Mechanism-based inactivation of cytochromes P450 2B1 and P450 2B6 by n-propylxanthate

n-Propylxanthate (nPX) inactivated the 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) O-deethylation activity of purified, reconstituted rat hepatic P450 2B1 or human P450 2B6 in a mechanism-based manner. The inactivation followed pseudo-first-order kinetics and was entirely dependent on both NADPH and nPX. The maximal rate constant for inactivation of P450 2B1 at 30 degrees C was 0.2 min-1. The apparent KI was 44 microM, and the half-time for inactivation was 4.1 min. Purified, reconstituted human P450 2B6 was also inactivated by nPX with a KI of 12 microM. The kinactivation for P450 2B6 was 0.06 min-1, and the t1/2 was 11 min. Incubations of P450 2B1 with nPX and NADPH for 20 min resulted in a 75% loss in enzymatic activity and a concurrent 25% loss of the enzyme's ability to form a reduced CO complex. Little loss in the absolute spectrum of nPX-inactivated P450 2B1 was observed. With P450 2B6, an 83% loss in enzymatic activity and a 12% loss in the CO-reduced spectra were observed. The extrapolated partition ratio for nPX with P450 2B1 was 32. P450 2B1 could be protected from inactivation by nPX by adding an alternate substrate to the reaction mixture. Removal of unbound nPX by dialysis did not reverse the inactivation. The alternate oxidant iodosobenzene was able to partially restore enzymatic activity to nPX-inactivated P450 2B1 samples. A stoichiometry for labeling of 1.2:1 for binding of radiolabeled nPX metabolite to P450 2B1 was seen. These results indicated that nPX inactivated P450 2B1 and P450 2B6 in a mechanism-based manner. P450 2B1 was inactivated primarily by a nPX reactive intermediate that bound to the apoprotein.     

Induction of cytochromes P450

Humans and rodents are exposed to many foreign compounds in their diet (e.g., herbal supplements such as St. John's wart), in their environment (e.g., organochlorine pesticides and polychorinated biphenyls), and as clinically prescribed drugs (e.g., rifampin and phenobarbital). In response to these exposures mammals have evolved mechanisms to induce proteins involved in xenobiotic detoxification. Metabolism by Phase I enzymes, particularly the heme containing monooxygenases cytochromes P450 is frequently the first line of defense against such xenobiotics.     

Induction of cytochromes P450

The induction of cytochromes P450 (CYPs) has been appreciated for some time but an understanding of the mechanisms involved has been poorly understood until recently. The discovery of the role of nuclear receptors such as the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) has provided a major trigger for research in this area. This work has provided an explanation for species differences in hepatic induction. The production of a PXR crystal structure in the presence and absence of known high affinity ligands has offered the possibility of predicting structures which may bind to the receptor and hence act as inducing agents in man. An improvement in the technology of hepatocyte culture, access to good quality human hepatocytes and the miniaturisation of cultured preparations has meant that the potential of this technique to predict induction in man has been realised. Molecular biological techniques have also proved essential in both the science and the quantitation of CYP induction. The use of transient transfection cell based systems coupled with reporter gene assays have meant that dose response curves can be generated for many chemicals. Assays have been developed to measure the increase of the corresponding CYP mRNAs in primary hepatocytes and some cell lines with a high degree of sensitivity and specificity (allowing the quantitation of closely related CYPs). Although CYP induction is not usually considered as a major drawback in drug development, the aim should be to eliminate or reduce the inducing effects of a new drug to a minimum. Thus, it is essential to increase our understanding of the complex mechanisms that regulate induction and to pay attention to both the dose and the physicochemical and structural properties of CYP inducing agents.     

Cholesterol-metabolizing cytochromes P450.

By catalyzing the first steps in different pathways of cholesterol degradation, cytochromes P450 (P450s) 7A1, 27A1, 11A1, and 46A1 play key roles in cholesterol homeostasis. CYP7A1 is a microsomal liver-specific enzyme that converts cholesterol to 7alpha-hydroxycholesterol. CYP27A1 is a ubiquitously expressed mitochondrial P450 that metabolizes cholesterol to 27-hydroxycholesterol. CYP11A1 also resides in mitochondria but is expressed mainly in steroidogenic tissues, where it catalyzes the conversion of cholesterol to pregnenolone. Finally, CYP46A1 is a brain-selective microsomal monooxygenase producing 24S-hydroxycholesterol from cholesterol. Catalytic efficiencies of cholesterol-metabolizing P450s vary significantly and probably reflect physiological requirements of different organs for the rate of cholesterol turnover. P450s 7A1, 27A1, 11A1, and 46A1 represent a unique system for elucidation of how different enzymes have adapted to fit their specific roles in cholesterol elimination. Studies of cholesterol-metabolizing P450s suggest that their activities could be modulated post-translationally and that they should also be considered as targets for regulation of cholesterol homeostasis.     

