Activation of procarcinogens by human cytochrome P450 enzymes

Enzymatic transformation of most chemical carcinogens is requisite to the formation of electrophiles that cause genotoxicity, and the cytochrome P450 (P450) enzymes are the most prominent enzymes involved in such activation reactions. During the past 15 years the human P450 enzymes have been extensively characterized. Considerable evidence exists that the variation in activity of these enzymes can have important consequences in the actions of drugs. Other studies have been concerned with the activation of procarcinogens by human P450s. Assignments of roles of particular P450s in the metabolism of chemical carcinogens are discussed, along with the current state of evidence for relationships of particular P450s with human cancer.     

Characterization of human cytochrome P450 enzymes.

Many biochemical approaches have been applied to the human cytochrome P450 enzymes, and more than 20 different gene products have been characterized with regard to their properties and catalytic specificities. The complement of the various cytochrome P450 enzymes in a given individual varies markedly, and dramatic differences may be seen in drug metabolism, pharmacological response, and susceptibility to toxic effects. An understanding of the nature of the individual cytochrome P450 enzymes and their regulation should be useful in determining the most suitable animal models, ascertaining risk from chemicals, and in avoiding undesirable drug interactions.     

Thermophilic cytochrome P450 enzymes

Thermophilic cytochrome P450 enzymes are of potential interest from structural, mechanistic, and biotechnological points of view. The structures and properties of two such enzymes, CYP119 and CYP175A1, have been investigated and provide the foundation for future work on thermophilic P450 enzymes.     

Targeting cytochrome P450 enzymes: a new approach in anti-cancer drug development

Cytochrome P450s (CYPs) represent a large class of heme-containing enzymes that catalyze the metabolism of multitudes of substrates both endogenous and exogenous. Until recently, however, CYPs have been largely overlooked in cancer drug development, acknowledged only for their role in phase I metabolism of chemotherapeutics. The first successful strategy targeting CYP enzymes in cancer therapy was the development of potent inhibitors of CYP19 (aromatase) for the treatment of breast cancer. Aromatase inhibitors ushered in a new era in hormone ablation therapy for estrogen dependent cancers, and have paved the way for similar strategies (i.e., inhibition of CYP17) that combat androgen dependent prostate cancer. Identification of CYPs involved in the inactivation of anti-cancer metabolites of vitamin D(3) and vitamin A has triggered development of agents that target these enzymes as well. The discovery of the over-expression of exogenous metabolizing CYPs, such as CYP1B1, in cancer cells has roused interest in the development of inhibitors for chemoprevention and of prodrugs designed to be activated by CYPs only in cancer cells. Finally, the expression of CYPs within tumors has been utilized in the development of bioreductive molecules that are activated by CYPs only under hypoxic conditions. This review offers the first comprehensive analysis of strategies in drug development that either inhibit or exploit CYP enzymes for the treatment of cancer.     

Oxidation of indole by cytochrome P450 enzymes

Indole is a product of tryptophan catabolism by gut bacteria and is absorbed into the body in substantial amounts. The compound is known to be oxidized to indoxyl and excreted in urine as indoxyl (3-hydroxyindole) sulfate. Further oxidation and dimerization of indoxyl leads to the formation of indigoid pigments. We report the definitive identification of the pigments indigo and indirubin as products of human cytochrome P450 (P450)-catalyzed metabolism of indole by visible, (1)H NMR, and mass spectrometry. P450 2A6 was most active in the formation of these two pigments, followed by P450s 2C19 and 2E1. Additional products of indole metabolism were characterized by HPLC/UV and mass spectrometry. Indoxyl (3-hydroxyindole) was observed as a transient product of P450 2A6-mediated metabolism; isatin, 6-hydroxyindole, and dioxindole accumulated at low levels. Oxindole was the predominant product formed by P450s 2A6, 2E1, and 2C19 and was not transformed further. A stable end product was assigned the structure 6H-oxazolo[3,2-a:4, 5-b']diindole by UV, (1)H NMR, and mass spectrometry, and we conclude that P450s can catalyze the oxidative coupling of indoles to form this dimeric conjugate. On the basis of these results, we propose that the P450/NADPH-P450 reductase system can catalyze oxidation of indole to a variety of products.     

