Regioselectivity Of Metabolism Research Articles

Selective cascade activation of polycyclic aromatic hydrocarbons in human cells: Role of enzyme's intrinsic electric field

Polycyclic aromatic hydrocarbons (PAHs) are major environmental organic pollutants. Some metabolites of PAHs show greater toxicity to humans while the others do not. It is highly important to decipher PAHs' regioselective activation mechanism and identify the major metabolites to accurately evaluate their public health risk. Here, we have performed a thorough computational study of benzo[a]anthracene (BA) metabolized by P450 1A1 by employing molecular docking, molecular dynamics simulations, quantum chemical calculation, and quantum mechanics/molecular mechanics calculations. Our findings show that highly-reactive species such as 3,4-epoxide, 8,9-epoxide, 3,4-diol-1,2-epoxide, and 8,9-diol-10,11-epoxide were major metabolites, which can efficiently react with guanine and damage DNA with extremely low energy barrier, therefore, supports the regioselective metabolism of BA. The origin of this selective activation is mainly contributed to both the oxygen‑carbon distance and previously overlooked enzyme's intrinsic electric field. Consequently, based on the resolved activation selectivity of BA. We built a high-throughput strategy to efficiently predict the metabolites of other PAHs. The accuracy of the strategy is validated by studying 16 PAHs on the priority control list. Hopefully this will aid the accurate evaluation of public health risks associated with PAH emissions.

Regioselective metabolism of cannabinoids by cytochrome P450 2J2.

Cannabichromene (CBC) is a major non-psychoactive phytocannabinoid extracted from the cannabis plant. CBC has been shown to have anti-inflammatory effects; hence it can serve as a non-narcotic alternative to tetrahydrocannabinol (THC). Nevertheless, the metabolic pathways and metabolites of CBC have not been fully characterized. Metabolism of phytocannabinoids typically occurs via a superfamily of hemoproteins called cytochrome P450s (CYP450s). Our liquid chromatography-mass spectrometry (LC/MS) experiments show that CBC is metabolized to three different products by various human CYP450s. We identified that CYP450 2J2 (CYP2J2) predominantly produces 8'-hydroxy-CBC, an allylic hydroxide, out of the three metabolites. To investigate the CBC metabolic regioselectivity in CYP2J2, we leveraged atomistic molecular dynamics (MD) and modeling of the system. Using 200 unique conformations of CYP2J2 obtained from MD, we performed ensemble molecular docking of CBC to reveal three putative binding modes. We then assessed the stability of the obtained CBC binding modes by simulating them individually for 1 μs. Our simulations captured an average distance of 4.8 Å between the CYP2J2 heme and the 8’ carbon atom on CBC for one of the predominant binding modes, at which the allylic hydroxide metabolite occurs experimentally. At the regioselective binding mode, we observe conserved hydrogen bonding networks, mainly between the CBC hydroxyl group and the backbone oxygen of I487, as well as N379, stabilized by a water molecule. We further corroborated the stability of the regioselective binding modes by calculating relative binding free energies using alchemical free energy perturbation calculations. In conjunction, our computational and experimental methodologies elucidate a likely mode for regioselective binding and metabolism of CBC by CYP2J2, furthering the way towards accessible plant-based non-psychoactive anti-inflammatory therapeutics.

Metabolic functional redundancy of the CYP9A subfamily members leads to P450-mediated lambda-cyhalothrin resistance in Cydia pomonella.

The evolution of insect resistance to pesticides poses a continuing threat to sustainable pest management. While much is known about the molecular mechanisms that confer resistance in model insects and few agricultural pests, far less is known about fruit pests. Field-evolved resistance to synthetic insecticides such as lambda-cyhalothrin has been widely documented in Cydia pomonella, a major invasive pest of pome fruit worldwide, and the increased production of cytochrome P450 monooxygenases (P450s) has been linked to resistance in field-evolved resistant populations. However, the underlying molecular mechanisms of P450-mediated insecticide resistance remain largely unknown. Here we found that functional redundancy and preference of metabolism by P450s genes in the CYP9A subfamily confer resistance to lambda-cyhalothrin in Cydia pomonella. A total of four CYP9A genes, including CYP9A61, CYP9A120, CYP9A121, and CYP9A122, were identified from Cydia pomonella. Among these, CYP9A120, CYP9A121, and CYP9A122 were predominantly expressed in the midgut of larvae. The expression levels of these P450 genes were significantly induced by a lethal dose that would kill 10% (LD10 ) of lambda-cyhalothrin and were overexpressed in a field-evolved lambda-cyhalothrin resistant population. Knockdown of CYP9A120 and CYP9A121 by RNA-mediated interference (RNAi) increased the susceptibility of larvae to lambda-cyhalothrin. In vitro assays demonstrated that recombinant P450s expressed in Sf9 cells can metabolize lambda-cyhalothrin, but with functional redundancy and divergence through regioselectivity of metabolism. CYP9A121 preferred to convert lambda-cyhalothrin to 2'-hydroxy-lambda-cyhalothrin, whereas CYP9A122 only generated 4'-hydroxy metabolite of lambda-cyhalothrin. Although possesses a relatively low metabolic capability, CYP9A120 balanced catalytic competence to generate both 2'- and 4'-metabolites. Collectively, these results reveal that metabolic functional redundancy of three members of the CYP9A subfamily leads to P450-mediated lambda-cyhalothrin resistance in Cydia pomonella, thus representing a potential adaptive evolutionary strategy during its worldwide expansion. © 2022 Society of Chemical Industry.

