Cytochrome P450 Enzymes and Drug Metabolism in Humans.
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ABSTRACT:
SUBMITTER: Zhao M
PROVIDER: S-EPMC8657965 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cytochrome P450 enzymes are found in all life forms. P450s play an important role in drug metabolism, and have potential uses as biocatalysts. Human P450s are membrane-bound proteins. However, the interactions between P450s and their membrane environment are not well-understood. To date, all P450 crystal structures have been obtained from engineered proteins, from which the transmembrane helix was absent. A significant number of computational studies have been performed on P450s, but the majority of these have been performed on the solubilised forms of P450s. Here we present a multiscale approach for modelling P450s, spanning from coarse-grained and atomistic molecular dynamics simulations to reaction modelling using hybrid quantum mechanics/molecular mechanics (QM/MM) methods. To our knowledge, this is the first application of such an integrated multiscale approach to modelling of a membrane-bound enzyme. We have applied this protocol to a key human P450 involved in drug metabolism: CYP3A4. A biologically realistic model of CYP3A4, complete with its transmembrane helix and a membrane, has been constructed and characterised. The dynamics of this complex have been studied, and the oxidation of the anticoagulant R-warfarin has been modelled in the active site. Calculations have also been performed on the soluble form of the enzyme in aqueous solution. Important differences are observed between the membrane and solution systems, most notably for the gating residues and channels that control access to the active site. The protocol that we describe here is applicable to other membrane-bound enzymes.
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Project description:Glyburide (GLB) is a widely used oral sulfonylurea for the treatment of gestational diabetes. The therapeutic use of GLB is often complicated by a substantial inter-individual variability in the pharmacokinetics and pharmacodynamics of the drug in human populations, which might be caused by inter-individual variations in factors such as GLB metabolism. Therefore, there has been a continued interest in identifying human cytochrome P450 (CYP) isoforms that play a major role in the metabolism of GLB. However, contrasting data are available in the present literature in this regard. The present study systematically investigated the contributions of various human CYP isoforms (CYP3A4, CYP3A5, CYP2C8, CYP2C9 and CYP2C19) to in vitro metabolism of GLB. GLB depletion and metabolite formation in human liver microsomes were most significantly inhibited by the CYP3A inhibitor ketoconazole compared with the inhibitors of other CYP isoforms. Furthermore, multiple correlation analysis between GLB depletion and individual CYP activities was performed, demonstrating a significant correlation between GLB depletion and the CYP3A probe activity in 16 individual human liver microsomal preparations, but not between GLB depletion and the CYP2C19, CYP2C8 or CYP2C9 probe activity. By using recombinant supersomes overexpressing individual human CYP isoforms, it was found that GLB could be depleted by all the enzymes tested; however, the intrinsic clearance (V(max)/K(m)) of CYP3A4 for GLB depletion was 4-17 times greater than that of other CYP isoforms. These results confirm that human CYP3A4 is the major enzyme involved in the in vitro metabolism of GLB.
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Project description:The cytochrome P450 (P450) enzymes are the predominant enzyme system involved in human drug metabolism. Alterations in the expression and/or activity of these enzymes result in changes in pharmacokinetics (and consequently the pharmacodynamics) of drugs that are metabolized by this set of enzymes. Apart from changes in activity as a result of drug-drug interactions (by P450 induction or inhibition), the P450 enzymes can exhibit substantial interindividual variation in basal expression and/or activity, leading to differences in the rates of drug elimination and response. This interindividual variation can result from a myriad of factors, including genetic variation in the promoter or coding regions, variation in transcriptional regulators, alterations in microRNA that affect P450 expression, and ontogenic changes due to exposure to xenobiotics during the developmental and early postnatal periods. Other than administering a probe drug or cocktail of drugs to obtain the phenotype or conducting a genetic analysis to determine genotype, methods to determine interindividual variation are limited. Phenotyping via a probe drug requires exposure to a xenobiotic, and genotyping is not always well correlated with phenotype, making both methodologies less than ideal. This article describes recent work evaluating the effect of some of these factors on interindividual variation in human P450-mediated metabolism and the potential utility of endogenous probe compounds to assess rates of drug metabolism among individuals.
