Testosterone, 7-benzyloxyquinoline, and 7-benzyloxy-40trifluoromethyl-coumarin bind to different domains within the active site of cytochrome P450 3A4

Testosterone, 7-benzyloxyquinoline, and 7-benzyloxy-4-trifluoromethyl-couma rin, marker substrates for cytochrome P450 3A4 are commonly used within the pharmaceutical industry to screen new chemical entities as inhibitors of C YP3A4 in a high-throughput manner to predict the potential for drug-drug in teractions. However, it has been observed that inhibition data obtained wit h a given CYP3A4 probe substrate may not correlate well with results from a different probe. As a consequence, the choice of the probe compound become s an important consideration in such screens. In the present study, kinetic interactions between either two of the above three substrates were evaluat ed, and three-dimensional nonlinear regression analysis was performed to un derstand the kinetic mechanisms of drug interaction. Our results demonstrat e that the kinetic interaction between each pair of substrates does not app ear to be competitive and that the interactions are characterized by an unc hanged or a decrease in both apparent K-m (a = 0.21-0.72, a change of K-m i n the absence of the effector) and V-max (alpha and beta = 0.09-0.75, chang es of V-max in the absence of the effector). These data suggest that 1) the three substrates bind to different domains; 2) at least two substrates can coexist in the active site of CYP3A4; and 3) the two bound substrates inte ract kinetically with each other (e.g., through steric hindrance), thereby leading to a change in both apparent kinetic parameters and partial inhibit ion. Selection of multiple substrates, which are shown not to be competitiv e, is necessary to accurately predict CYP3A4 inhibition and the potential f or drug-drug interaction.