Truncation of Huia versabilis Bowman-Birk inhibitor increases its selectivity, matriptase-1 inhibitory activity and proteolytic stability.

A gradual truncation of the primary structure of frog skin-derived Huia versabilis Bowman-Birk peptidic inhibitor (HV-BBI) resulted in 18-times stronger inhibitor of matriptase-1 (peptide 6, K = 8 nm) in comparison to the full-length HV-BBI (K = 155 nm). Analogous increase in the inhibitory activity in correlation with the peptide length reduction was not observed in case of other serine proteases, bovine trypsin (K = 151 nm for peptide 6 and K = 120 nm for HV-BBI) and plasmin (K = 120 nm for peptide 6 and 82 nm for HV-BBI). Weaker binding affinity to these enzymes emphasized an inhibitory specificity of peptide 6. Molecular dynamic analysis revealed that the observed variations in the binding affinity of peptide 6 and HV-BBI with matriptase-1 are associated with the entropic differences of the unbound peptides. Moreover, several aspects explaining differences in the inhibition of matriptase-1 by peptide 6 (bearing the C-terminal amide group) and its two analogues, peptide 6∗ (having the C-terminal carboxyl group, K = 473 nm) and cyclic peptide 6∗∗ (K = 533 nm), both exhibiting more than 50-fold reduced inhibitory potency, were discovered. It was also shown that peptide 6 presented significantly higher resistance to proteolytic degradation in human serum than HV-BBI. Additional investigations revealed that, in contrast to some amphibian-derived inhibitors, HV-BBI and its truncated analogues do not possess bactericidal activity, thus they cannot be considered as bifunctional agents.