Investigation of the molecular and mechanistic basis for the regioselective metabolism of midazolam by cytochrome P450 3A4.

Cytochrome P450 3A4 (CYP3A4) is the most important P450 enzyme for drug metabolism and drug-drug interaction, due to it being responsible for the biotransformation of approximately 50% of clinically used drugs. Advance knowledge of the molecular and mechanistic basis of CYP3A4 regioselective metabolism is beneficial for understanding the production of metabolites, and may allow personalized metabolic pathways or designing pathway-specific therapeutics. In this work, we focus on investigating the ligand-receptor interactions, substrate conformational transition, and key factors regulating the specificity of metabolic pathways using midazolam (MDZ) as a probe. Here, three types of substrate-binding conformations related to the diversity of MDZ metabolites are identified. The results also suggest that an allosteric site for MDZ is located near the F'-helix, A-anchor, and C-terminal loop of CYP3A4. The presence of an effector in the allosteric site can accelerate the conformational transition of the substrate via modulating a "sandwich" structure, and may affect the proportion of metabolites at high substrate concentration. We hope that the results can improve the understanding of the CYP3A4 structure and function, and provide a new perspective for drug development.

Binding and Metabolism of Brominated Flame Retardant β-1,2-Dibromo-4-(1,2-dibromoethyl)cyclohexane in Human Microsomal P450 Enzymes: Insights from Computational Studies.

The emerging brominated flame retardant, 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane (TBECH), has recently attracted strong interest due to its extensive detection in the environment and potential toxicological effects on humans. Previous in vitro experiments have shown that the technical mixture of TBECH and the pure β-isomer (β-TBECH) can be metabolized by cytochrome P450 enzymes (CYPs) into multiple metabolites, but the specific CYP isoforms involved in TBECH metabolism and the relevant metabolic regioselectivity remain unknown. Here, we, for the first time, investigated the binding patterns and affinities of β-TBECH in human CYPs 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4, through molecular dynamics (MD) simulations. The binding affinities of β-TBECH in CYPs, which are estimated by the calculated binding free energies, follow the order of 2A6 > 2C9 > 2B6 > 2E1 > 3A4 ≈ 2C19 ≈ 1A2 > 2D6. Although all CYPs are important β-TBECH receptors, only 2A6, 2C19, 2E1, and 3A4 are responsible for metabolizing β-TBECH. Specially, 2A6 and 2E1 may selectively hydroxylate the C1 and C7 sites of β-TBECH, while 2C19 and 3A4 show metabolic preference for C7- and C8-hydroxylations, respectively. The three hydroxylation routes proposed by the further density functional theory (DFT) calculations generate C1-, C7-, and C8-hydroxylated metabolites, while the latter two may further undergo debromination to yield the respective ketone and aldehyde as additional metabolites. The results provide meaningful insight into the binding and metabolism of β-TBECH by human CYPs, which is helpful for understanding the metabolic fate and toxicity mechanism of this chemical.

Quantitation of phenanthrene dihydrodiols in the urine of smokers and non-smokers by gas chromatography-negative ion chemical ionization-tandem mass spectrometry

