Enediynyl peptides and iso-coumarinyl methyl sulfones as inhibitors of proprotein convertases PCSK8/SKI-1/S1P and PCSK4/PC4: Design, synthesis and biological evaluations.

The proprotein convertases PCSK8 and PCSK4 are, respectively, the 8th and 4th members of Ca(+2)-dependent serine endoprotease of Proprotein Convertase Subtilisin Kexin (PCSK) super family structurally related to the bacterial subtilisin and yeast kexin. The membrane bound PCSK8 (also called SKI-1 or S1P) is implicated in sterol regulation and lipid synthesis via its role in the maturation of human (h) SREBP-2. It also plays role in cartilage formation, bone mineralization, as well as viral pathogenesis. On the other hand, PCSK4 has been linked to mammalian fertilization and placenta growth. Owing to these findings, interest has grown to develop specific inhibitors against these enzymes for potential biochemical and therapeutic applications. In this study we developed two types of small molecule inhibitors of PCSK8 and PCSK4 and demonstrated their anti-proteolytic activities in vitro cell-free and in vitro cell culture systems. These are isocoumarinyl methyl sulfone derivatives and enediyne amino acid containing peptides. Our in vitro data suggested that one of the 7 sulfone derivatives (methyl phenyl sulfone) inhibited PCSK8 with inhibition constant Ki ∼255μM. It also blocked PCSK8-mediated processing of hSREBP-2 in HepG2 cell in a concentration-dependent manner. However all 7 iso-coumarinyl methyl sulfones inhibited htrypsin with IC50 ranging from 2 to 165μM. In contrast, all our designed enediynyl peptides inhibited PCSK8 and PCSK4 activity with Ki and IC50 in low μM or high nM ranges. All compounds exhibited competitive inhibition as indicated by their enzyme kinetic plots and observed dependence of IC50 value on substrate concentration. Our study confirmed that incorporation at the substrate cleavage site of 'Enediyne amino acid' generates potent inhibitors of PCSK8 and PCSK4. This represents a novel approach for future development of inhibitors of PCSK or other enzymes.