Small molecule inhibitors of Apaf-1-related caspase- 3/-9 activation that control mitochondrial-dependent apoptosis

Abstract

Apoptosis is a biological process relevant to human disease states that is strongly regulated through protein-protein complex formation. These complexes represent interesting points of chemical intervention for the development of molecules that could modulate cellular apoptosis. The apoptosome is a holoenzyme multiprotein complex formed by cytochrome c-activated Apaf-1 (apoptotic proteaseactivating factor), dATP and procaspase-9 that link mitochondria disfunction with activation of the effector caspases and in turn is of interest for the development of apoptotic modulators. In the present study we describe the identification of compounds that inhibit the apoptosome-mediated activation of procaspase-9 from the screening of a diversityoriented chemical library. The active compounds rescued from the library were chemically optimised to obtain molecules that bind to both recombinant and human endogenous Apaf-1 in a cytochrome c-noncompetitive mechanism that inhibits the recruitment of procaspase-9 by the apoptosome. These newly identified Apaf-1 ligands decrease the apoptotic phenotype in mitochondrial-mediated models of cellular apoptosis. Keywords: apoptosis; apoptosome; Apaf-1; caspasa-3; caspasa-9; combinatorial libraries; inhibitor; molecular recognition; peptoid; protein-protein interactions; small molecule Abbreviations: Apaf-1, apoptotic protease-activating factor; DEVDase, hydrolysis of Ac-DEVD-afc; DTT, dithiothreitol; FCS, fetal-calf serum; MEFs, mouse embryo fibroblasts; MMP, mitochondrial membrane potential; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Ni-NTA, (Ni 2 þ -nitrilotriacetate)-agarose; peptoid f1a, 5 0 -6 0 carboxyfluorescein-labelled peptoid 1a; PBS, phosphate-buffered saline; PGA, poly-(L-glutamic acid); rApaf-1, recombinant Apaf-1

Polypeptide Modulators of Caspase Recruitment Domain (CARD)-CARD-mediated Protein-Protein Interactions

“…Recombinant Apaf-1 XL was obtained from a baculovirus expression system as described previously (14).…”

“…Cells were harvested after 24 h, and cytosolic extracts were obtained as described previously (14). The cell extract was protein-quantified, and 50-g protein equivalents of cell extract were supplemented with caspase assay buffer containing 20 M Ac-DEVD-afc substrate for caspase-3 activity evaluation.…”

Peptide-Based Polymer Therapeutics

“…Although pro-apoptotic approaches are related with cancer therapy, anti-apoptotic strategies have also applications in regenerative medicine [96]. Vicent and coworkers designed the first anti-apoptotic polymeric nanomedicine: a PGA-based Apaf-1 inhibitor conjugate [97,98], which has shown to promote regeneration in the course of inflammation-induced tissue injury [99].…”

Discovery of Inhibitors of Protein-Protein Interactions from Combinatorial Libraries

“…Our laboratory focused on the identification of compounds that inhibit the apoptosome-mediated activation of procaspase-9 from the screening of a positional scanning diversity-oriented combinatorial library of trimers of N-alkylglycines [99]. These type of molecules also known as peptoids present a series of characteristics that make them good candidates to cope with the disruption of protein-protein interactions.…”