Specificity of porcine pancreatic elastase, human leukocyte elastase and cathepsin G Inhibition with peptide chloromethyl ketones

Abstract

In order to design more effective elastase (EC 3.4.21.11) inhibitors, the specificity of porcine pancreatic elastase toward peptide substrates was analyzed by examining cleavages of peptides of known sequence which were reported in the literature. Elastase cleaved 99 peptide bonds in the sample of 419 bonds. The cleavage probability (number of peptide bonds cleaved/total peptide bonds in the sample) was highest for peptides with P 1 residue Ile, Val and Ala, the probability being Ile (0.42), Val (0.39) and Ala (0.35). No bonds with a P 3 Pro were cleaved. Based on the observed cleavage probabilities, several new chloromethyl ketones Ac-Ala-Ala-Pro-AACH 2Cl (AA = Ile, Val, Thr) were synthesized. Porcine pancreatic elastase was inhibited most rapidly by the Ile chloromethyl ketone, but there was little difference in rates of inhibition ( k obs/[I]) between the Ala, Val and Ile inhibitors. The Thr compound was poor. Human leukocyte elastase was inhibited more rapidly by the Ile and Val chloromethyl ketones compared to Ala inhibitor by factors of 49 and 40 respectively at pH 6.5. Excellent correlation was observed between rates of chloromethyl ketone inhibition, peptide nitroanilide hydrolysis and peptide bond cleavage for leukocyte elastase. The water soluble inhibitor MeO-Suc-Ala-Ala-Pro-ValCH 2Cl was found to be the most effective chloromethyl ketone inhibitor of leukocyte elastase yet reported. A number of chloromethyl ketones were surveyed as inhibitors for cathepsin G (a leukocyte chymotrypsin-like enzyme). The most effective was Z-Gly-Leu-PheCH 2Cl. Rates of reaction of several of chloromethyl ketones with bovine trypsin and chymotrypsin were determined to elucidate the specificity of the inhibitors. In addition, the rate of the reaction of MeO-Suc-Ala-Ala-Pro-ValCH 2Cl with glutathione was measured as a model for non-specific alkylation reactions which might occur when chloromethyl ketones are used in physiological systems. MeO-Suc-Ala-Ala-Pro-ValCH 2Cl reacted over 1770 times faster with leukocyte elastase than with glutathione and would not inhibit cathepsin G. Z-Gly-Leu-PheCH 2Cl inhibited cathepsin G, but not leukocyte elastase. The results demonstrate that it possible to construct highly reactive and selective inhibitors for use in physiological situations.