Assay of Endocannabinoid Oxidation by Cytochrome P450.

Cytochrome P450 enzymes are a large family of heme-containing proteins that have important functions in the biotransformation of xenobiotics, including pharmacologic and environmental agents, as well as endogenously produced chemicals with broad structural and functional diversity. Anandamide and 2-arachidonoylglycerol (2-AG) are substrates for P450s expressed in multiple tissues, leading to the production of a diverse set of mono- and di-oxygenated metabolites. This chapter describes tools and methods that have been used to identify major endocannabinoid metabolizing P450s and their corresponding products using subcellular tissue fractions, cultured cells, and purified recombinant enzymes in a reconstituted system.

Amphipol-facilitated elucidation of the functional tetrameric complex of full-length cytochrome P450 CYP2B4 and NADPH-cytochrome P450 oxidoreductase.

Interactions of membrane-bound mammalian cytochromes P450 (CYPs) with NADPH-cytochrome P450 oxidoreductase (POR), which are required for metabolism of xenobiotics, are facilitated by membrane lipids. A variety of membrane mimetics, such as phospholipid liposomes and nanodiscs, have been used to simulate the membrane to form catalytically active CYP:POR complexes. However, the exact mechanism(s) of these interactions are unclear because of the absence of structural information of full-length mammalian CYP:POR complexes in membranes.

The full-length cytochrome P450 enzyme CYP102A1 dimerizes at its reductase domains and has flexible heme domains for efficient catalysis.

The cytochrome P450 enzyme CYP102A1 from is a highly efficient hydroxylase of fatty acids, and there is a significant interest in using CYP102A1 for biotechnological applications. Here, we used size-exclusion chromatography-multiangle light scattering (SEC-MALS) analysis and negative-stain EM to investigate the molecular architecture of CYP102A1. The SEC-MALS analysis yielded a homogeneous peak with an average molecular mass of 235 ± 5 kDa, consistent with homodimeric CYP102A1.

Formation of Both Heme and Apoprotein Adducts Contributes to the Mechanism-Based Inactivation of Human CYP2J2 by 17-Ethynylestradiol.

17-Ethynylestradiol (EE), a major component of many oral contraceptives, affects the activities of a number of the human cytochrome P450 (P450) enzymes. Here, we characterized the effect of EE on CYP2J2, a major human P450 isoform that participates in metabolism of arachidonic acid. EE inactivated the hydroxyebastine carboxylation activity of CYP2J2 in a reconstituted system.

Functional characterization of the G162R and D216H genetic variants of human CYP17A1.

Cytochrome P450 17A1 (CYP17A1) is a dual-function enzyme catalyzing reactions necessary for cortisol and androgen biosynthesis. CYP17A1 is a validated drug target for prostate cancer as CYP17A1 inhibition significantly reduces circulating androgens and improves survival in castration-resistant prostate cancer. Germline CYP17A1 genetic variants with altered CYP17A1 activity manifesting as various endocrinopathies are extremely rare; however, characterizing these variants provides critical insights into CYP17A1 protein structure and function.

Heme Modification Contributes to the Mechanism-Based Inactivation of Human Cytochrome P450 2J2 by Two Terminal Acetylenic Compounds.

The mechanism-based inactivation of human CYP2J2 by three terminal acetylenic compounds: -(methylsulfonyl)-6-(2-propargyloxyphenyl)hexanamide (MS), 17-octadecynoic acid (OD), and danazol (DZ) was investigated. The loss of hydroxyebastine (OHEB) carboxylation activity in a reconstituted system was time- and concentration-dependent and required NADPH for MS and OD, but not DZ. The kinetic constants for the mechanism-based inactivation of OHEB carboxylation activity were: of 6.

Roles of Residues F206 and V367 in Human CYP2B6: Effects of Mutations on Androgen Hydroxylation, Mechanism-Based Inactivation, and Reversible Inhibition.

The crystal structures of human CYP2B6 indicate that Phe206 and Val367 are in close proximity to the substrate binding site and suggest that both residues may play important roles in substrate metabolism and inhibitor binding. To test this hypothesis, we investigated the effects of mutating these residues to Ala on the regiospecificity of CYP2B6 for the metabolism of testosterone and androstenedione. For testosterone metabolism, 16β-OH-testosterone formation by the F206A mutant was <5% of the wild type (WT), whereas the V367A mutant exhibited a doubling of 16α-OH-testosterone formation with a 50% decrease in 16β-OH-testosterone formation compared with the WT.

