Ligand-Based Site of Metabolism Prediction for Cytochrome P450 2D6.
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ABSTRACT:
SUBMITTER: Rydberg P
PROVIDER: S-EPMC4025842 | biostudies-literature | 2012 Jan
REPOSITORIES: biostudies-literature
Publications
Ligand-Based Site of Metabolism Prediction for Cytochrome P450 2D6.
ACS medicinal chemistry letters 20111107 1
A ligand-based method based on the SMARTCyp approach that predicts the sites of cytochrome P450 2D6-mediated metabolism of druglike molecules has been developed. The method uses only two descriptors besides the reactivity from SMARTCyp: the distance to a protonated nitrogen atom and the distance to the end of the molecule. Hence, the site of metabolism is predicted directly from the 2D structure of a molecule, without requiring calculation of electronic properties or generation of 3D structures. ...[more]
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Project description:The membrane-anchored enzyme Cytochrome P450 2D6 (CYP2D6) is involved in the metabolism of around 25% of marketed drugs and its metabolic performance shows a high interindividual variation. While it was suggested that ligands access the buried active site of the enzyme from the membrane, no proof from unbiased simulations has been provided to support this hypothesis. Laboratory experiments fail to capture the access process which is suspected to influence binding kinetics. Here, we applied unbiased molecular dynamics (MD) simulations to investigate the access of ligands to wild-type CYP2D6, as well as the allelic variant CYP2D6*53. In multiple simulations, substrates accessed the active site of the enzyme from the protein-membrane interface to ultimately adopt a conformation that would allow a metabolic reaction. We propose the necessary steps for ligand access and the results suggest that the increased metabolic activity of CYP2D6*53 might be caused by a facilitated ligand uptake.
| S-EPMC6848145 | biostudies-literature
Project description:Homology models of cytochrome P450 2D6 (CYP2D6) have identified serine 304 as an active-site residue and implicated a putative role for this residue in substrate enantioselectivity and the differential inhibition of enzyme activity by the diastereoisomers quinine and quinidine. The role of serine 304 in selectivity is thought to be achieved through a preferential hydrogen-bond interaction between the hydroxyl group of the residue and one of the stereoisomers of each ligand. We have tested this hypothesis by substituting serine 304 with alanine, a non-hydrogen-bonding residue, and compared the properties of the wild-type and mutant enzymes in microsomes prepared from yeast cells expressing the appropriate cDNA-derived enzyme. The Ser(304)Ala substitution did not alter the enantioselective oxidation of metoprolol; the O-demethylation reaction remained R-(+)-enantioselective (wild-type, R/S, 1.7; mutant, R/S, 1.6), whereas alpha-hydroxylation remained S-(-)-enantioselective (wild-type and mutant, R/S, 0.7). Similarly, the selective oxidation of the R-(+) and S-(-) enantiomers of propranolol to the major 4-hydroxy metabolite was identical with both wild-type and mutant forms of the enzyme (R/S 0.9), although the formation of minor metabolites (5-hydroxy and deisopropylpropranolol) did show some slight alteration in enantioselectivity. The differential inhibition of enzyme activity by quinine and quinidine was also identical with both forms of CYP2D6, the IC(50) values for each enzyme being approx. 10 microM and 0.1 microM for quinine and quinidine, respectively. The kinetics of formation of alpha-hydroxymetoprolol and 4-hydroxydebrisoquine by wild-type and the Ser(304)Ala mutant was also very similar. However, modest changes in the regioselective oxidation of metoprolol and debrisoquine were observed with the Ser(304)Ala mutant. The regio- and enantioselective oxidation of an analogue of metoprolol, in which the hydroxyl group attached to the chiral carbon was replaced by a methyl moiety, was again identical with both wild-type and Ser(304)Ala mutant. However, the observed selectivity was the reverse of that observed with metoprolol. Collectively, these data indicate that Ser(304) is unlikely to be a key ligand-binding residue, although the residue may indeed be located in the active-site cavity. The reversal of selectivity with the methyl analogue of metoprolol indicates that the hydroxyl group attached to the chiral centre of ligands, such as metoprolol, is important in defining the enzyme's selective properties, and that a hydrogen-bonding residue, other than Ser(304), may be involved in this interaction. Current homology models of the active site of CYP2D6 that predict a hydrogen-bond interaction between Ser(304) and specific ligands will need to be re-evaluated, and other candidate residues capable of such an interaction nominated and tested by site-directed mutagenesis studies.
