Inhibition of porcine pancreatic elastase by 7-substituted 4-chloro-3-ethoxyisocoumarins: structural characteristics of modeled noncovalent complexes relate to the measured inhibition kinetics.

Kinetic measurements for the inhibition of porcine pancreatic elastase by 7-substituted 4-chloro-3-ethoxyisocoumarins were performed. To obtain possible explanations for the kinetic results, structures resulting from energy minimizations of inhibitor-enzyme complex structures where each inhibitor was initially positioned in 64 locations within the active site were obtained. In keeping with solution NMR studies, a positive-charged His-57 was employed. The number of low energy complex structures with Ser-195 O gamma-inhibitor benzoyl ester carbonyl carbon distances < or = 2.9 A, Ser-195 O gamma-inhibitor benzoyl ester carbonyl carbon-inhibitor benzoyl ester carbonyl oxygen angles > 91 degrees, and the inhibitor in the oxyanion hole exhibits a direct linear relationship to ln(Ki/k3). The proportion of those structures that show 7-substituent H-bonding between the inhibitor and porcine pancreatic elastase exhibits a direct relationship to k3. Assuming a direct linear relationship to ln(k3) and k3, finer differences in k3 than are experimentally observed are expected. The relationship with k3 and that with Ki/k3 are shown to be useful tools for the design of more potent 7-substituted 4-chloro-3-ethoxyisocoumarins. A novel inhibitor of this class (4-chloro-3-ethoxy-7-[(2-methyl-2- butylcarbamoyl)amino]isocoumarin) expected to be more potent is synthesized and tested. Its potency within experimental error is as predicted. Although the relationship observed with Ki/k3 involves only a twofold increase in Ki/k3 (a statistically significant increase), results with the novel inhibitor show the relationship to be valid over a four- to fivefold increase.