Recombinant human cytochrome P450 monooxygenases for drug metabolite synthesis

Cytochrome P450 monooxygenases (CYPs) are important enzymes in the metabolism of xenobiotics. Therefore, several approaches to clone and overexpress the human isoforms have been made. In addition to microsomes or S9 preparations, these recombinant human isoforms have found diverse application in drug development. We discuss and give examples of the use of bacterial whole cell systems with rec. human CYPs for the preparative scale synthesis of drug metabolites. Biotechnol. Bioeng. 2010;106: 699–706. © 2010 Wiley Periodicals, Inc.     

Complex reactions catalyzed by cytochrome P450 enzymes

Cytochrome P450 (P450) enzymes are some of the most versatile redox proteins known. The basic P450 reactions include C-hydroxylation, heteroatom oxygenation, heteroatom release (dealkylation), and epoxide formation. Mechanistic explanations for these reactions have been advanced. A number of more complex P450 reactions also occur, and these can be understood largely in the context of the basic chemical mechanisms and subsequent rearrangements. The list discussed here updates a 2001 review and includes chlorine oxygenation, aromatic dehalogenation, formation of diindole products, dimer formation via Diels-Alder reactions of products, ring coupling and also ring formation, reductive activation (e.g., aristolochic acid), ring contraction (piperidine nitroxide radical), oxidation of troglitazone, cleavage of amino oxazoles and a 1,2,4-oxadiazole ring, bioactivation of a dihydrobenzoxathiin, and oxidative aryl migration.     

Inhibition of human drug metabolizing cytochrome P450 enzymes by plant isoquinoline alkaloids

The human cytochrome P450 (CYP) enzymes play a major role in the metabolism of endobiotics and numerous xenobiotics including drugs. Therefore it is the standard procedure to test new drug candidates for interactions with CYP enzymes during the preclinical development phase. The purpose of this study was to determine in vitro CYP inhibition potencies of a set of isoquinoline alkaloids to gain insight into interactions of novel chemical structures with CYP enzymes. These alkaloids (n = 36) consist of compounds isolated from the Papaveraceae family (n = 20), synthetic analogs (n = 15), and one commercial compound. Their inhibitory activity was determined towards all principal human drug metabolizing CYP enzymes: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4. All alkaloids were assayed in vitro in a 96-well plate format using pro-fluorescent probe substrates and recombinant human CYP enzymes. Many of these alkaloids inhibited the CYP3A4 form, with 30/36 alkaloids inhibiting CYP3A4 with at least moderate potency (IC₅₀ < 10 μM) and 15/36 inhibiting CYP3A4 potently (IC₅₀ < 1 μM). Among them corydine, parfumine and 8-methyl-2,3,10,11-tetraethoxyberbine were potent and selective inhibitors for CYP3A4. CYP2D6 was inhibited with at least moderate potency by 26/34 alkaloids. CYP2C19 was inhibited by 15/36 alkaloids at least moderate potently, whereas CYP1A2, CYP2B6, CYP2C8, and CYP2C9 were inhibited to a lesser degree. CYP2A6 was not significantly inhibited by any of the alkaloids. The results provide initial structure-activity information about the interaction of isoquinoline alkaloids with major human xenobiotic-metabolizing CYP enzymes, and illustrate potential novel structures as CYP form-selective inhibitors.     