| S-EPMC1220791 | biostudies-other
Project description:BackgroundThe prediction of sites and products of metabolism in xenobiotic compounds is key to the development of new chemical entities, where screening potential metabolites for toxicity or unwanted side-effects is of crucial importance. In this work 2D topological fingerprints are used to encode atomic sites and three probabilistic machine learning methods are applied: Parzen-Rosenblatt Window (PRW), Naive Bayesian (NB) and a novel approach called RASCAL (Random Attribute Subsampling Classification ALgorithm). These are implemented by randomly subsampling descriptor space to alleviate the problem often suffered by data mining methods of having to exactly match fingerprints, and in the case of PRW by measuring a distance between feature vectors rather than exact matching. The classifiers have been implemented in CUDA/C++ to exploit the parallel architecture of graphical processing units (GPUs) and is freely available in a public repository.ResultsIt is shown that for PRW a SoM (Site of Metabolism) is identified in the top two predictions for 85%, 91% and 88% of the CYP 3A4, 2D6 and 2C9 data sets respectively, with RASCAL giving similar performance of 83%, 91% and 88%, respectively. These results put PRW and RASCAL performance ahead of NB which gave a much lower classification performance of 51%, 73% and 74%, respectively.Conclusions2D topological fingerprints calculated to a bond depth of 4-6 contain sufficient information to allow the identification of SoMs using classifiers based on relatively small data sets. Thus, the machine learning methods outlined in this paper are conceptually simpler and more efficient than other methods tested and the use of simple topological descriptors derived from 2D structure give results competitive with other approaches using more expensive quantum chemical descriptors. The descriptor space subsampling approach and ensemble methodology allow the methods to be applied to molecules more distant from the training data where data mining would be more likely to fail due to the lack of common fingerprints. The RASCAL algorithm is shown to give equivalent classification performance to PRW but at lower computational expense allowing it to be applied more efficiently in the ensemble scheme.
| S-EPMC4047555 | biostudies-literature
Project description:Human cytochrome P450 2D6 contributes to the metabolism of >15% of drugs used in clinical practice. This study determined the structure of P450 2D6 complexed with a substrate and potent inhibitor, prinomastat, to 2.85 Å resolution by x-ray crystallography. Prinomastat binding is well defined by electron density maps with its pyridyl nitrogen bound to the heme iron. The structure of ligand-bound P450 2D6 differs significantly from the ligand-free structure reported for the P450 2D6 Met-374 variant (Protein Data Bank code 2F9Q). Superposition of the structures reveals significant differences for β sheet 1, helices A, F, F', G", G, and H as well as the helix B-C loop. The structure of the ligand complex exhibits a closed active site cavity that conforms closely to the shape of prinomastat. The closure of the open cavity seen for the 2F9Q structure reflects a change in the direction and pitch of helix F and introduction of a turn at Gly-218, which is followed by a well defined helix F' that was not observed in the 2F9Q structure. These differences reflect considerable structural flexibility that is likely to contribute to the catalytic versatility of P450 2D6, and this new structure provides an alternative model for in silico studies of substrate interactions with P450 2D6.
| S-EPMC3322812 | biostudies-literature
Project description:AIM:Over 90% of drugs are metabolized by the cytochrome P-450 (CYP) family of liver isoenzymes. The most important enzymes are CYP1A2, 3A4, 2C9/19, 2D6 and 2E1. Although CYP2D6 accounts for <2% of the total CYP liver enzyme content, it mediates metabolism in almost 25% of drugs. In order to study its enzymatic activity for drug metabolism, its cDNA was cloned and a HepG2 cell line stably expressing CYP2D6 was established. METHODS:Human CYP2D6 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR) from total RNA extracted from human liver tissue and cloned into pGEM-T vector. cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A cell line was established by transfecting the recombinant plasmid of pREP9-CYP2D6 to hepatoma HepG2 cells. Expression of mRNA was validated by RT-PCR. Enzyme activity of catalyzing dextromethorphan O-demethylation in postmitochondrial supernatant (S9) fraction of the cells was determined by high performance liquid chromatography (HPLC). RESULTS:The cloned cDNA had 4 base differences, e.g. 100 C-T, 336 T-C, 408 C-G and 1 457 G-C, which resulted in P34S, and S486T amino acid substitutions, and two samesense mutations were 112 F and 136 V compared with that reported by Kimura et al (GenBank accession number: M33388). P34S and S486T amino acid substitutions were the characteristics of CYP2D6*10 allele. The relative activity of S9 fraction of HepG2-CYP2D6*10 metabolized detromethorphan O-demethylation was found to be 2.31 +/- 0.19 nmol/min(-1)/mg(-1) S9 protein (n=3), but was undetectable in parental HepG2 cells. CONCLUSION:cDNA of human CYP2D6*10 can be successfully cloned. A cell line, HepG2-CYP2D6*10, expressing CYP2D6*10 mRNA and having metabolic activity, has been established.
