Novel circular, cyclic and acyclic Ψ(CH(2)O) containing peptide inhibitors of SKI-1/S1P: synthesis, kinetic and biochemical evaluations

Abstract

Background: Subtilisin Kexin Isozyme-1 (SKI-1)/Site1Protease (S1P) is a Ca(+2)-dependent membrane bound pyrolysin-type serine protease of mammalian subtilase super family Proprotein Convertases (PCs)/Proprotein Convertase Subtilisin Kexins (PCSKs). It cleaves precursor proteins at the carboxy terminus of a non basic amino acid characterized by the sequence Arg/Lys-θ-φ-Leu/Ser/Thr↓, where θ = any amino acid except Cys, φ = the alkyl side chain containing hydrophobic amino acid. SKI-1 cleaves pro-BDNF, pro-SREBP2, pro-ATF6, pro-somatostatin and viral glycoproteins to generate their active forms. As a result SKI-1 plays important roles in cartilage development, bone mineralization, cholesterol metabolism, fatty acid synthesis and infections caused by Arina viruses of hemorrhagic type. Interest has grown to develop inhibitors of SKI-1 that may find useful therapeutic and biochemical applications.

Objective: Our objective is to develop small molecule inhibitors of SKI-1/S1P and study their kinetic and biochemical properties.

Results: Peptide analogs were designed by inserting a protease resistant methylene-oxy (-CH(2)-O-) pseudoamide function at the cleavage site of (251)Asp-Ile-Tyr-Ile-Ser-Arg-Arg-Leu-Leu↓Gly-Thr-Phe-Thr(263), derived from SKI-1 processing site of Lassa virus glycoprotein. The synthesis was conducted by substituting Leu-Gly with previously made Leu-CH(2)-O-Gly. Flexible linear and conformationally constrained circular and disulphide bridged cyclic peptides were prepared by solid phase method. Circular and cyclic peptides inhibited SKI-1 more potently (K(i)~14-20 µM) than the corresponding acyclic peptide (K(i)~51 µM). They also blocked SKI-1-mediated processing of pro-h(human)SREBP2 into its mature form in HepG2 cells. Circular pseudopeptides designed from hATF6 and hSREBP2 also inhibited SKI-1. This is the first report of circular and cyclic Ψ(CH(2)-O) containing peptides as SKI-1 inhibitors with potential therapeutic applications in cholesterol synthesis.