Polycyclic aromatic hydrocarbons (PAH) are well-established environmental carcinogens likely to be causative agents for some human cancers. Bay-region diol epoxides are ultimate carcinogenic metabolites of multiple PAH. Dihydrodiols are the important intermediate products of this pathway and can be further oxidized to form diol epoxides. We quantified two dihydrodiol metabolites of phenanthrene (Phe), the simplest PAH with a bay-region, in the 6 h urine of smokers (N = 25) and non-smokers (N = 25) using a newly developed and validated analytical method. After hydrolysis by ß-glucuronidase and sulfatase, and solid phase extraction, the sample was silylated and analyzed by gas chromatography-negative ion chemical ionization-tandem mass spectrometry (GC-NICI-MS/MS). Levels (nmol/6h urine) of Phe-1,2-dihydrodiol (Phe-1,2-D) and Phe-3,4-dihydrodiol (Phe-3,4-D) were 2.04 ± 1.52 and 0.51 ± 0.35 , respectively, in smokers, significantly higher than those in non-smokers (1.35 ± 1.11 of Phe-1,2-D, p < 0.05; 0.27 ± 0.25 of Phe-3,4-D, p < 0.005). Cigarette smoking also influenced the regioselective metabolism of Phe, presenting as a significant difference in the urinary distribution pattern of Phe-1,2-D and Phe-3,4-D between smokers and non-smokers: the ratio Phe-3,4-D: Phe-1,2-D increased from 0.20 in non-smokers to 0.28 in smokers (p < 0.01), which can be explained by the induction of the phenanthrene metabolizing enzymes CYP1A2 and CYP1B1 by cigarette smoke. The method described here is the first example of facile quantitation of an intact human dihydrodiol metabolite of any PAH with three or more aromatic rings and will be applicable in clinical and molecular epidemiology studies of PAH metabolism and cancer susceptibility.

Molecular Basis for Metabolic Regioselectivity and Mechanism of Cytochrome P450s toward Carcinogenic 4-(Methylnitrosamino)-(3-pyridyl)-1-butanone.

As an abundantly present tobacco component, carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) has also been detected in atmospheric particulate matter, suggesting the ineluctable exposure risk of this contaminant. NNK metabolic activation by cytochrome P450 enzymes (CYPs) is a prerequisite to exerting its genotoxicity, but the metabolic regioselectivity and mechanism are still unknown. Here the binding feature and regioselectivity of CYPs 1A1, 1A2, 2A6, 2A13, 2B6, and 3A4 toward NNK are unraveled through molecular docking and molecular dynamics (MD) simulations. Binding mode analyses reveal that 1A2 and 2B6 have definite preferences for NNK α-methyl hydroxylation, while the other four CYPs preferentially catalyze α-methylene hydroxylation. The binding affinities between NNK and CYPs evaluated by the binding free energies follow the order 2A13 > 2B6 > 1A2 > 2A6 > 1A1 > 3A4. Density functional theory (DFT) calculations are further performed to characterize the mechanism of NNK biotransformation. Results show that the α-hydroxyNNK generated from α-hydroxylation may undergo nonenzymatic decomposition to form genotoxic diazohydroxide and aldehyde, and further oxidation by P450 to yield nitrosamide, which mainly contributes to NNK toxification capacity. Meanwhile the pyridine N-oxidation and denitrosation of Cα-radical intermediate play an important role in detoxifying NNK. Overall, the present study provides the molecular basis for CYP-catalyzed regioselectivity and mechanism of NNK biotransformation, which can enable the identification of metabolites for assessing the health risk of individual NNK exposure.

Expression of CYP2E1 gene in paint workers occupationally exposed to organic solvents