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Project description:Metoprolol is used for phenotyping of cytochrome P450 (CYP) 2D6, a CYP isoform considered not to be inducible by inducers of the CYP2C, CYP2B, and CYP3A families such as rifampicin. While assessing CYP2D6 activity under basal conditions and after pre-treatment with rifampicin in vivo, we surprisingly observed a drop in the metoprolol/?-OH-metoprolol clearance ratio, suggesting CYP2D6 induction. To study this problem, we performed in vitro investigations using HepaRG cells and primary human hepatocytes (before and after treatment with 20 ?M rifampicin), human liver microsomes, and CYP3A4-overexpressing supersomes. While mRNA expression levels of CYP3A4 showed a 15- to 30-fold increase in both cell models, mRNA of CYP2D6 was not affected by rifampicin. 1'-OH-midazolam formation (reflecting CYP3A4 activity) increased by a factor of 5-8 in both cell models, while the formation of ?-OH-metoprolol increased by a factor of 6 in HepaRG cells and of 1.4 in primary human hepatocytes. Inhibition studies using human liver microsomes showed that CYP3A4, 2B6, and 2C9 together contributed 19.0 ± 2.6% (mean ± 95%CI) to O-demethylation, 4.0 ± 0.7% to ?-hydroxylation, and 7.6 ± 1.7% to N-dealkylation of metoprolol. In supersomes overexpressing CYP3A4, metoprolol was ?-hydroxylated in a reaction inhibited by the CYP3A4-specific inhibitor ketoconazole, but not by the CYP2D6-specific inhibitor quinidine. We conclude that metoprolol is not exclusively metabolized by CYP2D6. CYP3A4, 2B6, and 2C9, which are inducible by rifampicin, contribute to ?-hydroxylation, O-demethylation, and N-dealkylation of metoprolol. This contribution is larger after CYP induction by rifampicin but is too small to compromise the usability of metoprolol ?-hydroxylation for CYP2D6 phenotyping.
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Project description:Cytochrome P450s (P450s) metabolize a large number of diverse substrates with specific regio- and stereospecificity. A number of compounds, including nicotine, cotinine, and aflatoxin B(1), are metabolites of the 94% identical CYP2A13 and CYP2A6 enzymes but at different rates. Phenacetin and 4-aminobiphenyl were identified as substrates of human cytochromes P450 1A2 and 2A13 but not of CYP2A6. The purpose of this study was to identify active site amino acids that are responsible for CYP2A substrate specificity using phenacetin as a structural probe. Ten amino acid residues that differ in the CYP2A13 and CYP2A6 active sites were exchanged between the two enzymes. Phenacetin binding revealed that the six substitution, CYP2A13 S208I, A213S, F300I, A301G, M365V, and G369S decreased phenacetin affinity. Although incorporation of individual CYP2A13 residues into CYP2A6 had little effect on this enzyme's very low levels of phenacetin metabolism, the combination of double, triple, and quadruple substitutions at positions 208, 300, 301, and 369 increasingly endowed CYP2A6 with the ability to metabolize phenacetin. Enzyme kinetics revealed that the CYP2A6 I208S/I300F/G301A/S369G mutant protein O-deethylated phenacetin with a K(m) of 10.3 muM and a k(cat) of 2.9 min(-1), which compare very favorably with those of CYP2A13 (K(m) of 10.7 muM and k(cat) of 3.8 min(-1)). A 2.15 A crystal structure of the mutant CYP2A6 I208S/I300F/G301A/S369G protein with phenacetin in the active site provided a structural rationale for the differences in phenacetin metabolism between CYP2A6 and CYP2A13.
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Project description:As a step toward exploring a targeted metabolomics approach to personalized warfarin (Coumadin) therapy, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method capable of quantifying specific enantiomeric (R and S) contributions of warfarin (WAR) and the corresponding hydroxywarfarins (OH-WAR) and glucuronides (-GLUC) generated by cytochrome P450s (CYP) and UDP-glucuronosyltransferases (UGTs), respectively. Evaluation of quality control samples and three commercially available human samples showed that our analytical approach has the ability to measure 24 unique WAR metabolites in human urine. Evaluation of the human data also provides new insights for evaluating WAR toxicity and begins characterizing important UGT metabolic pathways responsible for WAR detoxification. Data revealed the significance of specific metabolites among patients and the corresponding enzymatic capacity to generate these compounds, including the first report of direct WAR glucuronidation. On the basis of total OH-WAR levels, (S)-7-OH-WAR was the predominant metabolite indicating the significance of CYP2C9 in WAR metabolism, although other CYP2C enzymes also contributed to clearance of this isomer. (R)-WAR hydroxylation to OH-WARs was more diverse among the patients as reflected in varying contributions of CYP1A2 and multiple CYP2C enzymes. There was wide variation in the glucuronidation of WAR and the OH-WARs with respect to the compounds and patients. 6- and 7-OH-WAR were primarily (>70%) excreted as glucuronides unlike 4'-OH-WAR and 8-OH-WAR. For all patients, UGT1A1 is likely responsible for 6-O-GLUC production, although UGT1A10 may also contribute in one patient. 7-O-GLUC levels reflected contributions from potentially five different UGT1A enzymes. In all cases, WAR, 4'-OH-WAR, 8-OH-WAR, and the corresponding glucuronides were minor metabolites with respect to the others. Taken together, these data suggest that both P450 and UGT reactions contribute to the generation of excretable products in human urine, thereby generating complex metabolic networks.