Sulfaphenazole derivatives as tools for comparing cytochrome P450 2C5 and human cytochromes P450 2Cs: identification of a new high affinity substrate common to those enzymes

The inhibitory effects of a series of sulfaphenazole (SPA) derivatives were studied on two modified forms of rabbit liver cytochrome P450 2C5 (CYP2C5), CYP2C5dH, and structurally characterized CYP2C5/3LVdH and compared to the previously described effects of these compounds on human CYP2C8, 2C9, 2C18, and 2C19. SPA and other negatively charged compounds that potently inhibit CYP2C9 had very little effect on CYP2C5dH, whereas neutral, N-alkylated derivatives exhibited IC50 values between 8 and 22 microM. One of the studied compounds, 4, that derives from SPA by replacement of its NH(2) substituent with a methyl group and by N-methylation of its sulfonamide moiety, acted as a good substrate for all CYP2Cs used in this study. Hydroxylation of the benzylic methyl of 4 is the major reaction catalyzed by all of these CYP2C proteins, whereas hydroxylation of the N-phenyl group of 4 was observed as a minor reaction. CYP2C5dH, 2C5/3LVdH, 2C9, 2C18, and 2C19 are efficient catalysts for the benzylic hydroxylation of 4, with K(m) values between 5 and 13 microM and k(cat) values between 16 and 90 min(-1). The regioselectivity observed for oxidation of 4 by CYP2C5/3LVdH was easily interpreted on the basis of the existence of two different binding modes of 4 characterized in the experimentally determined structure of the complexes of CYP2C5/3LVdH with 4 described in the following paper [Wester, M. R. et al. (2003) Biochemistry 42, 6370-6379].     

Metabolism of retinol and retinoic acid by human liver cytochrome P450IIC8

Liver microsomes obtained from nine subjects were found to metabolize retinol to polar metabolites, including 4-hydroxyretinol. In a reconstituted monooxygenase system containing human liver P450IIC8, retinol was converted to 4-hydroxyretinol and other polar metabolites, with a Km of 0.071 mM and a Vmax of 1.73 nmol/min/nmol P450. Neither P450IIC9 nor P450IIE1, two other purified human P450s, displayed significant retinol hydroxylase activity. Immunoblots performed with a monospecific antibody directed against human P450IIC8 revealed that appreciable amounts of this enzyme were present in human liver microsomes. The same antibody significantly inhibited retinol metabolism in liver microsomes and in the system reconstituted with P450IIC8. The system reconstituted with P450IIC8 also converted retinoic acid to polar metabolites. Thus, this study shows, for the first time, metabolism of two physiologic substrates by a human liver cytochrome P450 related to a group of "constitutive" rodent P450s believed to participate in the metabolism of endogenous compounds. Through its involvement in vitamin A metabolism, P450IIC8 may participate in maintaining the balance between those vitamin A concentrations that promote cellular integrity (and oppose the development of cancer) and those concentrations that cause cellular toxicity.     

Dimemorfan N-demethylation by mouse liver microsomal cytochrome P450 enzymes

Dimemorfan (d-3-methyl-N-methylmorphinan), an analogue of dextromethorphan, is commonly used as a non-opioid antitussive. To clarify the contribution of cytochrome P450 (P450) in dimemorfan N-demethylation, effects of selective inducers and inhibitors were studied in ICR mice. Phenobarbital (PB)- and dexamethasone (Dex)-treatments caused 5-fold increases of liver microsomal dimemorfan N-demethylation activity. In untreated mouse liver microsomes, demethylation activity was strongly inhibited by a CYP3A inhibitor, ketoconazole. In PB-and Dex-treated mouse liver microsomes, ketoconazole caused strong inhibition, whereas orphenadrine caused a decrease of less than 20%. Pretreatment of control mouse liver microsomes with anti-CYP3A inhibited demethylation activity, whereas pre-treatment with anti-CYP2B had no effect. In PB-and Dex-treated mouse liver microsomes, the demethylation activity was inhibited by both anti-CYP3A and anti-CYP2B. In control mice, the intrinsic clearance of dimemorfan from N-demethylation was 5.8 microl min(-1)mg protein(-1). In PB- and Dex-treated mice, the correlation coefficient of fitting using one-enzyme and two-enzyme models were similar. The intrinsic clearances of induced mouse liver microsomes were similar. These results revealed that CYP3A played a major role in hepatic demethylation in untreated mice. Both CYP3A and CYP2B were involved in this demethylation in PB- and Dex-treated mice.     