Pharmacogenetics of the cytochromes P450

The cytochromes P450 are a family of heme-containing proteins with a major role in the oxidation of both xenobiotics (including prescribed drugs) and endogenous compounds. There are at least 57 human P450s (termed isoforms) which are all encoded by separate genes but only 10 of these contribute to drug metabolism, with the major contribution coming from only 3 isoforms, CYP3A4, CYP2D6 and CYP2C9. It is now well recognised that most cytochrome P450 genes are subject to genetic polymorphism and that therefore some individuals have sequence changes present that result in the production of an enzyme with altered catalytic activity or give rise to abnormal gene expression. This article describes the range of genetic polymorphisms now known to occur in the drug metabolizing cytochromes P450 with particular reference to their functional effects and the influence of ethnic origin on the frequency of variant alleles. The relevance of the various polymorphisms to drug response and toxicity is considered as well as the possibility that genotype for these polymorphisms may be a determinant for "personalized prescribing" in the future.     

Comparative modelling of cytochromes P450

The superfamily of enzymes known as the cytochromes P450 (P450s) comprises a wide-ranging class of proteins with diverse functions. They are known, amongst other things, to be involved in the hormonal regulation of metabolism and reproduction, as well as having a major clinical significance through their association with diseases such as cancer, diabetes and hepatitis. Knowledge of the three-dimensional (3D) structure of a protein gives insight into its function. The 3D structures of P450s are therefore of considerable scientific interest. A number of high-resolution structures of P450s have been determined by X-ray crystallography and studies of these structures have provided valuable insights into the mechanism of these enzymes. Only one of these structures is mammalian and as yet there is no structural information on human P450s in the public domain. Until such a structure is solved it is necessary to employ alternative methods to gain structural insight into how human P450s perform their biological function. Here we report on the use of comparative modelling to predict the structure of human P450s based on knowledge of their amino acid sequences plus the 3D structures of other (not human) P450s. As an illustrative example of these techniques we have modelled the structure of P450 2C5 using five bacterial P450 structures as templates. We examine the importance of selecting suitable templates, obtaining a good amino acid sequence alignment, and evaluating the models generated. To improve the quality of the models an iterative cycle of sequence alignment, model building, and model evaluation is employed. The result is a model with excellent stereochemistry, good amino acid side chain environment properties, and a Calpha trace similar to the crystal structure.     

Pharmacophore modeling of cytochromes P450

Understanding the binding of ligands in the active site of a membrane-bound protein is difficult in the absence of a crystal structure. When these proteins are the enzymes involved in drug metabolism, it leaves little option but to use site-directed mutagenesis and in vitro studies to provide critical information relating to determinants of binding affinity. Pharmacophore models and three-dimensional quantitative structure-activity relationships have been used either alone or in combination with protein homology models to provide this information for cytochrome P450s. At present, their application has been directed to the major enzymes but this may escalate in future as more in vitro data are generated for other P450s. The following review outlines the methodologies and models as well as future prospects for applying these technologies to P450s in the hope that future drugs will be selected with increased metabolic stability and fewer incidences of undesirable drug-drug interactions.     

Binding of flavonoids to staphylococcal enterotoxin B

Staphylococcal enterotoxins are metabolic products of Staphylococcus aureus that are responsible for the second-most-commonly reported type of food poisoning. Polyphenols are known to interact with proteins to form complexes, the properties of which depend on the structures of both the polyphenols and the protein. In the present study, we investigated the binding of four flavonoid polyphenols to Staphylococcal enterotoxin B (SEB) at pH 7.5 and 25 °C: (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), kaempferol-3-glucoside (KAM-G) and kaempferol (KAM). Fluorescence emission spectrometry and molecular docking were applied to compare experimentally determined binding parameters with molecular modeling. EGCG showed an order of magnitude higher binding constant (1.4 × 10⁵ M⁻¹) than the other studied polyphenols. Our blind-docking results showed that EGCG and similar polyphenolic ligands is likely to bind to the channel at the surface of SEB that is responsible for the recognition of the T-cell beta chain fragment and influence the adhesion of SEB to T cells.     

Small intestinal cytochromes P450.

