Here we developed small molecule inhibitors of SKI-1/S1P enzyme of the Proprotein Convertase family following two approaches. One involves the assembly of multi-branch peptides while the other utilizes the insertion of alkyloxy pseudo peptide bond at P1-P1 cleavage position. In first approach, 2 and 4-branch peptides were designed based on the human (h) SKI-1128-137 sequence, located N-terminal to its secondary activation site (K137fl L). The 4-branch peptide exhibited the highest SKI-1 inhibitory property (IC50 = 0.9 μM) with ∼8.6 and 1.3-fold more potency than the corresponding single and 2-branch peptides, respectively. In the second strategy, an oxymethylene containing unnatural amino acid such as aminooxy-acetic acid (Aoaa) or 8-amino-3, 6 dioxa-octanoic acid (Adoa) was introduced substituting P1, P1 or both residues of hSKI-1183-190 and hSKI-1178-190 segments. These domains contain the same primary hSKI-1 activation site L186φ R. Among those tested, P7-Tyr mutant [178GRYSSRRL(Adoa)AIP190] exhibited higher SKI-1 inhibitory activity (Ki in low μM). Circular dichroism (CD) spectra of SKI-1 inhibitors showed interactions of varying degrees between the enzyme and the inhibitor consistent with the observed inhibition profile. A 3D-homology model structure of SKI-1 catalytic domain indicated a broad catalytic pocket.
Keywords: Subtilisin Kexin Isozyme-1 (SKI-1), Site 1 Protease (S1P), Enzyme inhibitors, Pseudopeptide, Multibranch peptide, Isostere, Inhibition constant, Secondary structure, Circular dichroism, Mass spectrometry