The B cell lymphoma 2 (BCL2) proteins are a family of evolutionarily related proteins that act as positive or negative regulators of the intrinsic apoptosis pathway. Overexpression of anti-apoptotic BCL2 proteins in cells is associated with apoptotic resistance, which can result in cancerous phenotypes and pathogenic cell survival. Consequently, anti-apoptotic BCL2 proteins have attracted considerable interest as therapeutic targets. We recently reported the development of a novel class of synthetic protein based on scyllatoxin (ScTx) designed to mimic the helical BH3 interaction domain of the pro-apoptotic BCL2 protein Bax. These studies showed that the number and position of native disulfide linkages contained within the ScTx-Bax structure significantly influences the ability for these constructs to target anti-apoptotic BCL2 proteins in vitro. The goal of the present study is to investigate the contribution of two disulfide linkages in the folding and biological activity of ScTx-Bax proteins. Here, we report the full chemical synthesis of three ScTx-Bax sequence variants, each presenting two native disulfide linkages at different positions within the folded structure. It was observed that two disulfide linkages were sufficient to fold ScTx-Bax proteins into native-like architectures reminiscent of wild-type ScTx. Furthermore, we show that select (bis)disulfide ScTx-Bax variants can target Bcl-2 (proper) in vitro and that the position of the disulfide bonds significantly influences binding affinity. Despite exhibiting only modest binding to Bcl-2, the successful synthesis of ScTx-Bax proteins containing two disulfide linkages represents a viable route to ScTx-based BH3 domain mimetics that preserve native-like conformations. Finally, structural models of ScTx-Bax proteins in complex with Bcl-2 indicate that these helical mimetics bind in similar configurations as wild-type Bax BH3 domains. Taken together, these results suggest that ScTx-Bax proteins may serve as potent lead compounds that expand the repertoire of "druggable" protein-protein interactions.
Keywords: Anti-apoptotic BCL2 protein; BH3 domain mimetic; Disulfide linkage; Miniature protein; Orthogonal protecting group; Scyllatoxin.