Significant Improvement of Antithrombotic Responses to Clopidogrel by Use of a Novel Conjugate as Revealed in an Arterial Model of Thrombosis.

Clopidogrel is a prodrug that requires bioactivation by cytochrome P450 (P450) enzymes to a pharmacologically active metabolite for antiplatelet action. The clinical limitations of clopidogrel are in large part due to its poor pharmacokinetics resulting from inefficient bioactivation by P450s. In this study, we determined the pharmacokinetics and pharmacodynamics of a novel conjugate of clopidogrel, referred to as ClopNPT, in animal models and we evaluated its potential to overcome the limitations of clopidogrel.

Assay of Endocannabinoid Oxidation by Cytochrome P450.

Cytochrome P450 enzymes are a large family of heme-containing proteins that have important functions in the biotransformation of xenobiotics, including pharmacologic and environmental agents, as well as of endogenously produced chemicals with broad structural and functional diversity. Anandamide and 2-arachidonoylglycerol (2-AG) are substrates for P450s expressed in multiple tissues, leading to the production of a diverse set of mono- and di-oxygenated metabolites. This chapter describes tools and methods that have been used to identify major endocannabinoid-metabolizing P450s and their corresponding products, by using subcellular tissue fractions, cultured cells, and purified recombinant enzymes in a reconstituted system.

Oxidative hemoglobin reactions: Applications to drug metabolism.

Hb is a protein with multiple functions, acting as an O2 transport protein, and having peroxidase and oxidase activities with xenobiotics that lead to substrate radicals. However, there is a lack of evidence for intermediates involved in these reactions of Hb with redox-active compounds, including those with xenobiotics such as drugs, chemical carcinogens, and sulfides. In particular, questions exist as to what intermediates participate in reactions of either metHb or oxyHb with sulfides.

Metabolism of Anandamide by Human Cytochrome P450 2J2 in the Reconstituted System and Human Intestinal Microsomes.

According to the Centers for Disease Control and Prevention, the incidence of inflammatory bowel diseases (IBD) is about 1 in 250 people in the United States. The disease is characterized by chronic or recurring inflammation of the gut. Because of the localization of the endocannabinoid system in the gastrointestinal tract, it may be a potential pharmacologic target for the treatment of IBD and other diseases.

Mechanism-Based Inactivation of Human Cytochrome P450 2B6 by Chlorpyrifos.

Chlorpyrifos (CPS) is a commonly used pesticide which is metabolized by P450s into the toxic metabolite chlorpyrifos-oxon (CPO). Metabolism also results in the release of sulfur, which has been suggested to be involved in mechanism-based inactivation (MBI) of P450s. CYP2B6 was previously determined to have the greatest catalytic efficiency for CPO formation in vitro.

Hydroxylation and N-dechloroethylation of Ifosfamide and deuterated Ifosfamide by the human cytochrome p450s and their commonly occurring polymorphisms.

The hydroxylation and N-dechloroethylation of deuterated ifosfamide (d4IFO) and ifosfamide (IFO) by several human P450s have been determined and compared. d4IFO was synthesized with deuterium at the alpha and alpha' carbons to decrease the rate of N-dechloroethylation and thereby enhance hydroxylation of the drug at the 4' position. The purpose was to decrease the toxic and increase the efficacious metabolites of IFO.

CYP-independent inhibition of platelet aggregation in rabbits by a mixed disulfide conjugate of clopidogrel.

Dual antiplatelet therapy with clopidogrel and aspirin has been the standard of care in the United States for patients with acute coronary syndromes (ACS) and/or undergoing percutaneous coronary interventions (PCI). However, the effectiveness of clopidogrel varies significantly among different sub-populations due to inter-individual variability. In this study we examined the antiplatelet potential of a novel mixed disulfide conjugate of clopidogrel with the aim to overcome the inter-individual variability.

The effect of ritonavir on human CYP2B6 catalytic activity: heme modification contributes to the mechanism-based inactivation of CYP2B6 and CYP3A4 by ritonavir.

