Bivalent IAP antagonists, but not monovalent IAP antagonists, inhibit TNF-mediated NF-\u03baB signaling by degrading TRAF2-associated cIAP1 in cancer cells

Y Mitsuuchi,Gs Kapoor,Sc Beck,Sk Chunduru,Mr Arnone,Sm Condon,Ma Mckinlay,Cg Begley,Ca Benetatos,Y Deng,Me Seipel,T Haimowitz

doi:10.1038/cddiscovery.2016.46

Abstract

The inhibitor of apoptosis (IAP) proteins have pivotal roles in cell proliferation and differentiation, and antagonizing IAPs in certain cancer cell lines results in induction of cell death. A variety of IAP antagonist compounds targeting the baculovirus IAP protein repeat 3 (BIR3) domain of cIAP1have advanced into clinical trials. Here we sought to compare and contrast the biochemical activities of selected monovalent and bivalent IAP antagonists with the intent of identifying functional differences between these two classes of IAP antagonist drug candidates. The anti-cellular IAP1 (cIAP1) and pro-apoptotic activities of monovalent IAP antagonists were increased by using a single covalent bond to combine the monovalent moieties at the P4 position. In addition, regardless of drug concentration, treatment with monovalent compounds resulted in consistently higher levels of residual cIAP1 compared with that seen following bivalent compound treatment. We found that the remaining residual cIAP1 following monovalent compound treatment was predominantly tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2)-associated cIAP1. As a consequence, bivalent compounds were more effective at inhibiting TNF-induced activation of p65/NF-κB compared with monovalent compounds. Moreover, extension of the linker chain at the P4 position of bivalent compounds resulted in a decreased ability to degrade TRAF2-associated cIAP1 in a manner similar to monovalent compounds. This result implied that specific bivalent IAP antagonists but not monovalent compounds were capable of inducing formation of a cIAP1 E3 ubiquitin ligase complex with the capacity to effectively degrade TRAF2-associated cIAP1. These results further suggested that only certain bivalent IAP antagonists are preferred for the targeting of TNF-dependent signaling for the treatment of cancer or infectious diseases.

Highlights

The inhibitor of apoptosis (IAP) family of proteins is characterized by the presence of at least one baculovirus IAP protein repeat (BIR) domain
DISCUSSION cellular IAP1 (cIAP1) and cIAP2 are single-subunit really interesting new gene (RING)-containing E3 ubiquitin ligases and are recruited to TNFR complexes in order to modulate the receptor-mediated pathways leading to canonical p65/nuclear factor kappa-light-chainenhancer of activated B cells (NF-κB) activation.[7,8,10]
IAP antagonists bind to the cIAP1 baculovirus IAP protein repeat 3 (BIR3) domain and induce a conformational rearrangement, which promotes rapid RING domain-mediated dimerization, auto-ubiquitylation and cIAP1 degradation via the ubiquitin–proteasome system.[13,45]

Summary

INTRODUCTION

The inhibitor of apoptosis (IAP) family of proteins is characterized by the presence of at least one baculovirus IAP protein repeat (BIR) domain. Owing to the pleiotropic nature of the IAPs as critical proteins certain IAP proteins.[23,24,25,26] Crystallographic analysis has revealed for cell survival, and for immunoregulation and that the N-terminal tetrapeptide of SMAC, AVPI binds to a shallow groove on the BIR3 surface of both XIAP and cIAP1.26–29 inflammatory responses, the therapeutic utility of monovalent and bivalent IAP antagonists needs to be adjusted based on their. Both monovalent and bivalent small-molecule IAP antagonists biochemical properties. Have been designed to mimic the N-terminus of SMAC by interacting with the BIR3 domain of IAP proteins.[30,31] These IAP antagonists promote the auto-ubiquitylation and proteasomal

RESULTS

Findings

B3-EL2 B3-EL4 M3