Abstract IA03: Mechanistic studies of IAP inhibitors
Authors: Fairbrother Wayne J. 
Published: May 2017

Inhibitor of apoptosis (IAP) proteins are critical regulators of cell death, preventing apoptosis in response to a variety of signals. Overexpression of IAP proteins has been demonstrated to confer protection against a variety of pro apoptotic stimuli, including chemotherapies, and is a marker for poor prognosis in a variety of solid tumors and hematologic malignancies. Antagonism of the IAP protein mediated inhibition of caspases is required for caspase dependent cell death, and can be achieved by the mitochondrial protein SMAC/DIABLO, which is released into the cytoplasm in response to pro apoptotic stimuli. Structure-based design was used to identify small molecule SMAC mimetics that potently antagonize IAP protein interactions. These SMAC mimetics induce apoptosis in select cancer cell lines and have proven efficacious at inhibiting tumor growth in mouse xenograft models of cancer. Remarkably, binding of small-molecule SMAC mimetics to cIAP1 or 2 results in induction of auto-ubiquitination activity and rapid proteasomal degradation of these proteins. The cIAP proteins (cIAP1 and 2) comprise several domains, including three baculovirus IAP repeat (BIR1-3) domains, a ubiquitin-binding (UBA) domain, a caspase-associated recruitment domain (CARD), and a RING E3 ligase domain. Binding of SMAC mimetics to the cIAP1-BIR3 domain results in homodimerization of cIAP1 through its RING domain and activation of its ubiquitin E3 ligase activity. To understand how this long-range communication takes place, we solved the crystal structure of a truncated form of cIAP1, comprising the BIR3 through RING domains (cIAP1-B3R) and found that cIAP1-B3R adopts a compact tertiary structure in which the RING domain is sequestered on three sides by the BIR3 domain, the UBA domain, and the CARD. SMAC mimetic binding triggers E3 ligase activity in cIAP1 by disrupting the BIR3:RING interface, thereby allowing RING dimerization. However, this view of cIAP1 as a ‘binary switch' does not fully explain the mechanism of activation, since the compact structure of the inactive state precludes binding of peptides and SMAC mimetics. We have subsequently used NMR spectroscopy to study micro- and millisecond motions of specific domain interfaces in cIAP1 and have use time-resolved small-angle X-ray scattering to observe the global conformational changes necessary for activation. Although motions within each interface of the ‘closed' monomer are insufficient to activate cIAP1, they enable associations with catalytic partners and activation factors. Together these results suggest that cIAP1 internal motions are tuned to minimize the time required for the rate-limiting conformational rearrangements, priming the protein for rapid activation while maintaining the integrity of the closed monomer. The internal dynamics of cIAP1 present an intriguing picture of a “spring-loaded” cell death trigger, with activation speed balanced precisely against the critical need for message integrity.

Citation Format: Wayne J. Fairbrother. Mechanistic studies of IAP inhibitors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr IA03.