Mapping the binding site of tissue kallikrein: Preparation and testing of all possible substrate analog inhibitors homologous with the sequence of kininogen between Ser386 and Gln392

Article abstract of DOI:10.1021/jm00095a002

Programs aimed at converting peptide inhibitors of proteolytic enzymes into more traditional drug structures require an understanding of the role played by the individual amino acid residues in the inhibitor. To this end, all possible substrate analogues occurring within the sequence Ser³⁸⁶-Pro- Phe-Arg-Ser-Val-Gln³⁹² from bovine kininogen were synthesized and tested as inhibitors of tissue kallikrein (EC 3.4.21.35, β-PPK). Of the 21 sequences which can be formed from the heptapeptide, 11 have inhibitory constants which could be measured in the chromogenic assay employed in these studies. No dipeptide and only one tripeptide, Ac-Phe-Arg-Ser-NH₂ (K(i) = 718 μM), measurably inhibits the enzyme. All longer peptides inhibit β-PPK. The heptapeptide Ac-Ser-Pro-Phe-Arg-Ser-Val-Gln-NH₂ is the most effective inhibitor in this series (K(i) = 101 μM). Each amino acid residue in the sequence appears to alter binding in a relatively independent manner. The N- terminal seryl residue (P₄) and the prolyl residue (P₃) slightly improve the K(i) of the various inhibitors. The phenylalanyl residue at P₂ appears to have a more pronounced effect on K(i). The arginyl residue at P₁ and the seryl residue at P₁' appear to be the most important residues in the inhibitory sequence. They contribute approximately one-third and one-fourth of the binding energy to the interaction between the substrate analogues and β-PPK, respectively. The valyl residue at P₂', and the C-terminal glutaminyl residue improve K(i) of each of the peptides tested. Almost 80% of the binding energy of the substrate analogue inhibitors comes from the core sequence Phe-Arg-Ser which occurs between P₂ and P₁'. Molecular models developed from the Chen-Bode coordinates of the aprotinin-β-PPK complex have been used to interpret the results of these studies.