Abstract 3333: Birinapant, a novel bivalent Smac mimetic drug, is superior to monovalent Smac mimetics in inhibition of NF-kB by targeting TRAF2-bound cIAP1 and cIAP2.
Authors: Mitsuuchi Yasuhiro , Condon Stephen M. , Neiman Eric M. , Benetatos Christopher A. , Seipel Martin E. , Kapoor Gurpreet Singh , Mufalli Angeline C. , Yu Guangyao , Maguire Orla , Minderman Hans , Mckinlay Mark A. , Graham Martin , Weng David , Chunduru Srinivas 
Published: August 2014

Abstract
A variety of Smac-mimetic drugs have been developed to target the inhibitor of apoptosis (IAP) proteins including X chromosome-linked IAP (XIAP), cellular IAP proteins (cIAP1 and cIAP2) and ML-IAP whose gene mutations, amplifications and chromosomal translocations have been implicated in various malignancies. The IAPs also play an important role in multiple signaling pathways including the TNFα-mediated NF-kB and MAPK pathways (inflammatory responses) and pattern recognition receptor signaling pathways (innate immunity). Smac-mimetics have been designed to mimic the IAP-binding motif of the second mitochondria-derived activator of caspase (Smac). The IAP-binding motif consists of four amino acids (AVPI) that serve as an endogenous IAP antagonist. Structurally different Smac-mimetic compounds have been published and showed different binding affinity to IAP proteins. These differences in structure and binding have important consequences in terms of biological activity. Here, we present evidence that bivalent Smac-mimetics are superior to monovalent Smac-mimetics in their ability to inhibit NF-kB in cells stimulated with TNFα. Over 300 monovalent and 300 bivalent Smac-mimetics, including compounds whose structures have been published, were tested for ability to degrade cIAP1 and cIAP2, and inhibit NF-kB in cell-based assays. Inhibition of NF-kB by bivalent Smac-mimetics correlated (R²=0.78) with the degradation of cIAP1 over a range of four logs, while monovalent Smac-mimetics did not (R²=0.20). Furthermore, monovalent Smac-mimetics degraded TRAF2-bound cIAP1 and cIAP2 less effectively (1/10-1/100 fold) compared to bivalent Smac-mimetics. While bivalent Smac-mimetics effectively degraded cIAP1 and cIAP2, birinapant (TL32711), a bivalent Smac-mimetic currently in Phase 2 clinical trials, was unique in its ability to preferentially degrade TRAF2-bound cIAP1 and cIAP2. Inhibition of NF-kB by birinapant was further characterized by the ImageStream cytometry, which showed that the nuclear translocation of p65 in response to TNFα stimulation was blocked in both HeLa and HL-60 cells. These data demonstrate that bivalent Smac-mimetics are superior to monovalent Smac-mimetics in degrading TRAF2-bound cIAPs, in inhibition of NF-kB and efficiently mediating cell death. Furthermore, the unique profile of birinapant versus other bivalent Smac-mimetics may explain its preclinical and clinical safety profile.
Citation Format: Yasuhiro Mitsuuchi, Stephen M. Condon, Eric M. Neiman, Christopher A. Benetatos, Martin E. Seipel, Gurpreet Singh Kapoor, Angeline C. Mufalli, Guangyao Yu, Orla Maguire, Hans Minderman, Mark A. Mckinlay, Martin Graham, David Weng, Srinivas Chunduru. Birinapant, a novel bivalent Smac mimetic drug, is superior to monovalent Smac mimetics in inhibition of NF-kB by targeting TRAF2-bound cIAP1 and cIAP2. [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 3333. doi:10.1158/1538-7445.AM2013-3333