Atypical kinetic profiles observed with drug metabolizing enzymes, such as the cytochromes P450, can confound in vitro determinations of kinetic parameter estimates. During the development of new drug entities, inaccurate kinetic parameter estimates can lead to incorrect decisions as to the potential success of the drug compound. The long term goals of this research are to understand the biochemical mechanisms resulting in atypical kinetic profiles and develop computer models capable of predicting the occurrence of atypical kinetics. It is generally accepted that two (or more) substrate molecules can bind within the active site of cytochrome P450 enzymes and this phenomenon plays a role in atypical kinetics. NMR work from our laboratory has demonstrated two molecules present in the active site and the presence of an effector causes movement of the primary substrate closer to the heme. Increases in P450 reaction efficiency and reductions in reaction cycle uncoupling also have been observed during CYP2C9 heteroactivation, but preliminary data suggest that not all activator compounds act by the same mechanism. The structure-activity basis for these differential effects is unknown. More recently, we have observed that genetic variants of CYP2C9 exhibit greater degrees of heteroactivation than wild-type enzyme, suggesting that enzyme conformation may also be a factor in the observation of atypical kinetics. The current competing renewal builds upon the above findings and seeks to ascertain; a) the mechanism(s) by which effector molecules alter P450 reaction cycle efficiency and the structure-activity relationships of molecules causing these effects, b) the changes in substrate-heme iron distances and substrate orientation produced by different effectors, and c) how enzyme amino acid alterations affect substrate/effector-protein interactions and thus, activation. From these data, computer models will be developed to predict the occurrence of atypical kinetics and potential drug interactions to assist in the drug development process.
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