Bioactivation of xenobiotics to toxic intermediates through cytochrome P450 oxygenation mechanisms is a well recognized process. However, the production of electrophilic intermediates by several P450 enzymes (e.g. 1A2, 2B6, 2E1, 2F1, 3A4, and 4B1), through dehydrogenation pathways has only recently been investigated, and the mechanisms that govern selective dehydrogenation rather than oxygenation are not established. Several of the dehydrogenated intermediates are so reactive that they inactivate the P450 enzymes, generally through alkylation of active site nucleophilic residues. Research concerning the catalytic behavior of these specific P450 enzymes and their propensity to dehydrogenate rather than oxygenate substrates is vitally needed. The hypothesis of this research is: the unique catalytic mechanism(s) offacilitated electron transport that determines dehydrogenation by certain P450 enzymes results in xenobiotic-mediated injury and altered drug metabolism in humans. The specific goals of this application are to determine the characteristics of the enzyme active-site environment that direct dehydrogenation mechanisms of specific cytochrome P450 enzymes, and to define the substrate structural features that regulate selective dehydrogenation rather than oxygenation. These goals will be realized through the following aims: 1) To determine the structures of the reactive intermediates that are produced by dehydrogenation of prototypical substrates, and characterize enzyme preferences for dehydrogenation vs. oxygenation of the substrates; 2) To characterize the mechanisms of inactivation of each P450 enzyme by its specific inactivator; 3) To define the active-site parameters that control the mechanisms of dehydrogenation and bioactivation of toxicants by P450 enzymes; and 4) To use the dehydrogenation substrates that covalently modify the P450 apoproteins to elucidate critical active-site residues that direct the dehydrogenation mechanism, or that control inhibitor/substrate access channels, binding, or product release. The enzyme/substrate pairs are CYP2F3/3-methylindole, CYP3A4/zafirlukast, CYP2El/capsaicin, and CYP2B6/tamoxifen. The long-term goals of this research are to elucidate the mechanisms of cytochrome P450-mediated dehydrogenation of xenobiotics in processes that generate toxic electrophilic intermediates, to assess the potential harm engendered by these toxic intermediates to human health, and to utilize mechanistic information to predict dehydrogenation, and concomitant toxicities and/or enzyme inactivation (altered drug metabolism), of new drugs and xenobiotics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM074249-03
Application #
7338664
Study Section
Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
Program Officer
Okita, Richard T
Project Start
2006-01-01
Project End
2009-12-31
Budget Start
2008-01-01
Budget End
2008-12-31
Support Year
3
Fiscal Year
2008
Total Cost
$281,619
Indirect Cost
Name
University of Utah
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
009095365
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
Jia, Kunzhi; Li, Lei; Liu, Zhihua et al. (2014) Generation and characterization of a novel CYP2A13--transgenic mouse model. Drug Metab Dispos 42:1341-8
Roberts, Jessica K; Moore, Chad D; Ward, Robert M et al. (2013) Metabolism of beclomethasone dipropionate by cytochrome P450 3A enzymes. J Pharmacol Exp Ther 345:308-16
Moore, Chad D; Roberts, Jessica K; Orton, Christopher R et al. (2013) Metabolic pathways of inhaled glucocorticoids by the CYP3A enzymes. Drug Metab Dispos 41:379-89
Reilly, Christopher A; Henion, Fred; Bugni, Tim S et al. (2013) Reactive intermediates produced from the metabolism of the vanilloid ring of capsaicinoids by p450 enzymes. Chem Res Toxicol 26:55-66
Wei, Yuan; Wu, Hong; Li, Lei et al. (2012) Generation and characterization of a CYP2A13/2B6/2F1-transgenic mouse model. Drug Metab Dispos 40:1144-50
Shahrokh, Kiumars; Cheatham 3rd, Thomas E; Yost, Garold S (2012) Conformational dynamics of CYP3A4 demonstrate the important role of Arg212 coupled with the opening of ingress, egress and solvent channels to dehydrogenation of 4-hydroxy-tamoxifen. Biochim Biophys Acta 1820:1605-17
Zhou, Xin; D'Agostino, Jaime; Li, Lei et al. (2012) Respective roles of CYP2A5 and CYP2F2 in the bioactivation of 3-methylindole in mouse olfactory mucosa and lung: studies using Cyp2a5-null and Cyp2f2-null mouse models. Drug Metab Dispos 40:642-7
Shahrokh, Kiumars; Orendt, Anita; Yost, Garold S et al. (2012) Quantum mechanically derived AMBER-compatible heme parameters for various states of the cytochrome P450 catalytic cycle. J Comput Chem 33:119-33
Behrendorff, James B Y H; Moore, Chad D; Kim, Keon-Hee et al. (2012) Directed evolution reveals requisite sequence elements in the functional expression of P450 2F1 in Escherichia coli. Chem Res Toxicol 25:1964-74
Moore, Chad D; Reilly, Christopher A; Yost, Garold S (2010) CYP3A4-Mediated oxygenation versus dehydrogenation of raloxifene. Biochemistry 49:4466-75

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