The long-term objective of the proposed research is to determine the mechanistic basis for the atypical kinetics of substrate oxidation by human cytochromes P450 of the 3A subfamily. CYP3A4 is the major P450 in adult human liver and intestine, where it represents on average between 30 and 50% of the total P450. Because of the large number of therapeutic agents and environmental chemicals that can induce CYP3A4 expression and/or modulate the activity of the enzyme, a very significant potential for adverse drug reactions exists. Development of in vitro methods for predicting the pharmacokinetics and drug interaction potential of CYP3A4 substrates is often complicated by the non-Michaels Menten kinetics observed. Substantial work from this and other laboratories during the prior award periods has provided compelling evidence that such atypical results from simultaneous occupancy of a single active site by multiple ligands. At present, however, it is difficult to rationalize or predict interactions between two CYP3A4 substrates based on knowledge of their individual kinetic properties. Thus, a ligand can activate, inhibit, or have no effect on CYP3A4 depending on the particular substrate used in the catalytic assay and the specific product measured. In addition, the kinetic parameters, degree of cooperativity, and regioselectivity are influenced by such factors as NADPH-cytochrome P450 reductase, cytochrome b5, divalent cations, phospholipids, and salts. The central hypothesis is that conformational changes resulting from ligand binding and/or protein-protein interactions play a key role in atypical kinetics of CYP3A4-catalyzed oxidations. This will be tested by a variety of biophysical approaches including high-pressure perturbation spectroscopy, rapid kinetics, and fluorescence resonance energy transfer along with steady-state kinetics and binding assays.
The specific aims are to: 1) elucidate the role of conformational heterogeneity of CYP3A4 in the mechanisms of cooperativity; 2) probe the involvement of oligomerization of CYP3A4 in the mechanisms of cooperativity; 3) determine the basis of altered cooperativity in available active-site mutants of CYP3A4 and new mutants to be created by directed evolution. The mechanistic information obtained about the binding of substrates and modulators to CYP3A4 should provide the intellectual framework required for rational assessment of inhibition and drug interaction potential, and thereby enhance drug discovery and therapy.
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