| Abstract
| - A new method for identifying enzyme inhibitors is to conduct their synthesis in the presence ofthe targeted enzyme. Good inhibitors form in larger amounts than poorer ones because the binding eitherspeeds up synthesis (target-accelerated synthesis) or shifts the synthesis equilibrium (dynamic combinatoriallibraries). Several groups have successfully demonstrated this approach with simple systems, but applicationto larger libraries is challenging because of the need to accurately measure the amount of each inhibitor.In this report, we dramatically simplify this analysis by adding a reaction that destroys the unbound inhibitors.This works similar to a kinetic resolution, with the best inhibitor being the last one remaining. We demonstratethis method for a static library of several sulfonamide inhibitors of carbonic anhydrase. Four sulfonamide-containing dipeptides, EtOC-Phesa-Phe (4a), EtOC-Phesa-Gly (4b), EtOC-Phesa-Leu (4c) and EtOC-Phesa-Pro (4d), were prepared and their inhibition constants measured. These inhibitors migrated to the carbonicanhydrase compartment of a two-compartment vessel. Although higher concentrations of the better inhibitorswere observed in the carbonic anhydrase compartment, the concentration differences were small(1.83:1.71:1.54:1.46:1 for 4a:4b:4c:4d:5, where 5 is a noninhibiting dipeptide EtOC-Phe-Phe). Addition ofa protease rapidly cleaved the weaker inhibitors (4d and 5). Intermediate inhibitor 4c was cleaved at aslower rate, and at the end of the reaction, only 4a and 4b remained. In a separate experiment, the ratioof 4a to 4b was found to increase over time to a final ratio of nearly 4:1. This is greater than the ratio oftheir inhibition constants (approximately 2:1). The theoretical model predicts that these ratios would increaseeven further as the destruction proceeds. This removal of poorer inhibitors simplifies identification of thebest inhibitor in a complex mixture.
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