Benzene (B), toluene (T), ethylbenzene (E) and xylene (X) are used commonly in paint industry, so workers who are a high risk of exposure to organic solvent (VOCs). CYP2E1 gene encodes CYP2E1 which plays an important role in the metabolism and bio-activation of volatile organic compounds. When workers expose to VOCs, their body will have a mechanism to metabolize the toxic. The enhancement of mRNA expression of CYP2E1 is a very sensitive and accurate biological marker, which is the basis for the next study to propose the level of mRNA expression of CYP2E1 gene as a human biological indicator to monitor to workers occupationally exposed to VOCs. In this study, we studied the level of mRNA expression of CYP2E1 gene from 118 participants that including 73 workers of exposed group from the paint factories and 45 workers of non-exposed group from garment factories by using Realtime-PCR with SYBR Green – an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. The initial results, showed that the exposed group had a higher mRNA expression level of CYP2E1 than the non-exposed group approximately 10.47 times, and this difference was statistically significant (P value &lt; 0.05). And the age and the duration of exposure to organic solvents do not affect the expression level of CYP2E1.&#x0D; Keywords&#x0D; CYP2E1, the metabolisms of volatile organic compounds, mRNA expression of CYP2E1.&#x0D; References&#x0D; [1] A. Mendoza-Cantu, F. Castorena-Torres, M. Bermudez De Leon et al., Occupational toluene exposure induces cytochrome P450 2E1 mRNA expression in peripheral lymphocytes, Environmental Health Perspectives, 114 (2006) 494 - 499. https://doi.org/10.1289/ehp.8192.[2] J.H. Hartman, G. Boysen and G.P. Miller, CYP2E1 metabolism of styrene involves allostery, Drug Metabolism and Disposition, 40 (2012) 1976-1983. https://doi.org/10.1124/dmd. 112.046698. [3] S.M. Zhu, X.F. Ren, J.X. Wan et al., Evaluation in vinyl chloride monomer (VCM) - exposed workers and the relationship between liver lesions and gene polymorphisms of metabolic enzymes, World Journal of Gastroenterology,11 (2005) 5821 -5827. 10.3748/wjg.v11.i37.5821.[4] J. Wan, J. Shi, L. Hui et al., Association of genetic polymorphisms in CYP2E1, MPO, NQO1, GSTM1, and GSTT1 genes with benzene poisoning, Environmental Health Perspectives, 110 (2002), 1213-1218. https://doi.org/10.1289/ ehp.021101213.[5] U. Bernauer, B. Vieth, R. Ellrich et al., CYP2E1 - dependent benzene toxicity: the role of extrahepatic benzene metabolism, Archives of Toxicology, 73 (1999) 189 -196. https://doi.org/ 10.1007/s002040050605. [6] P.L. Sheets, G.S. Yost and G.P. Carlson. Benzene metabolism in human lung cell lines BEAS‐2B and A549 and cells overexpressing CYP2F1, Journal of Biochemical and Molecular Toxicology, 18 (2004) 92-99. https://doi.org/10. 1002/jbt.20010.[7] V. Nedelcheva, I. Gut, P. Souček et al., Metabolism of benzene in human liver microsomes: individual variations in relation to CYP2E1 expression, Archives of Toxicology, 73 (1999) 33-40. https://doi.org/10.1007/s0020400 50583.[8] W. Tassaneeyakul, D.J. Birkett, J.W. Edwards et al., Human cytochrome P450 isoform specificity in the regioselective metabolism of toluene and o-, m-and p-xylene, Journal of Pharmacology and Experimental Therapeutics, 276 (1996) 101 - 108. 10.1163/2211730x96x00063[9] A.-H. Wang, S.-M. Zhu, Y.-L. Qiu et al., CYP2E1 mRNA expression, genetic polymorphisms in peripheral blood lymphocytes and liver abnormalities in Chinese VCM-exposed workers, International Journal of Occupational Medicine and Environmental Health, 21 (2008), 141 - 146. 10.2478/v10001-008-0016-x.[10] J. Zhang, Y. Lihong, G. Liang et al., Detection of CYP2E1, a genetic biomarker of susceptibility to benzene metabolism toxicity in immortal human lymphocytes derived from the Han Chinese population, Biomedical and Environmental Sciences, 24 (2011) 300-309. https://doi.org/10. 3967/0895-3988.2011.03.014[11] American Conference of Industrial Hygienists, Guide to Occupational Exposeure Values, ACGIH, Cincinnati, 2018.[12] Bộ Y Tế, Quyết định của Bộ trưởng Bộ Y tế về việc ban hành 21 tiêu chuẩn vệ sinh lao động, 05 nguyên tắc và 07 thông số vệ sinh lao động, 2002.[13] American Conference of Industrial Hygienists, Threshold Limit Value for Chemical Substances and Physical Agents and Biological Exposure Indices, ACGIH, Cincinnati, 2018.[14] Bộ Y Tế, Thông tư 28/2006/TT- BYT hướng dẫn quản lý bệnh bệnh nghề nghiệp, 2016.[15] M. Al Zallouha, Y. Landkocz, J. Brunet et al., Usefulness of toxicological validation of VOCs catalytic degradation by air-liquid interface exposure system", Environmental Research, 152 (2017) 328-335. https://doi.org/10.1016/j.envres. 2016.10.027&#x0D;

Both Reactivity and Accessibility Are Important in Cytochrome P450 Metabolism: A Combined DFT and MD Study of Fenamic Acids in BM3 Mutants.

Cytochrome P450 102A1 from Bacillus megaterium (BM3) is a fatty acid hydroxylase that has one of the highest turnover rates of any mono-oxygenase. Recent studies have shown how mutants of BM3 can produce metabolites of known drug compounds similar to those observed in humans. Single-point mutations in the binding pocket change the regioselective metabolism of fenamic acids from aromatic hydroxylation to aliphatic hydroxylation. This study is concerned with the individual contribution from accessibility and reactivity for drug metabolism with a future goal to develop fast methods for prediction. For a BM3 M11 mutant as well as the M11 V87F and M11 V87I mutants, we studied the metabolism of the nonsteroidal anti-inflammatory drugs (NSAIDs) mefenamic acid, meclofenamic acid, tolfenamic acid, and diclofenac. Density functional theory (DFT; B3LYP and B3LYP-D3) calculations for all possible reactions were performed using a porphyrin model reacting with the four substrates. Molecular dynamics (MD) simulations were used to determine the potential sites of metabolism that are accessible. Finally, we combine reactivity and accessibility for each potential site to interpret the experimentally determined metabolism. Generally, the 3 and 5 positions (on the ring containing the acidic substituent) and the 2', 3', and 4' positions are most reactive, whereas 4, 5, 3', and 4' are most accessible. Combining reactivity and accessibility show that the 5, 3', and 4' positions are predicted to be most prone to be metabolized, in agreement with experimentally observed data. Reactivity seems to be the dominant factor in the CYP-mediated metabolism of these NSAIDs, which is consistent with previously published methods based solely on reactivity.

