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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM054995-06
Application #
6254768
Study Section
Pharmacology A Study Section (PHRA)
Program Officer
Okita, Richard T
Project Start
1997-02-01
Project End
2005-01-31
Budget Start
2001-02-01
Budget End
2002-01-31
Support Year
6
Fiscal Year
2001
Total Cost
$335,250
Indirect Cost
Name
University of Texas Medical Br Galveston
Department
Pharmacology
Type
Schools of Medicine
DUNS #
041367053
City
Galveston
State
TX
Country
United States
Zip Code
77555
Davydov, Dmitri R; Yang, Zhongyu; Davydova, Nadezhda et al. (2016) Conformational Mobility in Cytochrome P450 3A4 Explored by Pressure-Perturbation EPR Spectroscopy. Biophys J 110:1485-1498
Davydov, Dmitri R; Davydova, Nadezhda Y; Sineva, Elena V et al. (2015) Interactions among cytochromes P450 in microsomal membranes: oligomerization of cytochromes P450 3A4, 3A5, and 2E1 and its functional consequences. J Biol Chem 290:3850-64
Müller, Christian S; Knehans, Tim; Davydov, Dmitri R et al. (2015) Concurrent cooperativity and substrate inhibition in the epoxidation of carbamazepine by cytochrome P450 3A4 active site mutants inspired by molecular dynamics simulations. Biochemistry 54:711-21
Davydov, Dmitri R; Sineva, Elena V; Davydova, Nadezhda Y et al. (2013) CYP261 enzymes from deep sea bacteria: a clue to conformational heterogeneity in cytochromes P450. Biotechnol Appl Biochem 60:30-40
Sineva, Elena V; Rumfeldt, Jessica A O; Halpert, James R et al. (2013) A large-scale allosteric transition in cytochrome P450 3A4 revealed by luminescence resonance energy transfer (LRET). PLoS One 8:e83898
Davydov, Dmitri R; Davydova, Nadezhda Y; Sineva, Elena V et al. (2013) Pivotal role of P450-P450 interactions in CYP3A4 allostery: the case of ?-naphthoflavone. Biochem J 453:219-30
Davydov, Dmitri R; Rumfeldt, Jessica A O; Sineva, Elena V et al. (2012) Peripheral ligand-binding site in cytochrome P450 3A4 located with fluorescence resonance energy transfer (FRET). J Biol Chem 287:6797-809
Shimshoni, Jakob A; Roberts, Arthur G; Scian, Michele et al. (2012) Stereoselective formation and metabolism of 4-hydroxy-retinoic Acid enantiomers by cytochrome p450 enzymes. J Biol Chem 287:42223-32
Roberts, Arthur G; Yang, Jing; Halpert, James R et al. (2011) The structural basis for homotropic and heterotropic cooperativity of midazolam metabolism by human cytochrome P450 3A4. Biochemistry 50:10804-18
Fernando, Harshica; Rumfeldt, Jessica A O; Davydova, Nadezhda Y et al. (2011) Multiple substrate-binding sites are retained in cytochrome P450 3A4 mutants with decreased cooperativity. Xenobiotica 41:281-9

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