ABSTRACT:Silybin, a major constituent of the milk thistle, is used to treat several liver disorders. Silybin inactivated purified, recombinant cytochromes P450 (P450) 3A4 and 2C9 in a mechanism-based manner. The inactivations were time-, concentration-, and NADPHdependent. The inactivation of the 7-benzyloxy-4-(trifluoromethyl-)coumarin O-debenzylation activity (P450 3A4) was characterized by a K I of 32 M, a k inact of 0.06 min ؊1 , and a t 1/2 of 14 min.Testosterone metabolism to 6-␤-hydroxytestosterone (P450 3A4) was also inactivated with a K I of 166 M, a k inact of 0.08 min ؊1, and a t 1/2 of 9 min. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of purified human P450 2C9 was inactivated with a K I of 5 M, a k inact of 0.14 min ؊1, and a t 1/2 of 7 min. Parallel loss of heme was observed with both P450s. Activity of both P450 enzymes was not recovered after removal of silybin either by dialysis or by spin gel filtration. In addition, silybin inhibited the glucuronidation of 7-hydroxy-4-trifluoromethylcoumarin catalyzed by recombinant hepatic UDP-glucuronosyltransferases (UGTs) 1A1, 1A6, 1A9, 2B7, and 2B15, with IC 50 values of 1.4 M, 28 M, 20 M, 92 M, and 75 M, respectively. Silybin was a potent inhibitor of UGT1A1 and was 14-and 20-fold more selective for UGT1A1 than for UGT1A9 and UGT1A6, respectively. Thus, careful administration of silybin with drugs primarily cleared by P450s 3A4 or 2C9 is advised, since drug-drug interactions cannot be excluded. The clinical significance of in vitro UGT1A1 inhibition is unknown.

The P450 type cytochromes are responsible for the metabolism of a wide variety of xenobiotics and endogenous compounds. Although P450-catalyzed reactions are generally thought to lead to detoxication of xenobiotics, the reactions can also produce reactive intermediates that can react with cellular macromolecules leading to toxicity or that can react with the P450s that form them leading to irreversible (i.e., mechanism-based) inactivation. This perspective describes the fundamentals of mechanism-based inactivation as it pertains to P450 enzymes. The experimental approaches used to characterize mechanism-based inactivators are discussed, and the criteria required for a compound to be classified as a mechanism-based inactivator are outlined. The kinetic scheme for mechanism-based inactivation and the calculation of the relevant kinetic constants that describe a particular inactivation event are presented. The structural aspects and important functional groups of several classes of molecules that have been found to impart mechanism-based inactivation upon metabolism by P450s such as acetylenes, thiol-containing compounds that include isothiocyanates, thiazolidinediones, and thiophenes, arylamines, quinones, furanocoumarins, and cyclic tertiary amines are described. Emphasis throughout this perspective is placed on more recent findings with human P450s where the site of modification, whether it be the apoprotein or the heme moiety, and, at least in part, the identity of the reactive intermediate responsible for the loss in P450 activity are known or inferred. Recent advances in trapping procedures as well as new methods for identification of reactive intermediates are presented. A variety of clinically important drugs that act as mechanism-based inactivators of P450s are discussed. The irreversible inactivation of human P450s by these drugs has the potential for causing serious drug-drug interactions that may have severe toxicological effects. The clinical significance of inactivating human P450s for improving drug efficacy as well as drug safety is discussed along with the potential for exploiting mechanism-based inactivators of P450s for therapeutic benefits.

Bergamottin (BG), a component of grapefruit juice, is a mechanism-based inactivator of cytochromes P450 (P450) 2B6 and 3A5 in the reconstituted system. The inactivation of both P450s was NADPH-dependent and irreversible. The kinetic constants for the inactivation of the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of P450 2B6 were: K I , 5 M; k inact , 0.09 min Ϫ1 ; and t 1/2 , 8 min. The kinetic constants obtained for the inactivation of the testosterone 6␤-hydroxylation activity of P450 3A5 were: K I , 20 M; k inact , 0.045 min Ϫ1 ; and t 1/2 , 15 min. Incubations of P450s 2B6 and 3A5 with 20 M BG at 37°C for 20 min resulted in an ϳ60% loss in the catalytic activity that was accompanied by a significant loss in intact heme and a similar decrease in the reduced CO difference spectrum. The extrapolated partition ratios for BG with P450s 2B6 and 3A5 were ϳ2 and ϳ20, respectively. Liquid chromatography-mass spectroscopy analysis of the BG-inactivated samples showed that the mass of the inactivated apoprotein had increased by approximately 388 Da for both P450 2B6 and P450 3A5. SDSpolyacrylamide gel electrophoresis analysis demonstrated that [ 14 C]BG was irreversibly bound to the apoprotein in the BGinactivated samples. The stoichiometry of binding was ϳ0.5 mol BG metabolite/mol of each P450 inactivated. High-pressure liquid chromatography analysis of the metabolites of BG showed that P450 2B6 generated two major metabolites, whereas P450 3A5 generated three additional metabolites. Two of metabolites were identified as 6Ј,7Ј-dihydroxybergamottin and bergaptol.