| S-EPMC4717011 | biostudies-literature
Project description:CYP2D6 substrates are among the most highly prescribed medications in teenagers and also commonly associated with serious adverse events. To investigate the relative contributions of genetic variation, growth, and development on CYP2D6 activity during puberty, healthy children and adolescents 7-15 years of age at enrollment participated in a longitudinal phenotyping study involving administration of 0.3 mg/kg dextromethorphan (DM) and 4-h urine collection every 6 months for 3 years (7 total visits). At each visit, height, weight, and sexual maturity were recorded, and CYP2D6 activity was determined as the urinary molar ratio of DM to its metabolite dextrorphan (DX). A total of 188 participants completed at least one visit, and 102 completed all seven study visits. Following univariate analysis, only CYP2D6 activity score (p < 0.001), urinary pH (p < 0.001), weight (p = 0.018), and attention-deficit/hyperactivity disorder (ADHD) diagnosis (p < 0.001) were significantly correlated with log(DM/DX). Results of linear mixed model analysis with random intercept, random slope covariance structure revealed that CYP2D6 activity score had the strongest effect on log(DM/DX), with model-estimated average log(DM/DX) being 3.8 SDs higher for poor metabolizers than for patients with activity score 3. A moderate effect on log(DM/DX) was observed for sex, and smaller effects were observed for ADHD diagnosis and urinary pH. The log(DM/DX) did not change meaningfully with age or pubertal development. CYP2D6 genotype remains the single, largest determinant of variability in CYP2D6 activity during puberty. Incorporation of genotype-based dosing guidelines should be considered for CYP2D6 substrates given the prevalent use of these agents in this pediatric age group.
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Project description:Liver cancer morbidity and mortality rates differ among ethnic groups. In the United States, the burden of liver cancer in Asian Americans (AS) is higher compared to Caucasian Americans (CA). Research on liver cancer health disparities has mainly focused on environmental and socioeconomic factors yet has ignored the genotypic differences among various racial/ethnic groups. This lack of molecular level understanding has hindered the development of personalized medical approaches for liver cancer treatment. To understand the genetic heterogeneity of liver cancer between AS and CA, we performed a systematic analysis of RNA-seq data of AS and CA patients from The Cancer Genome Atlas (TCGA). We used four differential gene expression analysis packages; DESeq2, limma, edgeR, and Superdelta2, to identify the differentially expressed genes. Our analysis identified cytochrome P450-2D6 enzyme (CYP2D6) as the gene with the greatest differential expression with higher levels in AS compared to CA. To scrutinize the underlying mechanism of CYP2D6, Ingenuity Pathway Analysis (IPA) and Cytoscape were conducted and found hepatocyte nuclear factor-4α (HNF4A) and interleukin-6 (IL6) in direct association with CYP2D6. IL6 is downregulated in AS compared to CA, while HNF4A is not significantly different. Herein, we report that CYP2D6 may serve as a putative biomarker in liver cancer health disparities. Its negative association with IL6 proclaims an intricate relationship between CYP2D6 and inflammation in the ethnic differences seen in AS and CA liver cancer patients. The goal of the present study was to understand how genetic factors may contribute to the interethnic variability of liver cancer prevalence and outcomes in AS and CA patients. Identifying ethnic-specific genes may help ameliorate detection, diagnosis, surveillance, and treatments of liver cancer, as well as reduce disease-related incidence and mortality rates in the vulnerable population.