Small intestinal cytochromes P450 (P450) provide the principal, initial source of biotransformation of ingested xenobiotics. The consequences of such biotransformation are detoxification by facilitating excretion, or toxification by bioactivation. P450s occur at highest concentrations in the duodenum, near the pylorus, and at decreasing concentrations distally--being lowest in the ileum. Highest concentrations occur from midvillus to villous tip, with little or none occurring in the crypts of Lieberkuehn. Microsomal P4503A, 2C8-10, and 2D6 forms have been identified in human small intestine, and P450s 2B1, possibly 2B2, 2A1, and 3A1/2 were located in endoplasmic reticulum of rodent small intestine, while P4502B4 has been purified to electrophoretic homogeneity from rabbit intestine. Some evidence indicates a differential distribution of P450 forms along the length of the small intestine and even along the villus. Rat intestinal P450s are inducible by xenobiotics--with phenobarbital (PB) inducing P4502B1, 3-methylcholanthrene (3-MC) inducing P4501A1, and dexamethasone inducing two forms of P4503A. Induction is most effectively achieved by oral administration of the agents, and is rapid--aryl hydrocarbon hydroxylase (AHH) was increased within 1 h of administration of, for example, 3-MC. AHH, 7-ethoxycoumarin O-deethylase (ECOD), and 7-ethoxyresorufin O-deethylase (EROD) have been used most frequently as substrates to characterize intestinal P450s. Dietary factors affect intestinal P450s markedly--iron restriction rapidly decreased intestinal P450 to beneath detectable values; selenium deficiency acted similarly but was less effective; Brussels sprouts increased intestinal AHH activity 9.8-fold, ECOD activity 3.2-fold, and P450 1.9-fold; fried meat and dietary fat significantly increased intestinal EROD activity; a vitamin A-deficient diet increased, and a vitamin A-rich diet decreased intestinal P450 activities; and excess cholesterol in the diet increased intestinal P450 activity. The role of intestinal P450 in toxifying or detoxifying specific xenobiotics has been clearly demonstrated to only a limited extent. However, elevated intestinal P450 levels have been indirectly linked to gastrointestinal cancer. Intestinal metabolism of 2,2,2-trifluoroethanol produces intestinal lesions with consequent systemic bacterial infection.     

Characterization of orphan human cytochromes P450

Of the 57 human cytochromes P450 (P450) and 58 pseudogenes discovered to date, (http://drnelson.utmem.edu/CytochromeP450.html ), 1/4 still remain "orphans" in the sense that their function, expression sites, and regulation are still largely not elucidated. The post-human genome-sequencing project era has presented the research community with novel challenges. Despite many insights gathered about gene location and genetic variations in our human genome, we still lack important knowledge about these novel P450 enzymes and their functions in endogenous and exogenous metabolism, as well as their possible roles in the metabolism of toxicants and carcinogens. Our own list of such orphans currently consists of 13 members: P450 2A7, 2S1, 2U1, 2W1, 3A43, 4A22, 4F11, 4F22, 4V2, 4X1, 4Z1, 20A1, and 27C1. Some of the orphans, e.g. P450s 2W1 and 2U1, already have putative assigned functions in arachidonic acid metabolism and may activate carcinogens. However, at this point, for the majority of them more knowledge is available about their genes and single nucleotide polymorphisms than of their biological functions. It is noteworthy that most P450 orphans express high interspecies sequence conservation and have orthologs in rodents (e.g. CYP4X1/Cyp4x1, CYP4V2/Cyp4v3). This review summarizes recent knowledge about the P450 orphans and questions remaining about their specific roles in human metabolism.     

Inhibition of cytochromes P450 by antifungal imidazole derivatives.

The interactions of a panel of antifungal agents with cytochromes P450 (P450s), as a means of predicting potential drug-drug interactions, have not yet been investigated. The objective of this study was to evaluate the specificity and selectivity of five antifungal agents using selective probe reactions for each of the eight major P450s. The index reactions used were phenacetin O-deethylation (for CYP1A2), coumarin 7-hydroxylation (CYP2A6), diclofenac 4'-hydroxylation (CYP2C9), omeprazole 5-hydroxylation (CYP2C19), dextromethorphan O-demethylation (CYP2D6), 7-ethoxy-4-trifluoromethylcoumarin deethylation (CYP2B6), chlorzoxazone 6-hydroxylation (CYP2E1), and omeprazole sulfonation (CYP3A4). Five antifungal agents that include an imidazole moiety (clotrimazole, miconazole, sulconazole, tioconazole, and ketoconazole) were examined in cDNA-expressing microsomes from human lymphoblast cells or human liver microsomes. All inhibitors studied demonstrated nonselective inhibition of P450s. Ketoconazole seemed to be the most selective for CYP3A4, although it also inhibited CYP2C9. High-affinity inhibition was seen for CYP1A2 (sulconazole and tioconazole K(i), 0.4 microM), CYP2B6 (miconazole K(i), 0.05 microM; sulconazole K(i), 0.04 microM), CYP2C19 (miconazole K(i), 0.05 microM; sulconazole K(i), 0.008 microM; tioconazole K(i), 0.04 microM), CYP2C9 (sulconazole K(i), 0.01 microM), CYP2D6 (miconazole K(i), 0.70 microM; sulconazole K(i), 0.40 microM), CYP2E1 (tioconazole K(i), 0.4 microM), and CYP3A4 (clotrimazole K(i), 0.02 microM; miconazole K(i), 0.03 microM; tioconazole K(i), 0.02 microM). Therefore, this class of compounds is likely to result in significant drug-drug interactions in vivo.     