The cytochromes P450 superfamily of enzymes is a group of hemeproteins that catalyze the metabolism of an extensive series of compounds including drugs, chemical carcinogens, fatty acids, and steroids. They oxidize substrates ranging in size from ethylene to cyclosporin. Although significant efforts have been made to obtain structural information on the active sites of the microbial P450s, relatively little is currently known regarding the identities of the critical amino acid residues in the P450 active sites that are involved in substrate binding and catalysis. Since information on the crystal structures of the eukaryotic P450s has been relatively limited, investigators have used a variety of other techniques in attempts to elucide the structural features that play a role in the catalytic properties and substrate specificity at the enzyme active site. These include site-directed mutagenesis, natural mutations, homology modeling, mapping with aryl-iron complexes, affinity and photoaffinity labeling, and mechanism-based inactivators. A variety of different mechanism-based inactivators have proven to be useful in identifiying active site amino acid residues involved in substrate binding and catalysis. In this review we present a sampling of the types of studies that can be conducted using mechanism-based inactivators and highlight studies with several classes of compounds including acetylenes, isothiocyanates, xanthates, aminobenzotriazoles, phencyclidine, and furanocoumarins. Labeled peptides isolated from the inactivated proteins have been analyzed by N-terminal amino acid sequencing in conjunction with mass spectrometry to determine the sites of covalent modification. Mechanistic studies aimed at identifying the basis for the inactivation following adduct formation are also presented.

17␣-Ethynylestradiol (EE), a major constituent of many oral contraceptives, inactivated the testosterone 6␤-hydroxylation activity of purified P450 3A4 reconstituted with phospholipid and NADPH-cytochrome P450 reductase in a mechanismbased manner. The inactivation of P450 3A4 followed pseudo first order kinetics and was dependent on NADPH. The values for the K I and k inact were 18 M and 0.04 min Ϫ1 , respectively, and the t 1/2 was 16 min. Incubation of 50 M EE with P450 3A4 at 37°C for 30 min resulted in a 67% loss of testosterone 6␤-hydroxylation activity accompanied by a 35% loss of the spectral absorbance of the native protein at 415 nm and a 70% loss of the spectrally detectable P450-CO complex. The inactivation of P450 3A4 by EE was irreversible. Testosterone, an alternate substrate, was able to protect P450 3A4 from EEdependent inactivation. The partition ratio was ϳ50. (HPLC) analysis demonstrated that the inactivation resulting from EE metabolism led to the destruction of approximately half the heme with the concomitant generation of modified heme and EE-labeled heme fragments and produced covalently radiolabeled P450 3A4 apoprotein. Electrospray mass spectrometry demonstrated that the fraction corresponding to the major radiolabeled product of EE metabolism has a mass (M Ϫ H) Ϫ of 479 Da. HPLC and gas chromatography-mass spectometry analyses revealed that EE metabolism by P450 3A4 generated one major metabolite, 2-hydroxyethynylestradiol, and at least three additional metabolites. In conclusion, our results demonstrate that EE is an effective mechanism-based inactivator of P450 3A4 and that the mechanism of inactivation involves not only heme destruction, but also the irreversible modification of the apoprotein at the active site.

The endocannabinoid anandamide is an arachidonic acid derivative that is found in most tissues where it acts as an important signaling mediator in neurological, immune, cardiovascular, and other functions. Cytochromes P450 (P450s) are known to oxidize arachidonic acid to the physiologically active molecules hydroxyeicosatetraenoic acids (HETEs) and epoxyeicosatrienoic acids (EETs), which play important roles in blood pressure regulation and inflammation. To determine whether anandamide can also be oxidized by P450s, its metabolism by human liver and kidney microsomes was investigated. The kidney microsomes metabolized anandamide to a single monooxygenated product, which was identified as 20-HETE-ethanolamide (EA). Human liver microsomal incubations with anandamide also produced 20-HETE-EA in addition to 5,6-, 8,9-, 11-12, and 14,15-EET-EA. The EET-EAs produced by the liver microsomal P450s were converted to their corresponding dihydroxy derivatives by microsomal epoxide hydrolase. P450 4F2 was identified as the isoform that is most probably responsible for the formation of 20-HETE-EA in both human kidney and human liver, with an apparent K m of 0.7 M. The apparent K m values of the human liver microsomes for the formation of the EET-EAs were between 4 and 5 M, and P450 3A4 was identified as the primary P450 in the liver responsible for epoxidation of anandamide. The in vivo formation and biological relevance of the P450-derived HETE and EET ethanolamides remains to be determined.