Recent advances in the prediction of non‐CYP450‐mediated drug metabolism

Computational models of drug metabolism prediction have focused mainly on cytochrome P450 enzymes, because drug–drug interactions, reactive metabolite formation, hepatotoxicity, idiosyncratic adverse drug interactions, and/or loss of efficacy of many drugs were the results of interactions with CYP450s. Metabolic regioselectivity and isoform specificity prediction models for CYP450‐catalyzed reactions have reached approximately 95% accuracy. Thus, a new drug candidate is less likely to show unexpected metabolic profile due to metabolism via CYP450 pathways. For such candidates, secondary metabolic Phase I and II enzymes are likely to play an expected (or unexpected) role in drug metabolism. The importance of flavin monooxygenases (FMOs), aldehyde and alcohol dehydrogenase, monoamine oxidase from the Phase I and UDP‐glucuronosyltransferase (UGT), sulfotransferase, glutathione S‐transferase, and methyltransferase from Phase II has increased and United States Food and Drug Administration guidelines on NDA have specific recommendations for in vitro and in vivo testing against these enzymes. Thus, there is an urgent requirement of reliable predictive models for drug metabolism catalyzed by these enzymes. In this review, we have classified drug metabolism prediction models (site of metabolism, isoform specificity, and kinetic parameter) for these enzymes into Phase I and II. When such models are unavailable, we discuss the Quantitative Structure Activity Relationship (QSAR), pharmacophore, docking, dynamics, and reactivity studies performed for the prediction of substrates and inhibitors. Recently published models for FMO and UGT are discussed. The need for comprehensive, widely applicable, sequential primary and secondary metabolite prediction is highlighted. Potential difficulties and future prospectives in the development of such models are discussed. WIREs Comput Mol Sci 2017, 7:e1323. doi: 10.1002/wcms.1323This article is categorized under: Structure and Mechanism &gt; Reaction Mechanisms and Catalysis Computer and Information Science &gt; Chemoinformatics Software &gt; Molecular Modeling

Insights into regioselective metabolism of mefenamic acid by cytochrome P450 BM3 mutants through crystallography, docking, molecular dynamics, and free energy calculations.

Cytochrome P450 BM3 (CYP102A1) mutant M11 is able to metabolize a wide range of drugs and drug-like compounds. Among these, M11 was recently found to be able to catalyze formation of human metabolites of mefenamic acid and other nonsteroidal anti-inflammatory drugs (NSAIDs). Interestingly, single active-site mutations such as V87I were reported to invert regioselectivity in NSAID hydroxylation. In this work, we combine crystallography and molecular simulation to study the effect of single mutations on binding and regioselective metabolism of mefenamic acid by M11 mutants. The heme domain of the protein mutant M11 was expressed, purified, and crystallized, and its X-ray structure was used as template for modeling. A multistep approach was used that combines molecular docking, molecular dynamics (MD) simulation, and binding free-energy calculations to address protein flexibility. In this way, preferred binding modes that are consistent with oxidation at the experimentally observed sites of metabolism (SOMs) were identified. Whereas docking could not be used to retrospectively predict experimental trends in regioselectivity, we were able to rank binding modes in line with the preferred SOMs of mefenamic acid by M11 and its mutants by including protein flexibility and dynamics in free-energy computation. In addition, we could obtain structural insights into the change in regioselectivity of mefenamic acid hydroxylation due to single active-site mutations. Our findings confirm that use of MD and binding free-energy calculation is useful for studying biocatalysis in those cases in which enzyme binding is a critical event in determining the selective metabolism of a substrate.

Application of engineered cytochrome P450 mutants as biocatalysts for the synthesis of benzylic and aromatic metabolites of fenamic acid NSAIDs