Regioselective hydroxylation of steroid hormones by human cytochromes P450

This article reviews in vitro metabolic activities [including Michaelis constants (K_m), maximal velocities (V_max) and V_max/K_m] and drug–steroid interactions [such as induction and cooperativity (activation)] of cytochromes P450 (P450 or CYP) in human tissues, including liver and adrenal gland, for 14 kinds of endogenous steroid compounds, including allopregnanolone, cholesterol, cortisol, cortisone, dehydroepiandrosterone, estradiol, estrone, pregnenolone, progesterone, testosterone and bile acids (cholic acid). First, we considered the drug-metabolizing P450s. 6β-Hydroxylation of many steroids, including cortisol, cortisone, progesterone and testosterone, was catalyzed primarily by CYP3A4. CYP1A2 and CYP3A4, respectively, are likely the major hepatic enzymes responsible for 2-/4-hydroxylation and 16α-hydroxylation of estradiol and estrone, steroids that can contribute to breast cancer risk. In contrast, CYP1A1 and CYP1B1 predominantly metabolized estrone and estradiol to 2- and 4-catechol estrogens, which are endogenous ultimate carcinogens if formed in the breast. Some metabolic activities of CYP3A4, including dehydroepiandrosterone 7β-/16α-hydroxylation, estrone 2-hydroxylation and testosterone 6β-hydroxylation, were higher than those for polymorphically expressed CYP3A5. Next, we considered typical steroidogenic P450s. CYP17A1, CYP19A1 and CYP27A1 catalyzed steroid synthesis, including hydroxylation at 17α, 19 and 27 positions, respectively. However, it was difficult to predict which hepatic drug-metabolizing P450 or steroidogenic P450 will be mainly responsible for metabolizing each steroid hormone in vivo based on these results. Further research is required on the metabolism of steroid hormones by various P450s and on prediction of their relative contributions to in vivo metabolism. The findings collected here provide fundamental and useful information on the metabolism of steroid compounds.     

Mechanistic enzymology of oxygen activation by the cytochromes P450

The P450 cytochromes represent a universal class of heme-monooxygenases. The detailed mechanistic understanding of their oxidative prowess is a critical theme in the studies of metabolism of a wide range of organic compounds including xenobiotics. Integral to the O2 bond cleavage mechanism by P450 is the enzyme's concerted use of protein and solvent-mediated proton transfer events to transform reduced dioxygen to a species capable of oxidative chemistry. To this end, a wide range of kinetic, structural, and mutagenesis data has been accrued. A critical role of conserved acid-alcohol residues in the P450 distal pocket, as well as stabilized waters, enables the enzyme to catalyze effective monooxygenation chemistry. In this review, we discuss the detailed mechanism of P450 dioxygen scission utilizing the CYP101 hydroxylation of camphor as a model system. The application of low-temperature radiolytic techniques has enabled a structural and spectroscopic analysis of the nature of critical intermediate states in the reaction.     

Mechanism-based inactivation of cytochromes P450 2B1 and P450 2B6 by 2-phenyl-2-(1-piperidinyl)propane.