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Project description:Genetic diversity is greater in Africa than in other continental populations. Genetic variability in genes encoding drug metabolizing enzymes may contribute to the high numbers of adverse drug reactions reported in Africa. We reviewed publications (1995-April 2016) reporting frequencies of known cytochrome P450 (CYP) variants in African populations. Using principal components analysis (PCA) we identified CYP alleles of potential clinical relevance with a marked difference in distribution in Africa, compared with Asian and Caucasian populations. These were CYP2B6*6, CYP2C8*2, CYP2D6*3, CYP2D6*17, CYP2D6*29, CYP3A5*6, and CYP3A5*7. We show clearly that there is greater diversity in CYP distribution in Africa than in other continental populations and identify a need for optimization of drug therapy and drug development there. Further pharmacogenetic studies are required to confirm the CYP distributions we identified using PCA, to discover uniquely African alleles and to identify populations at a potentially increased risk of drug-induced adverse events or drug inefficacy.
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Project description:All-trans-retinoic acid (atRA), the major active metabolite of vitamin A, plays a role in many biological processes, including maintenance of epithelia, immunity, and fertility and regulation of apoptosis and cell differentiation. atRA is metabolized mainly by CYP26A1, but other P450 enzymes such as CYP2C8 and CYP3As also contribute to atRA 4-hydroxylation. Although the primary metabolite of atRA, 4-OH-RA, possesses a chiral center, the stereochemical course of atRA 4-hydroxylation has not been studied previously. (4S)- and (4R)-OH-RA enantiomers were synthesized and separated by chiral column HPLC. CYP26A1 was found to form predominantly (4S)-OH-RA. This stereoselectivity was rationalized via docking of atRA in the active site of a CYP26A1 homology model. The docked structure showed a well defined niche for atRA within the active site and a specific orientation of the ?-ionone ring above the plane of the heme consistent with stereoselective abstraction of the hydrogen atom from the pro-(S)-position. In contrast to CYP26A1, CYP3A4 formed the 4-OH-RA enantiomers in a 1:1 ratio and CYP3A5 preferentially formed (4R)-OH-RA. Interestingly, CYP3A7 and CYP2C8 preferentially formed (4S)-OH-RA from atRA. Both (4S)- and (4R)-OH-RA were substrates of CYP26A1 but (4S)-OH-RA was cleared 3-fold faster than (4R)-OH-RA. In addition, 4-oxo-RA was formed from (4R)-OH-RA but not from (4S)-OH-RA by CYP26A1. Overall, these findings show that (4S)-OH-RA is preferred over (4R)-OH-RA by the enzymes regulating atRA homeostasis. The stereoselectivity observed in CYP26A1 function will aid in better understanding of the active site features of the enzyme and the disposition of biologically active retinoids.
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Project description:SMARTCyp is an in silico method that predicts the sites of cytochrome P450-mediated metabolism of druglike molecules. The method is foremost a reactivity model, and as such, it shows a preference for predicting sites that are metabolized by the cytochrome P450 3A4 isoform. SMARTCyp predicts the site of metabolism directly from the 2D structure of a molecule, without requiring calculation of electronic properties or generation of 3D structures. This is a major advantage, because it makes SMARTCyp very fast. Other advantages are that experimental data are not a prerequisite to create the model, and it can easily be integrated with other methods to create models for other cytochrome P450 isoforms. Benchmarking tests on a database of 394 3A4 substrates show that SMARTCyp successfully identifies at least one metabolic site in the top two ranked positions 76% of the time. SMARTCyp is available for download at http://www.farma.ku.dk/p450.
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