ABSTRACT:The potent antioxidants licorice root extract and glabridin, an isoflavan purified from licorice root extract, were tested for their ability to modulate the activities of several cytochrome P450 (P450) enzymes. P450 3A4, the major human drug metabolizing P450 enzyme, was inactivated by licorice root extract and by glabridin in a time-and concentration-dependent manner. The inactivation was NADPH-dependent and was not reversible by extensive dialysis. Further analysis showed that the loss in enzymatic activity correlated with a loss in the P450-reduced CO spectrum and with a loss of the intact heme moiety. In contrast, incubations of P450 3A4 with similar concentrations of 2,4-dimethylglabridin and NADPH did not lead to inactivation of P450 3A4. P450 2B6 was also inactivated by glabridin in a time-and concentration-dependent manner. The majority of the glabridin-inactivated P450 2B6 was able to form a reduced CO spectrum suggesting that the heme was not modified with this isoform. High-performance liquid chromatography analysis of the P450 heme confirmed that incubations with glabridin and NADPH did not result in the destruction of the heme moiety. The activity of P450 2C9 was competitively inhibited by glabridin, whereas P450 2D6 and P450 2E1 were virtually unaffected. The data show that glabridin can serve as a substrate for at least three human P450 enzymes and that depending on the isoform, metabolism of glabridin can lead to mechanism-based inactivation or inhibition of the P450. Heme and reduced CO spectral analysis also indicated that glabridin inactivated P450s 2B6 and 3A4 by different mechanisms. Liver microsomal P4501 enzymes are involved in the metabolism of endogenous substrates such as fatty acids, cholesterol, and steroids. These enzymes also carry out an important function in the catalysis and ultimate clearance of many structurally distinct xenobiotics such as drugs, pesticides, carcinogens, and environmental pollutants (Porter and Coon, 1991; Rendic and DiCarlo, 1997). P450 3A4, the major hepatic and intestinal P450 enzyme in humans, metabolizes more than 50% of clinically used drugs such as cyclosporine A, dihydropyridines, ethynylestradiol, midazolam, terfenadine, and triazolam. On average, P450 2B6 comprises approximately 0.2% of human liver P450 and is responsible for the metabolism of roughly 3% of all drugs such as ketamine (Yanagihara et al., 2001), orphenadrine, secobarbital, phenobarbital, dexamethasone, and rifampin (Chang et al., 1997). The P450 2C family comprises approximately 18% of the P450 enzymes found in human liver and are responsible for the metabolism of at least 25% of all drugs including tolbutamide, diclofenac, (S)-warfarin, phenytoin, and hexobarbital.

17-␣-Ethynylestradiol (17EE) inactivated purified, reconstituted rat hepatic cytochrome P450 (P450) 2B1 and human P450 2B6 in a mechanism-based manner. Little or no inactivation was observed when P450s 2B2 or 2B4 were incubated with 17EE. The inactivation of P450s 2B1 and 2B6 was entirely dependent on both NADPH and 17EE and followed pseudo-first order kinetics. The maximal rate constants for the inactivation of P450s 2B1 and 2B6 at 30°C were 0.2 and 0.03 min Ϫ1 , respectively. For P450s 2B1 and 2B6 the apparent K I was 11 and 0.8 M, respectively. Incubation of P450 2B1 with 17EE and NADPH for 20 min resulted in a 75% loss in enzymatic activity and a concurrent 20 to 25% loss of the enzyme's ability to form a reduced CO complex. With P450 2B6, an 83% loss in enzymatic activity and a 5 to 10% loss in the CO reduced spectrum were observed. The extrapolated partition ratios for 17EE with P450 2B1 and 2B6 were 21 and 13, respectively. Simultaneous incubation of an alternate substrate together with 17EE protected both enzymes from inactivation. A 1.3:1 stoichiometry of labeling for binding of the radiolabeled 17EE to P450 2B1 and 2B6 was seen. These results indicate that 17EE inactivates P450s 2B1 and 2B6 in a mechanism-based manner, primarily by the binding of a reactive intermediate of 17EE to the apoprotein. Analysis of the 17EE metabolites showed that 2B enzymes that become inactivated differ primarily by their ability to generate two metabolites that were not produced by P450s 2B2 or 2B4.