2-Phenyl-2-(1-piperidinyl)propane (PPP), an analog of phencyclidine, was tested for its ability to inactivate cytochrome P450s (P450s) 2B1 and 2B6. PPP inactivated the 7-(benzyloxy)resorufin O-dealkylation activity of liver microsomes obtained from phenobarbital-induced rats with a K(I) of 11 microM. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of purified rat liver P450 2B1 and expressed human P450 2B6 was inactivated by PPP in a reconstituted system containing NADPH-cytochrome P450 reductase and lipid. In the presence of NADPH, the loss of activity was time- and concentration-dependent, and followed pseudo first order kinetics. The rate of inactivation for P450 2B1 was 0.3 min(-1), and the concentration of PPP required to achieve half-maximal inactivation was 12 microM. The time for 50% of the P450 2B1 to become inactivated at saturating concentrations of PPP was 2.5 min. P450 2B6 was inactivated with a k(inact) of 0.07 min(-1), a K(I) of 1.2 microM, and a t(1/2) of 9.5 min. The inactivated P450s 2B1 and 2B6 lost about 25 and 15%, respectively, of their ability to form a CO-reduced complex, suggesting that the loss of activity was caused by a PPP modification of the apoprotein rather than the heme. The estimated partition ratio for P450s 2B1 and 2B6 with PPP was 31 and 15, respectively. The inactivation was not reversible and reductase activity was not affected. Coincubation of P450 2B1 and 2B6 with PPP and NADPH in the presence of an alternate substrate protected both enzymes from inactivation. The exogenous nucleophile GSH did not affect the rate of inactivation. PPP-inactivated P450s 2B1 and 2B6 were recognized on Western blots by an antibody generated to phencyclidine that had been conjugated to BSA. Stoichiometries of 1.4:1 and 0.7:1 were determined for the binding of a [3H]PPP metabolite to P450 2B1 and 2B6, respectively.     

Metabolic activation of polycyclic aromatic hydrocarbons and other procarcinogens by cytochromes P450 1A1 and P450 1B1 allelic variants and other human cytochromes P450 in Salmonella typhimurium NM2009.

A variety of polycyclic aromatic hydrocarbons and their dihydrodiol derivatives, arylamines, heterocyclic amines, and nitroarenes, were incubated with cDNA-based recombinant (Escherichia coli or Trichoplusia ni) systems expressing different forms of human cytochrome P450 (P450 or CYP) and NADPH-P450 reductase using Salmonella typhimurium tester strain NM2009, and the resultant DNA damage caused by the reactive metabolites was detected by measuring expression of umu gene in the cells. Recombinant (bacterial) CYP1A1 was slightly more active than any of four CYP1B1 allelic variants, CYP1B1*1, CYP1B1*2, CYP1B1*3, and CYP1B1*6, in catalyzing activation of chrysene-1,2-diol, benz[a]anthracene-trans-1,2-, 3,4-, 5,6-, and 8,9-diol, fluoranthene-2,3-diol, dibenzo[a,l]pyrene, benzo[c]phenanthrene, and dibenz[a,h]anthracene and several arylamines and heterocyclic amines, whereas CYP1A1 and CYP1B1 enzymes had essentially similar catalytic specificities toward other procarcinogens, such as (+)-, (-)-, and (+/-)-benzo[a]pyrene-7,8-diol, 5-methylchrysene-1,2-diol, 7,12-dimethylbenz[a]anthracene-3,4-diol, dibenzo[a,l]pyrene-11,12-diol, benzo[b]fluoranthene-9,10-diol, benzo[c]chrysene, 5,6-dimethylchrysene-1,2-diol, benzo[c]phenanthrene-3,4-diol, 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene, 5-methylchrysene, and benz[a]anthracene. We also determined activation of these procarcinogens by recombinant (T. ni) human P450 enzymes in S. typhimurium NM2009. There were good correlations between activities of procarcinogen activation by CYP1A1 preparations expressed in E. coli and T. ni cells, although basal activities with three lots of CYP1B1 in T. ni cells were very high without substrates and NADPH in our assay system. Using 14 forms of human P450s (but not CYP1B1) (in T. ni cells), we found that CYP1A2, 2C9, 3A4, and 2C19 catalyzed activation of several of polycyclic aromatic hydrocarbons at much slower rates than those catalyzed by CYP1A1 and that other enzymes, including CYP2A6, 2B6, 2C8, 2C18, 2D6, 2E1, 3A5, 3A7, and 4A11, were almost inactive in the activation of polycyclic aromatic hydrocarbons examined here.     

The human drug metabolizing cytochromes P450

The superfamily of heme-thiolate proteins known as the cytochromes P450 is responsible for the oxidative metabolism of the majority of drugs. Thus, the phenotypes of individuals with respect to their levels of catalytically active cytochromes P450 determines to a large part the substantial interindividual variation observed in the metabolic clearance of drugs. Over the past 10 years 15 different human cytochromes P450 involved in drug metabolism have been isolated and characterized to varying degrees. This brief review discusses the characterization of these cytochromes P450 and how this knowledge has been used by the pharmaceutical industry to aid in the development of new drugs.