 PDX-10 PDX-8 PDX-9 PDX-11 PDX-13 PDX-14 AT-IAP AT-IAP + 1ng TNF-a pIC 50 Ex Vivo PDX Cells (colony formation) • Balanced cIAP1/XIAP profile • Non peptidomimetic • Non alanine warhead • Potent cellular activity • Oral activity in in vivo models Assay Description EC 50 (nM) XIAP Cell Assay HEK293-XIAP-Caspase-9 (I.P) 5.1 ML-IAP Cell Assay HEK293-ML-IAP-SMAC (I.P) 11.0 cIAP1 Cell Assay MDA-MB-231 (cIAP1 degradation) 0.32 Cell Proliferation Assays EVSA-T MDA-MB-231 HCT-116 (insensitive control) 0.83 4.4 >10,000 • AT-IAP induces cIAP1 inhibition and up-regulation of apoptosis marker in vivo. • In vivo single agent efficacy can be demonstrated in the A375 melanoma and MDA-MB-231 breast cancer xenograft models. SK-MEL28 A375 DMSO +TNFa 10nM AT-IAP + TNFa 100nM AT-IAP + TNFa 6 h Veh. Ctr. AT-IAP 50 mg/kg p.o. 24 h cIAP1 Cleaved PARP Cleaved Caspase-3 48 h 0 1 2 3 4 0 20 40 60 80 100 120 CTR 6 h 24 h 48 h Tumour [AT-IAP] μM cIAP1 % Veh. CTR cIAP1 level [AT-IAP] μM PK/PD Data A375 melanoma xenograft MDA-MB-231 breast cancer xenograft HEK293 transfected with FLAG-XIAP MDA-MB-231 breast cancer cell line • XIAP-Caspase-9 interaction disrupted 2h after AT-IAP addition