Template‐assisted rational design of peptide inhibitors of furin using the lysine fragment of the mung bean trypsin inhibitor

Tao, Hu; Zhang, Zhen; Shi, Jiahao; Shao, Xiao‐xia; Cui, Dafu; Chi, Cheng‐wu

Highly active, small‐molecule furin inhibitors are attractive drug candidates to fend off bacterial exotoxins and viral infection. Based on the 22‐residue, active Lys fragment of the mung bean trypsin inhibitor, a series of furin inhibitors were designed and synthesized, and their inhibitory activity towards furin and kexin was evaluated using enzyme kinetic analysis. The most potent inhibitor, containing 16 amino acid residues with a K (i) value of 2.45 × 10(−9) m for furin and of 5.60 × 10(−7) m for kexin, was designed with three incremental approaches. First, two nonessential Cys residues in the Lys fragment were deleted via a Cys‐to‐Ser mutation to minimize peptide misfolding. Second, residues in the reactive site of the inhibitor were replaced by the consensus substrate recognition sequence of furin, namely, Arg at P(1), Lys at P(2), Arg at P(4) and Arg at P(6). In addition, the P(7) residue Asp was substituted with Ala to avoid possible electrostatic interference with furin inhibition. Finally, the extra N‐terminal and C‐terminal residues beyond the doubly conjugated disulfide loops were further truncated. However, all resultant synthetic peptides were found to be temporary inhibitors of furin and kexin during a prolonged incubation, with the scissile peptide bond between P(1) and P(1)′ being cleaved to different extents by the enzymes. To enhance proteolytic resistance, the P(1)′ residue Ser was mutated to d‐Ser or N‐methyl‐Ser. The N‐methyl‐Ser mutant gave rise to a K (i) value of 4.70 × 10(−8) m for furin, and retained over 80% inhibitory activity even after a 3 h incubation with the enzyme. By contrast, the d‐Ser mutant was resistant to cleavage, although its inhibitory activity against furin drastically decreased. Our findings identify a useful template for the design of potent, specific and stable peptide inhibitors of furin, shedding light on the molecular determinants that dictate the inhibition of furin and kexin.