Cytochrome P450 BM3 mutants are promising biocatalysts for the production of drug metabolites. In the present study, the ability of cytochrome P450 BM3 mutants to produce oxidative metabolites of structurally related NSAIDs meclofenamic acid, mefenamic acid and tolfenamic acid was investigated. A library of engineered P450 BM3 mutants was screened with meclofenamic acid (1) to identify catalytically active and selective mutants. Three mono-hydroxylated metabolites were identified for 1. The hydroxylated products were confirmed by NMR analysis to be 3′-OH-methyl-meclofenamic acid (1a), 5-OH-meclofenamic acid (1b) and 4′-OH-meclofenamic acid (1c) which are human relevant metabolites. P450 BM3 variants containing V87I and V87F mutation showed high selectivity for benzylic and aromatic hydroxylation of 1 respectively. The applicability of these mutants to selectively hydroxylate structurally similar drugs such as mefenamic acid (2) and tolfenamic acid (3) was also investigated. The tested variants showed high total turnover numbers in the order of 4000–6000 and can be used as biocatalysts for preparative scale synthesis. Both 1 and 2 could undergo benzylic and aromatic hydroxylation by the P450 BM3 mutants, whereas 3 was hydroxylated only on aromatic rings. The P450 BM3 variant M11 V87F hydroxylated the aromatic ring at 4′ position of all three drugs tested with high regioselectivity. Reference metabolites produced by P450 BM3 mutants allowed the characterisation of activity and regioselectivity of metabolism of all three NSAIDs by thirteen recombinant human P450s. In conclusion, engineered P450 BM3 mutants that are capable of benzylic or aromatic hydroxylation of fenamic acid containing NSAIDs, with high selectivity and turnover numbers have been identified. This shows their potential use as a greener alternative for the generation of drug metabolites.

RS-Predictor Models Augmented with SMARTCyp Reactivities: Robust Metabolic Regioselectivity Predictions for Nine CYP Isozymes

RS-Predictor is a tool for creating pathway-independent, isozyme-specific, site of metabolism (SOM) prediction models using any set of known cytochrome P450 (CYP) substrates and metabolites. Until now, the RS-Predictor method was only trained and validated on CYP 3A4 data, but in the present study, we report on the versatility the RS-Predictor modeling paradigm by creating and testing regioselectivity models for substrates of the nine most important CYP isozymes. Through curation of source literature, we have assembled 680 substrates distributed among CYPs 1A2, 2A6, 2B6, 2C19, 2C8, 2C9, 2D6, 2E1, and 3A4, the largest publicly accessible collection of P450 ligands and metabolites released to date. A comprehensive investigation into the importance of different descriptor classes for identifying the regioselectivity mediated by each isozyme is made through the generation of multiple independent RS-Predictor models for each set of isozyme substrates. Two of these models include a density functional theory (DFT) reactivity descriptor derived from SMARTCyp. Optimal combinations of RS-Predictor and SMARTCyp are shown to have stronger performance than either method alone, while also exceeding the accuracy of the commercial regioselectivity prediction methods distributed by Optibrium and Schrödinger, correctly identifying a large proportion of the metabolites in each substrate set within the top two rank-positions: 1A2 (83.0%), 2A6 (85.7%), 2B6 (82.1%), 2C19 (86.2%), 2C8 (83.8%), 2C9 (84.5%), 2D6 (85.9%), 2E1 (82.8%), 3A4 (82.3%), and merged (86.0%). Comprehensive datamining of each substrate set and careful statistical analyses of the predictions made by the different models revealed new insights into molecular features that control metabolic regioselectivity and enable accurate prospective prediction of likely SOMs.

Regioselective Sulfation and Glucuronidation of Phenolics: Insights into the Structural Basis

The phase II metabolism sulfation and glucuronidation, mediated by sulfotransferases (SULTs) and UDP-glucuronosyltransferases (UGTs) respectively, are significant metabolic pathways for numerous endo-and xenobiotics. Understanding of SULT/UGT substrate specificity including regioselectivity (i.e., position preference) is of great importance in predicting contribution of sulfation/ glucuronidation to drug and metabolite disposition in vivo. This review summarizes regioselective sulfation and glucuronidation of phenolic compounds with multiple hydroxyl (OH) groups as the potential conjugation sites. The strict regioselective patterns are highlighted for several SULT and UGT isoforms towards flavonoids, a large class of natural polyphenols. To seek for a molecular-level explanation, the enzyme structures (i.e., SULT crystal structures and a homology-modeled UGT structure) combined with molecular docking are employed. In particular, the structural basis for regioselective metabolism of flavonoids by SULT1A3 and UGT1A1 is discussed. It is concluded that the regioselective nature of these phase II enzymes is determined by the size and shape of the binding pocket. While the molecular structures of the enzymes can be used to explain regioselective metabolism regarding the binding property, predicting the turnover at different positions remains a particularly difficult task.

