The long-term objective of the research described in this proposal is to elucidate the structural basis for the substrate specificity and cooperativity of human cytochromes P450 3A. These enzymes are very versatile catalysts and play a crucial role in the metabolism of a wide variety of compounds of pharmacological and toxicological interest. CYP3A4 is the most highly expressed P450 in the liver of most humans, is responsible for the metabolism of more clinically used drugs than any other P450, and is the locus of numerous serious drug-drug interactions. CYP3A5 is expressed in the liver of approximately one in four individuals. 3A4 and 3A5 exhibit 84 percent amino acid sequence identity and metabolize many of the same substrates. However, each enzyme produces a distinct pattern of metabolites of certain drugs such as cyclosporin A and midazolam. An intriguing question is how these enzymes can accept so many structurally diverse substrates yet exhibit remarkable regio- and stereoselectivity towards a single compound. CYP3A4 and 3A5 also exhibit positive cooperativity with certain substrates, which manifests itself as autoactivation (homotropic cooperativity) or activation by a second compound, such as alpha-naphthoflavone (heterotropic cooperativity). In other cases, two substrates can be accommodated by CYP3A4 with no apparent effect on each others' metabolism. Results generated during the current award period have allowed us to identify many of the amino acid residues responsible for substrate specificity and cooperativity of CYP3A4. The central hypothesis of the proposed studies is that atypical interactions (activation, partial inhibition, no inhibition) between two CYP3A4 substrates reflect simultaneous occupancy of two or more preferred locations within a single large binding pocket. This will be tested by a combination of site-directed mutagenesis functional analysis with a variety of substrates and effectors, nuclear magnetic resonance (NMR) spectroscopy, and 3-D molecular modeling.
The Specific Aims are to: 1) determine the structural basis for homotropic and heterotropic cooperativity of CYP3A4; 2) determine the structural basis for oxidation of prototypical drug substrates by human CYP3A enzymes; 3) determine substrate orientation in the CYP3A4 active site by NMR; 4) determine the structural basis for CYP3A inhibition by selected compounds. Knowledge of the molecular basis of human P450 3A function should allow the prediction of substrates, activators, and inhibitors of these enzymes, making it possible to minimize drug-drug interactions and interindividual differences in drug metabolism.
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