Allosteric peptides bind a caspase zymogen and mediate caspase tetramerization

Abstract

The caspases are a family of cytosolic proteases with essential roles in inflammation and apoptosis. Drug discovery efforts have focused on developing molecules directed against the active sites of caspases, but this approach has proved challenging and has not yielded any approved therapeutics. Here we describe a new strategy for generating inhibitors of caspase-6, a potential therapeutic target in neurodegenerative disorders, by screening against its zymogen form. Using phage display to discover molecules that bind the zymogen, we report the identification of a peptide that specifically impairs the function of caspase-6 in vitro and in neuronal cells. Remarkably, the peptide binds at a tetramerization interface that is uniquely present in zymogen caspase-6, rather than binding into the active site, and acts via a new allosteric mechanism that promotes caspase tetramerization. Our data illustrate that screening against the zymogen holds promise as an approach for targeting caspases in drug discovery.

Zinc-mediated Allosteric Inhibition of Caspase-6

“…Very recently, a novel allosteric peptide, pep419, which binds near the 90s helix in caspase-6 inducing a tetrameric state, has been reported to function via this second class of allosteric mechanism. The authors state the following (70): "The effect of pep419 is not the transmission of a specific conformational change from the peptide-binding site to the active site but rather a more global effect on protein dynamics whereby peptide binding induces tetramerization and, most likely, a structural rigidity that prevents catalytic turnover." Of course in both classes of allosteric inhibition, the same fundamental thermodynamic properties control the equilibrium, and it is simply the ability to visualize these pathways that differs between the two classes.…”

Structural Basis of the Interaction of MbtH-like Proteins, Putative Regulators of Nonribosomal Peptide Biosynthesis, with Adenylating Enzymes

“…Therefore, the hypothesis should be considered that the small MbtH-like proteins may function as allosteric regulators of adenylating enzymes. Allosteric regulation of proteins by small peptides is a well established mechanism, utilized both in natural regulation processes and in the development of artificial regulators (47,48). Allosteric regulation may occur both in oligomeric and in monomeric proteins (49), e.g.…”

Identification of a Small Peptide That Inhibits PCSK9 Protein Binding to the Low Density Lipoprotein Receptor

“…Construction of Phage-displayed Peptide Libraries-Two groups of phage-displayed peptide libraries, the linear peptide library called Linear-lib and cysteine-restrained cyclic library called Cyclic-lib, were constructed by fusing randomized peptides to the N terminus of M13 major coat protein p8 (25). Linear-lib consisted of random peptides with 8, 10, 12, 14, or 16 amino acids, and Cyclic-lib consisted of 14-mer random peptides with varied length between two invariant cysteines.…”

“…Two types of phage-displayed naive peptide libraries, Linear-lib and Cyclic-lib (25), were used to select for PCSK9 binding peptides by solution sorting against biotinylated PCSK9 in calciumfree buffer (26). After four rounds of panning, several clones gave modest binding signals.…”