Molecular Basis of Human Cytochrome P450 3A Function

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Program Director/Principal Investigator (Last, First, Middle): Halpert, James R. PROJECT SUMMARY (See instructions): The long-term objective of the proposed research is to determine the mechanistic basis for the atypical kinetics of substrate oxidation by human CYP3A4, the major P450 in adult liver and intestine. This enzyme and the related CYP3A5 are of particular pharmacological and toxicological significance due to their ability to metabolize a vast array of therapeutic and environmental agents of diverse structures, sizes, and shapes. The non-Michaelis-Menten behavior exhibited with a number of substrates is a major confounding factor in vitro-in vivo extrapolations and predictions of drug-drug interactions. The central hypothesis is that CYP3A4 cooperativity reveals a true allosteric regulatory mechanism that involves modulation of the tertiary structure of the enzyme, its oligomerization, and interactions with redox partners. Building on new concepts and methodologies developed during the current award period, the molecular mechanisms of CYP3A4 cooperativity will be further tested by a variety of biophysical approaches including pressure perturbation spectroscopy, rapid kinetics, fluorescence resonance energy transfer, and time-resolved fluorescence spectroscopy along with steady-state kinetics and advanced binding assays. These approaches will be applied to purified CYP3A4wild-type, key active site mutants, and new mutants allowing site-directed incorporation of fluorescent probes. The knowledge of the mechanistic basis and physiological role of allosteric regulation of CYP3A4 will provide key information necessary for understanding the mechanisms of adverse drug effects and drug-drug interactions. The individual specific aims are: 1) To probe the molecular mechanisms of substrate- and effector-induced conformational transitions in CYP3A4 involved in the mechanisms of cooperativity 2) To investigate the modulation of protein-protein interactions of CYP3A4, including its oligomerization, by drug substrates and effectors 3) To probe the effect of reduced glutathione and other potential physiological effectors on cooperativity, catalytic efficiency, and functional heterogeneity of CYP3A4 in microsomes and model membranes RELEVANCE (See instructions): Cytochromes P450 3A break down a wide variety of compounds to which humans are exposed, including drugs, environmental contaminants, and industrial chemicals. The research will enable us to understand how P450s bind and metabolize compounds of very different chemical structure. The information gained will help avoid drug interactions and help predict individual response to medications. PROJECT/