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--Φ-Leu/Ser/Thr↓, where = 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 (-CH2-O-) pseudoamide function at the cleavage site of 251Asp-Ile-Tyr-Ile-Ser-Arg-Arg-Leu-Leu↓Gly-Thr-Phe-Thr263, derived from SKI-1 processing site of Lassa virus glycoprotein. The synthesis was conducted by substituting Leu-Gly with previously made Leu-CH2-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 (Ki∼14-20 μM) than the corresponding acyclic peptide (Ki7sim;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(CH2-O) containing peptides as SKI-1 inhibitors with potential therapeutic applications in cholesterol synthesis.

Keywords: Subtilisin Kexin Isozyme-1 (SKI-1), Site 1 Protease (S1P), Methylene-oxy pseudopeptides, Enzyme inhibitors, Intramolecularly quenched fluorogenic peptide, Circular peptide, Disulfide bridged cyclic peptide, Inhibition constant (Ki), Proprotein processing