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: N-(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: KI of 6.1 µM and kinact of 0.22 min-1 for MS and KI of 2.5 µM and kinact of 0.05 min-1 for OD. The partition ratios for MS and OD were ∼10 and ∼20, respectively. Inactivation of CYP2J2 by MS or OD resulted in a loss of the native heme spectrum and a similar decrease in the reduced CO difference spectrum. A heme adduct was observed in the MS-inactivated CYP2J2. The possible reactive metabolite which covalently modified the prosthetic heme was characterized by analysis of the glutathione conjugates formed by MS or OD following oxygenation of the ethynyl moiety. Liquid chromatography-mass spectrometry showed that inactivation by MS or OD did not lead to modification of apoprotein. Interaction of CYP2J2 with DZ produced a type II binding spectrum with a Ks of 2.8 µM and the IC50 for loss of OHEB carboxylation activity was 0.18 µM. In conclusion, heme modification by MS and OD was responsible for the mechanism-based inactivation of CYP2J2. The results suggest that the ethynyl moiety of MS and OD faces the heme iron, whereas the isoxazole ring of DZ is preferentially oriented toward the heme iron of CYP2J2.

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. Fatty acid amide hydrolase (FAAH) is a potential candidate because it is upregulated in IBD. FAAH hydrolyzes and, as a consequence, inactivates anandamide (AEA), a prominent endocannabinoid. Inhibition of FAAH would lead to increases in the amount of AEA oxidized by cytochrome P450s (P450s). CYP2J2, the major P450 epoxygenase expressed in the heart, is also expressed in the intestine and has previously been reported to oxidize AEA. We have investigated the possibility that it may play a role in AEA metabolism in the gut and have demonstrated that purified human CYP2J2 metabolizes AEA to form the 20-hydroxyeicosatetraenoic acid ethanolamide (HETE-EA) and several epoxygenated products, including the 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid ethanolamides (EET-EAs), in the reconstituted system. Kinetic studies suggest that the KM values for these products range from approximately 10 to 468 µM and the kcat values from 0.2 to 23.3 pmol/min per picomole of P450. Human intestinal microsomes, which express CYP2J2, metabolize AEA to give the 5,6-, 8,9-, and 11,12-EET-EAs, as well as 20-HETE-EA. Studies using specific P450 inhibitors suggest that although CYP2J2 metabolizes AEA, it is not the primary P450 responsible for AEA metabolism in human intestines.

Oxidation of the endogenous cannabinoid arachidonoyl ethanolamide by the cytochrome P450 monooxygenases: physiological and pharmacological implications.

Arachidonoyl ethanolamide (anandamide) is an endogenous amide of arachidonic acid and an important signaling mediator of the endocannabinoid system. Given its numerous roles in maintaining normal physiological function and modulating pathophysiological responses throughout the body, the endocannabinoid system is an important pharmacological target amenable to manipulation directly by cannabinoid receptor ligands or indirectly by drugs that alter endocannabinoid synthesis and inactivation. The latter approach has the possible advantage of more selectivity, thus there is the potential for fewer untoward effects like those that are traditionally associated with cannabinoid receptor ligands. In that regard, inhibitors of the principal inactivating enzyme for anandamide, fatty acid amide hydrolase (FAAH), are currently in development for the treatment of pain and inflammation. However, several pathways involved in anandamide synthesis, metabolism, and inactivation all need to be taken into account when evaluating the effects of FAAH inhibitors and similar agents in preclinical models and assessing their clinical potential. Anandamide undergoes oxidation by several human cytochrome P450 (P450) enzymes, including CYP3A4, CYP4F2, CYP4X1, and the highly polymorphic CYP2D6, forming numerous structurally diverse lipids, which are likely to have important physiological roles, as evidenced by the demonstration that a P450-derived epoxide of anandamide is a potent agonist for the cannabinoid receptor 2. The focus of this review is to emphasize the need for a better understanding of the P450-mediated pathways of the metabolism of anandamide, because these are likely to be important in mediating endocannabinoid signaling as well as the pharmacological responses to endocannabinoid-targeting drugs.

C331A mutant of neuronal nitric-oxide synthase is labilized for Hsp70/CHIP (C terminus of HSC70-interacting protein)-dependent ubiquitination.

