Utility of Microtiter Plate Assays for Human Cytochrome P450 Inhibition Studies in Drug Discovery: Application of Simple Method for Detecting Quasi-irreversible and Irreversible Inhibitors

Abstract

  In this study, a simple in vitro method for detecting human P450 (CYP) quasi-irreversible and irreversible inhibitors was evaluated. For the method, cDNA-expressed CYPs were applied to microtiter plate assays, CYP inhibitors were co-incubated with fluorometric substrates, and IC₅₀ were continuously measured (without stopping enzyme reactions). The typical reversible inhibitors (sulfaphenazole, tranylcypromine, quinidine, ketoconazole) showed constant IC₅₀ throughout the reaction. In contrast, the typical quasi-irrversible inhibitors (isosafrole, erythromycin, troleandomycin, diltiazem) and the typical irreversible inhibitors (furafylline, propranolol, mifepristone) showed time-dependent decreases in IC₅₀. For CYP3A4 inhibition studies, two substrates, 7-benzyloxyresorufin (BzRes) and 7-benzyloxy-4-trifluoromethyl-coumarin (BFC), were used. The IC₅₀ of the CYP3A4 inhibitors were dependent on the substrate. However, the quasi-irreversible and irreversible inhibitors could be detected by examining changes in the IC₅₀, regardless of the substrate. Further, the detection method was applied to josamycin and bergamottin. Josamycin did not show definite time-dependent decreases in IC₅₀ for CYP 3A4, suggesting that josamycin is neither a quasi-irrversible nor an irreversible inhibitor of CYP3A4. On the other hand, bergamottin showed time-dependent decreases in IC₅₀ for CYP1A2, CYP 2C9, CYP 2C19, CYP 2D6 and CYP 3A4, suggesting that bergamottin is a quasi-irrversible or an irreversible inhibitor of the 5 CYP isoforms. This method provides more rapid and reliable detection of quasi-irreversible and irreversible inhibitors and may be useful in drug discovery.