Design of kallidin-releasing tissue kallikrein inhibitors based on the specificities of the enzyme's binding subsites

The tissue kallikrein inhibitors reported in the present work were derived by selectively replacing residues in N-alpha-substituted arginine- or phenylalanine-pNA (where pNA is p-nitroanilide), and in peptide substrates for these enzymes. Phenylacetyl-Arg-pNA was found to be an efficient inhibitor of human tissue kallikrein (K-i 0.4 mu M) and was neither a substrate nor an inhibitor of plasma kallikrein. The peptide inhibitors having phenylalanine as the P-1 residue behaved as specific inhibitors for kallidin-releasing tissue kallikreins, while plasma kallikrein showed high affinity for inhibitors containing (p-nitro)phenylalanine at the same position. The K-i value of the most potent inhibitor developed, Abz-Phe-Arg-Arg-Pro-Arg-EDDnp [where Abz is o-aminobenzoyl and EDDnp is N-(2,4-dinitrophenyl)ethylenediamine], was 0.08 mu M for human tissue kallikrein. Progress curve analyses of the inhibition of human tissue kallikrein by benzoyl-Arg-pNA and phenylacetyl-Phe-Ser-Arg-EDDnp indicated a single-step mechanism for reversible formation of the enzyme-inhibitor complex.UNIV FED SAO PAULO,ESCOLA PAULISTA MED,DEPT BIOPHYS,BR-04044020 SAO PAULO,BRAZILUNIV SHEFFIELD,DEPT MOL BIOL & BIOTECHNOL,SHEFFIELD S10 2UH,S YORKSHIRE,ENGLANDUNIV FED SAO PAULO,ESCOLA PAULISTA MED,DEPT BIOPHYS,BR-04044020 SAO PAULO,BRAZILWeb of Scienc