Use of bioconjugation with cytochrome P450 enzymes

Bioconjugation, defined as chemical modification of biomolecules, is widely employed in biological and biophysical studies. It can expand functional diversity and enable applications ranging from biocatalysis, biosensing and even therapy. This review summarizes how chemical modifications of cytochrome P450 enzymes (P450s or CYPs) have contributed to improving our understanding of these enzymes. Genetic modifications of P450s have also proven very useful but are not covered in this review. Bioconjugation has served to gain structural information and investigate the mechanism of P450s via photoaffinity labeling, mechanism-based inhibition (MBI) and fluorescence studies. P450 surface acetylation and protein cross-linking have contributed to the investigation of protein complexes formation involving P450 and its redox partner or other P450 enzymes. Finally, covalent immobilization on polymer surfaces or electrodes has benefited the areas of biocatalysis and biosensor design. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.     

Probing the world of cytochrome P450 enzymes

Variability in drug response can be attributed in part to variability in the activity of drug-metabolizing enzymes. One of the most important drugmetabolizing enzyme systems in humans is the cytochrome P450 (CYP) enzyme family, which is responsible for the oxidative metabolism of numerous endogenous compounds and xenobiotics. The clinical relevance of factors that influence CYP-mediated metabolism can be appreciated by estimating in vivo enzyme activity (i.e., the phenotype) through the use of "probe drugs," which are drugs predominately or exclusively metabolized by an individual CYP enzyme. Thus, the use of probe drugs alone or in combination (i.e., the cocktail approach) can provide an invaluable tool to explore the clinical relevance of genetic and nongenetic factors that affect CYP enzyme activity and thereby contribute substantially to variability in response to therapeutic drugs.     

Characterization of the alkane-inducible cytochrome P450 (P450alk) gene from the yeast Candida tropicalis: identification of a new P450 gene family

The P450alk gene, which is inducible by the assimilation of alkane in Candida tropicalis, was sequenced and characterized. Structural features described in promoter and terminator regions of Saccharomyces yeast genes are present in the P450alk gene and some particular structures are discussed for their possible role in the inducibility of this gene. Expression of the P450alk gene was achieved in Saccharomyces cerevisiae using the yeast alcohol dehydrogenase expression system after removal of the P450alk gene flanking regions. The resultant expressed protein had a molecular mass slightly greater than that of P450alk from C. tropicalis. This alteration did not prevent the function and the localization of P450alk expressed in S. cerevisiae, as this organism showed an acquired microsome-bound activity for the terminal hydroxylation of lauric acid. The deduced P450alk amino acid sequence was compared with members of the nine known P450 gene families. These comparisons indicated that P450alk had a low relationship with these members and was therefore the first member (A1) of a new P450 gene family (LII).     

Distinction between human cytochrome P450 (CYP) isoforms and identification of new phosphorylation sites by mass spectrometry

In mammals, Cytochrome P450 (CYP) enzymes are bound to membranes of the endoplasmic reticulum and mitochondria, where they are responsible for the oxidative metabolism of many xenobiotics as well as organic endogenous compounds. In humans, 57 isoforms were identified which are classified based on sequence homology. In the present work, we demonstrate the performance of a mass spectrometry-based strategy to simultaneously detect and differentiate distinct human Cytochrome P450 (CYP) isoforms including the highly similar CYP3A4, CYP3A5, CYP3A7, as well as CYP2C8, CYP2C9, CYP2C18, CYP2C19, and CYP4F2, CYP4F3, CYP4F11, CYP4F12. Compared to commonly used immunodetection methods, mass spectrometry overcomes limitations such as low antibody specificity and offers high multiplexing possibilities. Furthermore, CYP phosphorylation, which may affect various biochemical and enzymatic properties of these enzymes, is still poorly analyzed, especially in human tissues. Using titanium dioxide resin combined with tandem mass spectrometry for phosphopeptide enrichment and sequencing, we discovered eight human P450 phosphorylation sites, seven of which were novel. The data from surgical human liver samples establish that the isoforms CYP1A2, CYP2A6, CYP2B6, CYP2E1, CYP2C8, CYP2D6, CYP3A4, CYP3A7, and CYP8B1 are phosphorylated in vivo. These results will aid in further investigation of the functional significance of protein phosphorylation for this important group of enzymes.     