Metabolites of puerarin identified by liquid chromatography tandem mass spectrometry: Similar metabolic profiles in liver and intestine of rats

Puerarin is a major active ingredient of Pueraria radix. Puerarin may exert its medicinal functions in part via its metabolites. In this study, we identified these metabolites to better understand and elucidate puerarin's metabolic pathway. Puerarin was intravenously administered to rats and then metabolites in plasma samples were identified by rapid resolution liquid chromatography electrospray ionization-collision induced dissociation tandem mass spectrometry (RRLC-ESI-CID-MS/MS). Chromatography was conducted on a Zorbax SB C18 column (2.1x100 mm, 1.8 microm) at 30 degrees C, with a gradient mobile phase consisting of 0.05% formic acid and acetonitrile, a flow rate of 0.2 mL min(-1), and a total run time of 14 min. MS/MS acquisition parameters were as follows: positive ionization mode, dry gas: nitrogen, 10 L min(-1), dry temperature: 350 degrees C, nebulizer: 40 psi, capillary: -3500 V, scan range: 250-800. The autoMS, manual, or multiple reaction monitoring mode was selected as required. Two glucuronidated metabolites of puerarin (M1 and M2) were detected. M1 and M2 are presumed to be puerarin-7-O-glucuronide and puerarin-4'-O-glucuronide, respectively, and M2 likely is suspected to be the major metabolite because it represented the predominate peak. Kinetic studies of metabolites demonstrated that M1 and M2 were detected in rat plasma at 5 min after intravenous administration of puerarin, the levels of M1 and M2 then reached their peaks at 10-15 and 15-30 min, respectively. The metabolic profiles were similar in rat liver and intestine investigated by in situ liver and intestine perfusion, indicating that no metabolic regioselectivity of puerarin occurs in the two organs.

Metabolic activation of phenanthrene by human and mouse cytochromes P450 and pharmacokinetics in CYP1A2 knockout mice

In the present study V79 Chinese hamster cells were genetically engineered for stable expression of the cytochromes P450 1A1, 1A2, 1B1, and 2E1 from man and mouse to investigate species-specific differences in the regioselective metabolism and toxicity of phenanthrene (Phe), the simplest polycyclic aromatic hydrocarbon (PAH) forming a bay-region. Phe is present in various environmental samples and serves as a model substrate for PAH exposure in human biomonitoring studies. For this reason we explored metabolite profiles and metabolite-dependent cytotoxic activities in vitro. The total turnover of CYP-mediated transformation of Phe was as follows: human CYP1B1 > CYP1A1 > CYP1A2 ≫ CYP2E1, and for mouse CYP1A2 ≫ CYP2E1 > CYP1A1. Striking species differences were seen as mouse CYP1B1 did not activate Phe at all, but human CYP1B1 exhibited a significant metabolic turnover comparable to CYP1A1 and CYP1A2. In vivo studies monitoring the whole blood Phe elimination in CYP1A2 knockout and wild-type mice after oral administration confirmed involvement of CYP1A2 in the bioactivation of Phe, but other processes must contribute also. Our data suggest that in humans not only CYP1A2 expressed solely in the liver plays a crucial role in Phe metabolism, but also constitutively expressed extrahepatic CYP1B1 in tissues such as lung, kidney or intestine. This finding will substantially improve the validity of human biomonitoring studies using individual Phe metabolites for the assessment of PAH exposure.

Taxane's Substituents at C3′ Affect Its Regioselective Metabolism: Different in Vitro Metabolism of Cephalomannine and Paclitaxel

To investigate how taxane's substituents at C3' affect its metabolism, we compared the metabolism of cephalomannine and paclitaxel, a pair of analogs that differ slightly at the C3' position. After cephalomannine was incubated with human liver microsomes in an NADPH-generating system, two monohydroxylated metabolites (M1 and M2) were detected by liquid chromatography/tandem mass spectrometry. C4'' (M1) and C6alpha (M2) were proposed as the possible hydroxylation sites, and the structure of M1 was confirmed by (1)H NMR. Chemical inhibition studies and assays with recombinant human cytochromes P450 (P450s) indicated that 4''-hydroxycephalomannine was generated predominantly by CYP3A4 and 6alpha-hydroxycephalomannine by CYP2C8. The overall biotransformation rate between paclitaxel and cephalomannine differed slightly (184 vs. 145 pmol/min/mg), but the average ratio of metabolites hydroxylated at the C13 side chain to C6alpha for paclitaxel and cephalomannine varied significantly (15:85 vs. 64:36) in five human liver samples. Compared with paclitaxel, the major hydroxylation site transferred from C6alpha to C4'', and the main metabolizing P450 changed from CYP2C8 to CYP3A4 for cephalomannine. In the incubation system with rat or minipig liver microsomes, only 4''-hydroxycephalomannine was detected, and its formation was inhibited by CYP3A inhibitors. Molecular docking by AutoDock suggested that cephalomannine adopted an orientation in favor of 4''-hydroxylation, whereas paclitaxel adopted an orientation favoring 3'-p-hydroxylation. Kinetic studies showed that CYP3A4 catalyzed cephalomannine more efficiently than paclitaxel due to an increased V(m). Our results demonstrate that relatively minor modification of taxane at C3' has major consequence on the metabolism.

