Using Nanodiscs, we quantitate the heterotropic interaction between two different drugs mediated by monomeric CYP3A4 incorporated into a nativelike membrane environment. The mechanism of this interaction is deciphered by global analysis of multiple-turnover experiments performed under identical conditions using the pure substrates progesterone (PGS) and carbamazepine (CBZ) and their mixtures. Activation of CBZ epoxidation and simultaneous inhibition of PGS hydroxylation are measured and quantitated through differences in their respective affinities for both a remote allosteric site and the productive catalytic site near the heme iron. Preferred binding of PGS at the allosteric site and a stronger preference for CBZ binding at the productive site give rise to a nontrivial drug-drug interaction. Molecular dynamics simulations indicate functionally important conformational changes caused by PGS binding at the allosteric site and by two CBZ molecules positioned inside the substrate binding pocket. Structural changes involving Phe-213, Phe-219, and Phe-241 are thought to be responsible for the observed synergetic effects and positive allosteric interactions between these two substrates. Such a mechanism is likely of general relevance to the mutual heterotropic effects caused by biologically active compounds that exhibit different patterns of interaction with the distinct allosteric and productive sites of CYP3A4, as well as other xenobiotic metabolizing cytochromes P450 that are also involved in drug-drug interactions. Importantly, this work demonstrates that a monomeric CYP3A4 can display the full spectrum of activation and cooperative effects that are observed in hepatic membranes. (Chemical Equation Presented).
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