| S-EPMC8486079 | biostudies-literature
Project description:Based on the X-ray crystal structures of 4-(4-chlorophenyl)imidazole (4-CPI)- and bifonazole (BIF)-bound P450 2B4, eight active site mutants at six positions were created in an N-terminal modified construct termed 2B4dH and characterized for enzyme inhibition and catalysis. I363A showed a >4-fold decrease in differential inhibition by BIF and 4-CPI (IC(50,BIF)/IC(50,4-CPI)). F296A, T302A, I363A, V367A, and V477A showed a 2-fold decreased k(cat) for 7-ethoxy-4-trifluoromethylcoumarin O-deethylation, whereas V367A and V477F showed an altered K(m). T302A, V367L, and V477A showed >4-fold decrease in total testosterone hydroxylation, whereas I363A, V367A, and V477F showed altered stereo- and regioselectivity. Interestingly, I363A showed a 150-fold enhanced k(cat)/K(m) with testosterone, and yielded a new metabolite. Furthermore, testosterone docking into three-dimensional models of selected mutants based on the 4-CPI-bound structure suggested a re-positioning of residues 363 and 477 to yield products. In conclusion, our results suggest that the 4-CPI-bound 2B4dH/H226Y crystal structure is an appropriate model for predicting enzyme catalysis.
| S-EPMC1773018 | biostudies-literature
Project description:The cytochrome P450 (CYP) 2D6 enzyme exhibits large interindividual differences in metabolic activity. Patients are commonly assigned a CYP2D6 phenotype based on their CYP2D6 genotype, but there is a lack of consensus on how to translate genotypes into phenotypes, causing inconsistency in genotype-based dose recommendations. The aim of this study was to quantify and compare the impact of different CYP2D6 genotypes and alleles on CYP2D6 metabolism using a large clinical data set. A population pharmacokinetic (popPK) model of tedatioxetine and its CYP2D6-dependent metabolite was developed based on pharmacokinetic data from 578 subjects. The CYP2D6-mediated metabolism was quantified for each subject based on estimates from the final popPK model, and CYP2D6 activity scores were calculated for each allele using multiple linear regression. The activity scores estimated for the decreased function alleles were 0.46 (CYP2D6*9), 0.34 (CYP2D6*10), 0.01 (CYP2D6*17), 0.65 (CYP2D6*29), and 0.21 (CYP2D6*41). The CYP2D6*17 and CYP2D6*41 alleles were thus associated with the lowest CYP2D6 activity, although only the difference to the CYP2D6*9 allele was shown to be statistically significant (p = 0.02 and p = 0.05, respectively). The study provides new in vivo evidence of the enzyme function of different CYP2D6 genotypes and alleles. Our findings suggest that the activity score assigned to CYP2D6*41 should be revisited, whereas CYP2D6*17 appears to exhibit substrate-specific behavior. Further studies are needed to confirm the findings and to improve the understanding of CYP2D6 genotype-phenotype relationships across substrates.
| S-EPMC8452298 | biostudies-literature
Project description:BackgroundCytochrome P450 2D6 is one of the important enzymes involved in the metabolism of many widely used drugs. Genetic polymorphisms of CYP2D6 can affect its activity. Therefore, an efficient method for identifying CYP2D6 polymorphisms is clinically important.MethodsWe developed a high-resolution melting (HRM) analysis to investigate CYP2D6 polymorphisms. Genomic DNA was extracted from peripheral blood samples from 71 healthy individuals. All nine exons of the CYP2D6 gene were sequenced before screening by HRM analysis. This method can detect the most genotypes (*1, *2, *4, *10, *14, *21 *39, and *41) of CYP2D6 in Chinese.ResultsAll samples were successfully genotyped. The four most common mutant CYP2D6 alleles (*1, *2, *10, and *41) can be genotyped. The single nucleotides polymorphism (SNP) frequencies of 100C > T (rs1065852), 1039C > T (rs1081003), 1661G > C (rs1058164), 2663G > A (rs28371722), 2850C > T (rs16947), 2988G > A (rs28371725), 3181A > G, and 4180G > C (rs1135840) were 58%, 61%, 73%, 1%, 13%, 3%, 1%, 73%, respectively. We identified 100% of all heterozygotes without any errors. The two homozygous genotypes (1661G > C and 4180G > C) can be distinguished by mixing with a known genotype sample to generate an artificial heterozygote for HRM analysis. Therefore, all samples could be identified using our HRM method, and the results of HRM analysis are identical to those obtained by sequencing. Our method achieved 100% sensitivity, specificity, positive prediction value and negative prediction value.ConclusionHRM analysis is a nongel resolution method that is faster and less expensive than direct sequencing. Our study shows that it is an efficient tool for typing CYP2D6 polymorphisms.
| S-EPMC6806731 | biostudies-literature