Modulators of proprotein convertase, Subtilisin Kexin Isozyme-1 (SKI-1) Site 1 Protease (SIP): Design, synthesis and in vitro evaluation

Abstract

Subtilisin Kexin Isozyme-1 (SKI-I) also called Site1 Protease (S1P) is a member of mammalian subtilisin family that is involved in cholesterol metabolism, lipid synthesis and viral infections. It is considered as a target for intervention of these diseases or disorders. Herein, vaccinia virus transfected HEK 293 expression system was used in the production of ∼98 kDa C-terminally truncated soluble human hSKI-1 enzyme in enzymatically active form. Partial purification of recombinant (rec) hSKI-1 enzyme was achieved by using modified cell culture condition and affinity column chromatography. In addition, gel filtration size exclusion chromatography was also attempted as an alternative to column chromatography method to purify the enzyme. Purification ofrec-hSKI-1 led to significant dimerization of the enzyme which results the partial loss of protease activity. Nonetheless, it was used to design novel SKI-I inhibitors for biochemical and therapeutic applications. Oxymethylene (-OCH2-) based pseudo and multibranch peptide strategies were used for the design of new hSKI-1 inhibitors. The peptide sequences used in these approaches were obtained from SKI-I prodomain near its autocatalytic cleavage sites. Depending on the length of the peptide sequence, competitive and mixed type inhibition was observed for the Oxymethylene based pseudo peptide inhibitors. However, most of them showed competitive inhibition of hSKI-1 activity, suggesting their interactions with the catalytic domain of SKI-I enzyme. It also indicated that Oxymethylene function might be a good mimicry of peptidyl amide bond (-CONH-). In the pseudopeptide series, ∼5 fold more inhibition was observed for the P7- Tyr mutant peptide. This suggested the preference of aromatic residue at P7 position of the substrate is important for hSKI-1 enzyme recognition. In the multibranch peptide series, the 2- and 4- branch peptides displayed ∼8.6 and ∼13 fold increased inhibition of hSKI-1 enzyme respectively compared to the single branch linear peptide. This suggests that increasing the number of inhibitory peptide chain within a core molecule may be an interesting concept for the design of enzyme inhibitors and should be explored not only for convertase but also for other protease inhibition as well.