The mechanism-based inactivation of human CYP2B6 by ritonavir (RTV) in a reconstituted system was investigated. The inactivation is time, concentration, and NADPH dependent and exhibits a K(I) of 0.9 μM, a k(inact) of 0.

Role of the highly conserved threonine in cytochrome P450 2E1: prevention of H2O2-induced inactivation during electron transfer.

A highly conserved threonine in the I-helix of cytochrome P450s has been suggested to play an important role in dioxygen activation, a critical step for catalytic turnover. However, subsequent studies with some P450s in which this highly conserved threonine was replaced by another residue such as alanine showed that significant catalytic activities were still retained when the variants were compared with the wild type enzymes. These results make the role of this residue unclear.

High-throughput fluorescence assay for cytochrome P450 mechanism-based inactivators.

The mechanism-based inactivation (MBI) of the human cytochrome P450 (P450 or CYP) drug-metabolizing enzymes may lead to adverse drug-drug interactions, especially for drugs with narrow therapeutic windows. Unlike reversible inhibitors of P450, drug-drug interactions originating from MBI may persist in patients for some time after the body eliminates the offending drug because P450 enzymatic activity can be recovered only after de novo synthesis of the P450. In a pharmaceutical setting, a substantial amount of effort is often expended to understand the potential for mechanism-based inactivation and its possible contribution to the drug-drug interaction profile of drug candidates.

The inactivation of human CYP2E1 by phenethyl isothiocyanate, a naturally occurring chemopreventive agent, and its oxidative bioactivation.

Phenethylisothiocyanate (PEITC), a naturally occurring isothiocyanate and potent cancer chemopreventive agent, works by multiple mechanisms, including the inhibition of cytochrome P450 (P450) enzymes, such as CYP2E1, that are involved in the bioactivation of carcinogens. PEITC has been reported to be a mechanism-based inactivator of some P450s. We describe here the possible mechanism for the inactivation of human CYP2E1 by PEITC, as well as the putative intermediate that might be involved in the bioactivation of PEITC.

Formation, reactivity, and antiplatelet activity of mixed disulfide conjugates of clopidogrel.

In this work, we investigated the formation, reactivity, and antiplatelet activity of various mixed disulfide conjugates of clopidogrel. Our results showed that the production of the active metabolite (AM) from 2-oxoclopidogrel by human liver microsomes (HLMs) is greatly affected by the thiol reductants used. Among the 10 thiol compounds tested, glutathione (GSH) is most efficient in producing the AM at a rate of 167 pmoles AM/min/mg HLM.

Potent mechanism-based inactivation of cytochrome P450 2B4 by 9-ethynylphenanthrene: implications for allosteric modulation of cytochrome P450 catalysis.

The mechanism-based inactivation of cytochrome P450 2B4 (CYP2B4) by 9-ethynylphenanthrene (9EP) has been investigated. The partition ratio and k(inact) are 0.2 and 0.

Interactions between CYP2E1 and CYP2B4: effects on affinity for NADPH-cytochrome P450 reductase and substrate metabolism.

Studies in microsomal and reconstituted systems have shown that the presence of one cytochrome P450 isoform can significantly influence the catalytic activity of another isoform. In this study, we assessed whether CYP2E1 could influence the catalytic activity of CYP2B4 under steady-state turnover conditions. The results show that CYP2E1 inhibits CYP2B4-mediated metabolism of benzphetamine (BNZ) with a K(i) of 0.

Reaction of human cytochrome P450 3A4 with peroxynitrite: nitrotyrosine formation on the proximal side impairs its interaction with NADPH-cytochrome P450 reductase.

The reaction of peroxynitrite (PN) with purified human cytochrome P450 3A4 (CYP3A4) resulted in the loss of the reduced-CO difference spectrum, but the absolute absorption spectrum of the heme was not significantly altered. The loss of 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) O-debenzylation activity of CYP3A4 was concentration-dependent with respect to PN, and the loss of BFC activity supported by NADPH-cytochrome P450 reductase (CPR) was much greater than that supported by tert-butyl hydroperoxide. Moreover, the PN-treated CYP3A4 exhibited a reduced-CO spectrum when reduced by CPR that was much smaller than when it was reduced by dithionite.