Structural Insight into Inhibitor of Apoptosis Proteins Recognition by a Potent Divalent Smac-Mimetic

Federica Cossu,Serena Grassi,Mario Milani,Patrice Vachette,Eloise Mastrangelo,Pierfausto Seneci,Francesca Malvezzi,Carmelo Drago,Daniele Lecis,Martino Bolognesi,Domenico Delia,Srinivasa M Srinivasula

doi:10.1371/journal.pone.0049527

Abstract

Genetic alterations enhancing cell survival and suppressing apoptosis are hallmarks of cancer that significantly reduce the efficacy of chemotherapy or radiotherapy. The Inhibitor of Apoptosis Protein (IAP) family hosts conserved proteins in the apoptotic pathway whose over-expression, frequently found in tumours, potentiates survival and resistance to anticancer agents. In humans, IAPs comprise eight members hosting one or more structural Baculoviral IAP Repeat (BIR) domains. Cellular IAPs (cIAP1 and 2) indirectly inhibit caspase-8 activation, and regulate both the canonical and the non-canonical NF-κB signaling pathways. In contrast to cIAPs, XIAP (X chromosome-linked Inhibitor of Apoptosis Protein) inhibits directly the effector caspases-3 and -7 through its BIR2 domain, and initiator caspase-9 through its BIR3 domain; molecular docking studies suggested that Smac/DIABLO antagonizes XIAP by simultaneously targeting both BIR2 and BIR3 domains. Here we report analytical gel filtration, crystallographic and SAXS experiments on cIAP1-BIR3, XIAP-BIR3 and XIAP-BIR2BIR3 domains, alone and in the presence of compound 9a, a divalent homodimeric Smac mimetic. 9a is shown to bind two BIR domains inter- (in the case of two BIR3) and intra-molecularly (in the case of XIAP-BIR2BIR3), with higher affinity for cIAP1-BIR3, relative to XIAP-BIR3. Despite the different crystal lattice packing, 9a maintains a right handed helical conformation in both cIAP1-BIR3 and XIAP-BIR3 crystals, that is likely conserved in solution as shown by SAXS data. Our structural results demonstrate that the 9a linker length, its conformational degrees of freedom and its hydrophobicity, warrant an overall compact structure with optimal solvent exposure of its two active moieties for IAPs binding. Our results show that 9a is a good candidate for pre-clinical and clinical studies, worth of further investigations in the field of cancer therapy.

Highlights

Apoptosis is a process of programmed cell death essential for homeostasis maintenance in multicellular organisms, which is regulated by a subset of caspases (Cysteine-dependent ASPartylspecific proteASES) in charge of propagating, once activated, the apoptotic signal to the nucleus [1]
Cellular Cytotoxicity Preliminary cytotoxicity tests of 9a after 72 hours of treatment versus MDA-MB-231, HL60, and PC-3 cells, were addressed [14]. 9a showed nanomolar cytotoxicity both in MDA-MB-231 and HL60 cell lines, whereas it was inactive against the PC-3 cell line, as expected
This suggests that the two BIR2 and BIR3 structured domains are well separated in the absence of 9a, likely to be mobile around a flexible linker, while the divalent Smac-mimetic brings them into close proximity, resulting in the narrower distance distribution observed

Summary

Introduction

Apoptosis is a process of programmed cell death essential for homeostasis maintenance in multicellular organisms, which is regulated by a subset of caspases (Cysteine-dependent ASPartylspecific proteASES) in charge of propagating, once activated, the apoptotic signal to the nucleus [1]. Since the structural details of IBM interactions with XIAP and cIAPs have been previously described [4,5], the IBM peptides provide a natural basis for the design of Smac-mimetics These compounds have been shown to displace caspases 3, 7 and 9 from XIAP-BIR2 and –BIR3 inhibitory pockets, and to induce auto-ubiquitination and degradation of cIAPs by perturbing BIR3/RING domain interaction [6,7]. 9a displayed significant potency as a single agent in reducing the development of solid tumours in mice injected subcutaneously with a human ovarian cancer cell line, and increased the median survival time of mice in a human ovarian ascites model [14] In this communication we present biochemical, biophysical and structural characterization of 9a in its complexes with XIAP-BIR3, XIAP-BIR2BIR3 and cIAP1-BIR3. All the experimental evidences here reported suggest that 9a is one of the most powerful divalent Smac-mimetics known to date; the structural analysis of its recognition patterns, here presented, is the basis for further optimization in terms of target affinity and bioavailability

Results

Discussion

Experimental Procedures