Inhibition of caspase‐9 by stabilized peptides targeting the dimerization interface

Abstract

Caspases comprise a family of dimeric cysteine proteases that control apoptotic programmed cell death and are therefore critical in both organismal development and disease. Specific inhibition of individual caspases has been repeatedly attempted, but has not yet been attained. Caspase-9 is an upstream or initiator caspase that is regulated differently from all other caspases, as interaction with natural inhibitor XIAP-BIR3 occurs at the dimer interface maintaining caspase-9 in an inactive monomeric state. One route to caspase-9-specific inhibition is to mimic this interaction, which has been localized to the α5 helix of XIAP-BIR3. We have developed three types of stabilized peptides derived from the α5 helix, using incorporation of amino-isobutyric acid, the avian pancreatic polypeptide-scaffold or aliphatic staples. The stabilized peptides are helical in solution and achieve up to 32 µM inhibition, indicating that this allosteric site at the caspase-9 dimerization interface is regulatable with low-molecular weight synthetic ligands and is thus a druggable site. The most potent peptides against caspase-9 activity are the avian pancreatic polypeptide-scaffolded peptides. Other caspases, which are not regulated by dimerization, should not be inactivated by these peptides. Given that all of the peptides attain helical structures but cannot recapitulate the high-affinity inhibition of the intact BIR3 domain, it has become clear that interactions of caspase-9 with the BIR3 exosite are essential for high-affinity binding. These results explain why the full XIAP-BIR3 domain is required for maximal inhibition and suggest a path forward for achieving allosteric inhibition at the dimerization interface using peptides or small molecules.

Caspase Substrates and Inhibitors

“…Sometimes, experiments of nature often give reasonable starting points, and to this end the dimer interface provides a point of leverage. Thus, the BIR3 domain of the endogenous caspase-9 inhibitor XIAP acts by preventing the activating dimerization of the enzyme (Shiozaki et al, 2003), and to this end peptides mimicking this interaction have been described as allosteric caspase inhibitors (Huber et al, 2012). The dimerization interface of caspases-3 and 7 contains pockets that can be utilized to block the active conformation, by an allosteric mechanism (Hardy et al, 2004).…”

Dimeric Switch of Hakai-truncated Monomers during Substrate Recognition

“…The findings from this study suggest that the dimeric architecture of the HYB domain is necessary to engage the Tyr(P) ligand, which is in sharp contrast to all the other known Tyr(P)-binding domains that predominantly function as monomers. Selectively targeting the dimeric interface of therapeutically important enzymes has emerged as an attractive method of allosteric inhibition (62)(63)(64)(65). The importance of the dimeric architecture of HYB in Tyr(P) substrate binding demonstrated by the present study makes it an ideal target for the design of selective allosteric inhibitors that abrogate HYB dimerization and potentially act as novel therapeutic interventions against cancer.…”

A Multipronged Approach for Compiling a Global Map of Allosteric Regulation in the Apoptotic Caspases

“…Inhibition of procaspase-3 by zinc was defined upon exploration of the mechanism of activation by the chemical ligand PAC-1 (Peterson, et al, 2009) which directly competes with the caspase-3 active site for zinc. We have shown that synthetic peptide ligands can be used to modulate dimerization of caspase-9 (Huber, Ghosh, & Hardy, 2012) (exosite B, Fig. 4.0) and a small peptide, Pep419, has been developed at Genentech, which inhibits caspase-6 allosterically (Stanger, et al, 2012) (exosite G, Fig.…”

“…Due to low sequence homology of residues comprising the dimerization interface, the size and nature of the cavity formed at this interface also varies, allowing small molecules to differentially act on this region; whether by preventing the formation of a properly-formed active site (e.g. caspase-7 (Hardy, et al, 2004a)) or by inhibiting dimerization (caspase-9 (Huber, et al, 2012) (Feldman, et al, 2012) and -8 (Feldman, et al, 2012)) (see section 4).…”

“…Three classes of BIR3-mimicking peptides produced by novel synthetic schemes developed are shown. Figure adapted from (Huber, et al, 2012) with permission.…”