Investigation of the genotoxicity of dibenzo[c,p]chrysene in human carcinoma MCF-7 cells in culture.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants that have been linked to certain human cancers. The fjord region PAH dibenzo[a,l]pyrene exhibits the highest levels of carcinogenic activity of all PAH as yet tested in rodent tumor models. Another hexacyclic aromatic hydrocarbon, dibenzo[c,p]chrysene (DBC), is a unique PAH that possesses one bay region and two fjord regions within the same molecule. Due to its structure, which is a merger of the fjord region PAHs benzo[c]phenanthrene, benzo[c]chrysene, and benzo[g]chrysene, DBC is of considerable research interest. In order to investigate the pathway of regioselective metabolism we have studied the cytotoxicity, metabolic activation and DNA adduct formation of DBC in human mammary carcinoma MCF-7 cells in culture. The cytotoxicity assay indicated undisturbed cell proliferation even at concentrations as high as 4.5 microM (1.5 micro g/ml) DBC. Concurrently, DNA adducts were detected in MCF-7 cells treated with DBC only in low amounts (0.6 pmol adducts/mg DNA). On the contrary, exposure to anti-DBC-1,2-diol-3,4-epoxide and anti-DBC-11,12-diol-13,14-epoxide, two putatively genotoxic metabolites of DBC, resulted in high levels of DNA adducts (33 and 51 pmol adducts/mg DNA, respectively). Although DBC was not efficiently transformed into DNA-reactive metabolites in MCF-7 cells in culture, the results from our study indicate that the two fjord region diol-epoxide derivatives of DBC may serve as ultimate genotoxic metabolites once they are enzymatically generated under certain circumstances in vitro or in vivo.

Regioselective metabolism of taxoids by human CYP3A4 and 2C8: structure-activity relationship.

Paclitaxel and docetaxel are metabolized by liver microsomal monooxygenases into inactive metabolites further eliminated from the body via the bile route. In spite of their close chemical structure, the two drugs are oxidized by two different enzymes; CYP2C8 catalyzes the 6-hydroxylation on the taxane ring of paclitaxel, whereas CYP3A4 oxidizes docetaxel on the tert-butyl group of the lateral chain in C13. Since paclitaxel and docetaxel differ only by two substitutions, the role of individual modifications was investigated; the regioselectivity of hydroxylation was assessed by high-pressure liquid chromatography/mass spectrometry, and enzymes implicated in individual reactions were identified using human liver microsomes and recombinant P450 expressed in Ad293 cells. The biotransformation of docetaxel, 10-deacetylpaclitaxel, and 10-deacetylbaccatin III was steadily increased (2- to 5-fold) by the addition of an acetyl group in position 10, suggesting that the presence of a hydrophobic group in position 10 stimulated hydroxylation by P450 proteins. The absence of the lateral chain at C13 in baccatin III severely impaired the metabolism supported by CYP3A4. The presence of a tert-butyl group in the lateral chain of docetaxel favored the hydroxylation on the tert-butyl by CYP3A4, whereas the presence of a phenyl group in the lateral chain facilitated the oxidation on the taxane ring by CYP2C8. Collectively, these data strongly suggested that the structure of the lateral chain and the nature of substituent in position 10 play an important role in determining the regioselective oxidation by P450 proteins and modulate the reaction rate by human liver microsomes.

The effect of gender, sexual maturation and xenobiotic treatment on the formation of hydroxymethyl metabolites from 7,12-dimethylbenz[a]anthracene in rat liver microsomes

The effects of age, gender, strain, phenobarbital (PB) treatment and pituitary influence on the regioselective metabolism of 7,12-dimethylbenz[a]anthracene to hydroxmethyl metabolites were investigated. Studies used hepatic microsomal membranes from immature and mature Long Evans (LE) rats and adult Hooded Lister (HL) animals. Hydroxymethyl metabolites were resolved by both normal and reverse phase HPLC with on-line diode array detection. The CYP isoform(s) responsible for oxidation at the 12 methyl position exhibited no gender or developmental regulation and the rate of formation was not altered following hypophysectomy. PB-treatment of adult rats caused a significant increase in the rate of formation of both male and female animals (29 and 41-fold, respectively) suggesting a major contribution from a PB-inducible isoform, such as CYP2B. The rate of formation of 7OHMe12MBA exhibited no gender dependency in immature animals but was 2-fold greater than that observed for 12OHMe7MBA suggesting that steric hindrance resulting from the adjacent 1,2 benzyl ring favours substrate oxidation at the 7-methyl position. Male predominant formation of 7OHMe12MBA was apparent following sexual maturation of the LE rats and was significantly reduced upon hypophysectomy suggesting the involvement of a male-specific GH dependent isoform e.g. CYP2C11.