DNA-expressed human cytochrome P450s: a new age of molecular toxicology and human risk assessment

It has long been recognized that a large degree of species differences exists among drug and carcinogen metabolizing enzymes. In particular, differences in cytochrome P450s, the principal enzymes of metabolic activation of procarcinogens, are widespread and may determine species and individual susceptibility to cancer causing chemicals. Although species differences in both the regulation and catalytic activities of P450s are quite large, roden-based systems are mainly used as the means to determine the degree of hazard of environmental pollutants, pesticides, drugs and other environmental chemicals to humans. During recent years, a large effort has been expended on analyzing directly the structure, properties and catalytic activities of P450s from human tissues. In vitro mutagen testing systems, based on activation by human P450s, are being developed that will supplement other test systems in order to more accurately predict human risk to chemical exposure.     

Isolation of a new human fetal liver cytochrome P450 cDNA clone: evidence for expression of a limited number of forms of cytochrome P450 in human fetal livers

From a human fetal liver cDNA library, a new cDNA clone (lambda HFL10) was isolated using an antiserum to P450 HFLa, which has been isolated from livers of human fetuses. Cytochrome P450 cDNAs, namely lambda hPA6, lamda hP2-1, and lambda hPD4 which were highly homologous to cDNA clones, pHY13, Hp1-1, and phP450j, respectively, were also isolated from the cDNA library of human adult livers. Using these cDNA clones as probes together with Lambda HFL10, Northern blot analysis was conducted to determine whether all of these cytochromes were expressed in human fetal livers. The results clearly showed that only P450 HFL10 mRNA was detected in human fetal livers. This result supports the allegation that there is a much more limited number of forms of cytochrome P450 in human fetal livers than in adult livers.     

Oxidation of human cytochrome P450 1A2 substrates by Bacillus megaterium cytochrome P450 BM3

Cytochrome P450 enzymes (P450s or CYPs) are good candidates for biocatalysis in the production of fine chemicals, including pharmaceuticals. Despite the potential use of mammalian P450s in various fields of biotechnology, these enzymes are not suitable as biocatalysts due to their low stability, low catalytic activity, and limited availability. Recently, wild-type and mutant forms of bacterial P450 BM3 (CYP102A1) from Bacillus megaterium have been found to metabolize various. It has therefore been suggested that CYP102A1 may be used to generate the metabolites of drugs and drug candidates. In this report, we show that the oxidation reactions of typical human CYP1A2 substrates (phenacetin, ethoxyresorufin, and methoxyresorufin) are catalyzed by both wild-type and mutant forms of CYP102A1. In the case of phenacetin, CYP102A1 enzymes show only O-deethylation product, even though two major products are produced as a result of O-deethylation and 3-hydroxylation reactions by human CYP1A2. Formation of the metabolites was confirmed by HPLC analysis and LC–MS to compare the metabolites with the actual biological metabolites produced by human CYP1A2. The results demonstrate that CYP102A1 mutants can be used for cost-effective and scalable production of human CYP1A2 drug metabolites. Our computational findings suggest that a conformational change in the cavity size of the active sites of the mutants is dependent on activity change. The modeling results further suggest that the activity change results from the movement of several specific residues in the active sites of the mutants.