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

Public Health Relevance

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/

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM054995-17
Application #
8227984
Study Section
Special Emphasis Panel (NSS)
Program Officer
Okita, Richard T
Project Start
1997-02-01
Project End
2015-02-28
Budget Start
2012-03-01
Budget End
2013-02-28
Support Year
17
Fiscal Year
2012
Total Cost
$545,752
Indirect Cost
$176,376
Name
University of California San Diego
Department
None
Type
Schools of Pharmacy
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
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
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
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
Davydov, Dmitri R (2011) Microsomal monooxygenase as a multienzyme system: the role of P450-P450 interactions. Expert Opin Drug Metab Toxicol 7:543-58
Kumar, Santosh (2010) Engineering cytochrome P450 biocatalysts for biotechnology, medicine and bioremediation. Expert Opin Drug Metab Toxicol 6:115-31
Davydov, Dmitri R; Sineva, Elena V; Sistla, Srinivas et al. (2010) Electron transfer in the complex of membrane-bound human cytochrome P450 3A4 with the flavin domain of P450BM-3: the effect of oligomerization of the heme protein and intermittent modulation of the spin equilibrium. Biochim Biophys Acta 1797:378-90
Sineva, Elena V; Davydov, Dmitri R (2010) Cytochrome P450 from Photobacterium profundum SS9, a piezophilic bacterium, exhibits a tightened control of water access to the active site. Biochemistry 49:10636-46
Sun, Hao; Moore, Chad; Dansette, Patrick M et al. (2009) Dehydrogenation of the indoline-containing drug 4-chloro-N-(2-methyl-1-indolinyl)-3-sulfamoylbenzamide (indapamide) by CYP3A4: correlation with in silico predictions. Drug Metab Dispos 37:672-84

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