It is established that suicide inactivation of neuronal nitric-oxide synthase (nNOS) by drugs and other xenobiotics leads to ubiquitination and proteasomal degradation of the enzyme. The exact mechanism is not known, although it is widely thought that the covalent alteration of the active site during inactivation triggers the degradation. A mechanism that involves recognition of the altered nNOS by Hsp70 and its cochaperone CHIP, an E3-ubiquitin ligase, has been proposed. To further address how alterations of the active site trigger ubiquitination of nNOS, we examined a C331A nNOS mutant, which was reported to have impaired ability to bind L-arginine and tetrahydrobiopterin. We show here that C331A nNOS is highly susceptible to ubiquitination by a purified system containing ubiquitinating enzymes and chaperones, by the endogenous ubiquitinating system in reticulocyte lysate fraction II, and by intact HEK293 cells. The involvement of the altered heme cleft in regulating ubiquitination is confirmed by the finding that the slowly reversible inhibitor of nNOS, N(G)-nitro-L-arginine, but not its inactive D-isomer, protects the C331A nNOS from ubiquitination in all these experimental systems. We also show that both Hsp70 and CHIP play a major role in the ubiquitination of C331A nNOS, although Hsp90 protects from ubiquitination. Thus, these studies further strengthen the link between the mobility of the substrate-binding cleft and chaperone-dependent ubiquitination of nNOS. These results support a general model of chaperone-mediated protein quality control and lead to a novel mechanism for substrate stabilization based on nNOS interaction with the chaperone machinery.

tert-Butylphenylacetylene is a potent mechanism-based inactivator of cytochrome P450 2B4: inhibition of cytochrome P450 catalysis by steric hindrance.

We have demonstrated that 4-(tert-butyl)-phenylacetylene (tBPA) is a potent mechanism-based inactivator for cytochrome P450 2B4 (P450 2B4) in the reconstituted system. It inactivates P450 2B4 in a NADPH- and time-dependent manner with a K(I) of 0.44 microM and k(inact) of 0.12 min(-1). The partition ratio was approximately zero, indicating that inactivation occurs without the reactive intermediate leaving the active site. Liquid chromatography-mass spectrometry analyses revealed that tBPA forms a protein adduct with a 1:1 stoichiometry. Peptide mapping of the tBPA-modified protein provides evidence that tBPA is covalently bound to Thr302. This is consistent with results of molecular modeling that show the terminal carbon of the acetylenic group is only 3.65 A away from Thr302. To characterize the effect of covalent modification of Thr302, tBPA-modified P450 2B4 was purified to homogeneity from the reconstituted system. The Soret band of tBPA-modified protein is red-shifted by 5 to 422 nm compared with unmodified protein. Benzphetamine binding to the modified P450 2B4 causes no spin shift, indicating that substrate binding and/or the heme environment has been altered by covalently bound tBPA. Cytochrome P450 reductase reduces the unmodified and tBPA-modified P450s at approximately the same rate. However, addition of benzphetamine stimulates the rate of reduction of unmodified P450 2B4 by approximately 20-fold but only marginally stimulates reduction of the tBPA-modified protein. This large discrepancy in the stimulation of the first electron transfer by benzphetamine strongly suggests that the impairment of P450 catalysis is due to inhibition of benzphetamine binding to the tBPA-modified P450 2B4.

Targeting Iron Homeostasis in Acute Kidney Injury.

Iron is an essential metal involved in several major cellular processes required to maintain life. Because of iron's ability to cause oxidative damage, its transport, metabolism, and storage is strictly controlled in the body, especially in the small intestine, liver, and kidney. Iron plays a major role in acute kidney injury and has been a target for therapeutic intervention. However, the therapies that have been effective in animal models of acute kidney injury have not been successful in human beings. Targeting iron trafficking via ferritin, ferroportin, or hepcidin may offer new insights. This review focuses on the biology of iron, particularly in the kidney, and its implications in acute kidney injury.

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.

Cocaine modulates locomotion behavior in C. elegans.

Cocaine, a potent addictive substance, is an inhibitor of monoamine transporters, including DAT (dopamine transporter), SERT (serotonin transporter) and NET (norepinephrine transporter). Cocaine administration induces complex behavioral alterations in mammals, but the underlying mechanisms are not well understood. Here, we tested the effect of cocaine on C. elegans behavior. We show for the first time that acute cocaine treatment evokes changes in C. elegans locomotor activity. Interestingly, the neurotransmitter serotonin, rather than dopamine, is required for cocaine response in C. elegans. The C. elegans SERT MOD-5 is essential for the effect of cocaine, consistent with the role of cocaine in targeting monoamine transporters. We further show that the behavioral response to cocaine is primarily mediated by the ionotropic serotonin receptor MOD-1. Thus, cocaine modulates locomotion behavior in C. elegans primarily by impinging on its serotoninergic system.