Abstract
Members of the Inhibitor of APoptosis (IAP) protein family suppress apoptosis within tumor cells, particularly in the context of immune cell-mediated killing by the tumor necrosis factor (TNF) superfamily cytokines. Most IAPs are opposed endogenously by the second mitochondrial activator of caspases (SMAC), which binds to selected baculovirus IAP repeat (BIR) domains of IAPs to displace interacting proteins. The development of SMAC mimetics as novel anticancer drugs has gained impetus, with several agents now in human clinical trials. To further understand the cellular mechanisms of SMAC mimetics, we focused on IAP family members cIAP1 and cIAP2, which are recruited to TNF receptor complexes where they support cell survival through NF-κB activation while suppressing apoptosis by preventing caspase activation. We established fluorescence polarization (FP) assays for the BIR2 and BIR3 domains of human cIAP1 and cIAP2 using fluorochrome-conjugated SMAC peptides as ligands. A library of SMAC mimetics was profiled using the FP assays to provide a unique structure activity relationship (SAR) analysis compared to previous assessments of binding to XIAP. Potent compounds displayed mean inhibitory binding constants (Ki) of 9 to 27 nM against the BIR3 domains of cIAP1 and cIAP2, respectively. Selected compounds were then characterized using cytotoxicity assays in which a cytokine-resistant human tumor cell line was sensitized to either TNF or lymphotoxin-α (LT-α). Cytotoxicity correlated closely with cIAP1 and cIAP2 BIR3 binding activity with the most potent compounds able to reduce cell viability by 50%. Further testing demonstrated that active compounds also inhibit RIP1 binding to BIR3 of cIAP1 and cIAP2 in vitro and reduce steady-state cIAP1 protein levels in cells. Altogether, these data inform the SAR for our SMAC mimetics with respect to cIAP1 and cIAP2, suggesting that these IAP family members play an important role in tumor cell resistance to cytotoxicity mediated by TNF and LT-α.
Highlights
Defects in the regulation of apoptosis underlie many disease processes, including cancer [1]
We examined the effects on second mitochondrial activator of caspases (SMAC)-rhodamine peptide binding to cIAP1 and cIAP2 BIR2 and BIR3 domains using a library of synthesized compounds that we have previously described in detail, and characterized with respect to XIAP binding [21,22,23]
Most efforts to generate apoptosis-promoting anticancer compounds based on mimicking SMAC have focused on XIAP as a target, due to both the technical ease of producing abundant quantities of recombinant SMAC peptide-binding BIR3 domain of XIAP, and the prominent functional role of XIAP in blocking caspase activation induced by tumor necrosis factor (TNF) family members Fas (CD95) and TNF-related apoptosis-inducing ligand (TRAIL) Receptors (CD261 and CD262)
Summary
Defects in the regulation of apoptosis underlie many disease processes, including cancer [1]. A family of intracellular cysteine proteases, are the effectors of apoptosis [2]. These proteases are present as inactive zymogens in essentially all mammalian cells. Some caspases are inhibited by members of the inhibitor of apoptosis proteins (IAP) family [3]. IAPs contain a structural motif called the baculovirus IAP repeat (BIR) domain that participates in the binding of active caspases. Certain IAPs bind via their BIR domains to other classes of protein targets, including proteins involved in signal transduction pathways leading to activation of NF-κB and the stress kinases of the MAPK pathway [4, 5]
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