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

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.

Regulatory effects of peptides from the pro and catalytic domains of proprotein convertase subtilisin/kexin 9 (PCSK9) on low-density lipoprotein receptor (LDL-R).

BACKGROUND: Proprotein convertase subtilisin/kexin 9 (PCSK9) is a proteinase K subtype of mammalian subtilases collectively called PCSKs. PCSK9 upregulates plasma-cholesterol level by degrading low-density lipoprotein receptor (LDL-R). As a result, PCSK9 is a major target for intervention of hypercholesterolemia and in this regard PCSK9- inhibitors may find useful therapeutic and biochemical applications. OBJECTIVE: Our objective is to develop short peptide based PCSK9 inhibitors from its own pro and/or catalytic domains. RESULTS: Using human (h) hepatic HepG2 and Huh7 cells we showed that the acidic N-terminal hPCSK(931-60), 31-40 and the mid-basic hPCSK(991-120) peptides derived from hPCSK9-prodomain significantly enhanced LDL-R level without altering PCSK9 content. Moreover, the physiologically relevant phoshpho-Ser47 and sulpho-Y38 containing hPCSK(931-60) peptides diminished LDL-R level suggesting that such posttranslational modifications in the prodomain lead to gain of PCSK9- functional activity. These modifications are thus expected to lead to even higher level of plasma cholesterol. As expected, addition of purified recombinant-PCSK9 to the culture medium decreased LDL-R level which can be restored back by exogenous addition of hPCSK(931-40), (31-60) or (91-120) peptides. Using a series of truncated peptides, we identified the most potent LDL-R promoting activity to reside within the prodomain sequence hPCSK(931-37). Two catalytic domain peptides hPCSK(9181-200) and hPCSK(9368-390), containing proposed LDL-R interacting sites have been shown to diminish LDL-R level. CONCLUSION: Our study concludes that specific peptides from pro- and catalytic domains of hPCSK9 can regulate LDL-R in cell based assay and may be useful for development of novel therapeutics for cholesterol regulation.