Introduction of non-natural amino acid residues into the substrate-specific P 1 position of trypsin inhibitor SFTI-1 yields potent chymotrypsin and cathepsin G inhibitors

Abstract

A series of trypsin inhibitor SFTI-1compounds modified in substrate-specific P 1 position was synthesized by the solid-phase method. Lys5 present in the wild inhibitor was replaced by Phe derivatives substituted in para position of the phenyl ring, l-pyridylalanine and N-4 -nitrobenzylgycine. Their inhibitory activities with bovine α-chymotrypsin and cathepsin G were estimated by determination of association equilibrium constants ( K a). All analogues inhibited bovine α-chymotrypsin. The highest inihbitory activity displayed peptides with the fluorine, nitro and methyl substituents. They were 13–15-fold more active than [Phe 5]SFTI-1 used as a reference. They are the most potent chymotrypsin inhibitors of this size. Substitution of Lys5 by Phe did not change the cathepsin G inhibitory activity. Introduction of Phe( p-F), Phe( p-NH 2) and Phe( p-CH 3) in this position retained the affinity towards this proteinase, whereas Phe( p-guanidine) gave an inhibitor more than twice as active, which appeared to be stable in human serum. On the other hand, a peptomeric analogue with N-4-nitrobenzylglycine failed to inhibit cathepsin G. Despite the fact the introduced amino acids were non-coded, the peptide bonds formed by them were hydrolyzed by chymotrypsin. We postulate that additional interaction of para-substitutents with the enzyme are responsible for the enhanced inhibitory activity of the analogues.