Drug metabolizing enzyme activities versus genetic variances for drug of clinical pharmacogenomic relevance

Enzymes are critically important in the transportation, metabolism, and clearance of most therapeutic drugs used in clinical practice today. Many of these enzymes have significant genetic polymorphisms that affect the enzyme's rate kinetics. Regarding drug metabolism, specific polymorphisms to the cytochrome (CYP) P450 enzyme family are linked to phenotypes that describe reaction rates as "ultra", "intermediate", and "poor," as referenced to "extensive" metabolizers that are assigned to wildtype individuals. Activity scores is an alternate designation that provides more genotype-to-phenotype resolution. Understanding the relative change in enzyme activities or rate of clearance of specific drugs relative to an individual's genotypes is an important component in the interpretation of pharmacogenomic data for personalized medicine. Currently, the most relevant drug metabolizing enzymes are CYP 2D6, CYP 2C9, CYP 2C19, thiopurine methyltransferase (TPMT) and UDP-glucuronosyltransferase (UGT). Each of these enzymes is reactive to a host of different drug substrates. Pharmacogenomic tests that are in routine clinical practice include CYP 2C19 for clopidogrel, TPMT for thiopurine drugs, and UDP-1A1 for irinotecan. Other tests where there is considerable data but have not been widely implemented includes CYP 2C9 for warfarin, CYP 2D6 for tamoxifen and codeine, and CYP 2C19 for the proton pump inhibitors.     

Prevalence of CYP2C19 polymorphisms in the Lebanese population

Clopidogrel is one of the most commonly prescribed drugs, as its combination with low-dose aspirin is the recommended oral anti-platelet therapy, to prevent ischaemic events following coronary syndromes or stent placement. Numerous recent studies have shown that polymorphisms in the gene encoding the cytochrome P450 (CYP450) 2C19 enzyme (CYP2C19) contribute to variability in response to clopidogrel; patients with certain common genetic variants of CYP2C19 (*2, *3) have a reduced metabolism of clopidogrel and have a higher rate of cardiovascular events or stent thrombosis compared to patients with the CYP2C19 (*1) allele. CYP2C19*2 is most common in Caucasians, Africans and Asians while CYP2C19*3 has been found mostly in Asians. Since the prevalence of these variants in the Lebanese population has not yet been reported, our aim was to determine the genotypes of CYP2C19 in our population. CYP2C19 (*1/*2/*3) variants were assessed by Polymerase Chain Reaction-Restriction Length Polymorphism (PCR–RFLP) assays in a representative sample of 161 unrelated healthy Lebanese volunteers. The allele frequencies of CYP2C19 *2 and *3 were 0.13 and 0.03. Carriers of the CYP2C19 *2 or *3 represented 24.2% of the subjects. Our data show no significant difference in the frequency of CYP2C19 allelic variants when compared to Caucasian populations and demonstrate that the application of the recent FDA recommendations would also be beneficial in Lebanon, allowing physicians to identify patients at high risk for atherothrombotic events, and eventually advising them to consider other antiplatelet medications or alternative dosing strategies in poor metabolizers.     

Recent advances in the pharmacogenetics of clopidogrel

Clopidogrel has been used to prevent recurrent ischemic events after acute coronary syndrome and/or coronary stent implantation. An impaired platelet response to this drug (residual high platelet reactivity) has been identified as a risk factor for recurrent ischemic events. The platelet response to clopidogrel is highly heritable (73%) suggesting a substantial genetic component. Two sequential cytochrome P450-dependent oxidative steps are required to convert clopidogrel to its active metabolite. The first step leads to the formation of 2-oxo-clopidogrel, which is then metabolized to the active metabolite. Cytochrome P450s are large highly polymorphic family of mono-oxygenases. Many alleles have been reported, and some of these are able to modify the activity of proteins, reducing or increasing the concentration of active metabolites and the drug effect. Loss-of-function variants in the hepatic cytochrome 2C19 (mainly *2 allele) system have been found to be the predominant genetic mediators of clopidogrel response. Variant carriers have higher treatment platelet reactivity and higher risk of adverse cardiac events including stent thrombosis, myocardial infarction, and death. Although value of CYP2C19 genotyping has been demonstrated in ACS population treated with PCI, there is still a wide interindividual variability within each genotype to systematically advocate this genetic testing in clinical practice. The CYP2C19*2 variant only explained 12% of the platelet response to clopidogrel. In the near future, it is highly probable that additional gene variants or epigenetic phenomenon will emerge as significant contributors to clopidogrel response that will allow recommending genetic testing for routine use. The purpose of this review is to discuss the contribution of individual genetic differences responsible for variations of action and clopidogrel efficacy.     

Molecular genetics and epigenetics of the cytochrome P450 gene family and its relevance for cancer risk and treatment

The cytochromes P450 (CYPs) are very efficient catalysts of foreign compound metabolism and are responsible for the major part of metabolism of clinically important drugs. The enzymes are important in cancer since they (a) activate dietary and environmental components to ultimate carcinogens, (b) activate or inactivate drugs used for cancer treatment, and (c) are potential targets for anticancer therapy. The genes encoding the CYP enzymes active in drug metabolism are highly polymorphic, whereas those encoding metabolism of precarcinogens are relatively conserved. A vast amount of literature is present where investigators have tried to link genetic polymorphism in CYPs to cancer susceptibility, although not much conclusive data have hitherto been obtained, with exception of CYP2A6 polymorphism and tobacco induced cancer, to a great extent because of lack of important functional polymorphisms in the genes studied. With respect to anticancer treatment, the genetic CYP polymorphism is of greater importance, where treatment with tamoxifen, but also with cyclophosphamide and maybe thalidomide is influenced by CYP genetic variants. In the present review we present updates on CYP genetics, cancer risk and treatment and also epigenetic aspects of interindividual variability in CYP expression and the use of these enzymes as targets for cancer therapy. We conclude that the CYP polymorphism does not predict cancer susceptibility to any large extent but that this polymorphism might be an important factor for optimal cancer therapy using selected anticancer agents.     

Polymorphic Cytochrome P450 Enzymes (CYPs) and Their Role in Personalized Therapy

The cytochrome P450 (CYP) enzymes are major players in drug metabolism. More than 2,000 mutations have been described, and certain single nucleotide polymorphisms (SNPs) have been shown to have a large impact on CYP activity. Therefore, CYPs play an important role in inter-individual drug response and their genetic variability should be factored into personalized medicine. To identify the most relevant polymorphisms in human CYPs, a text mining approach was used. We investigated their frequencies in different ethnic groups, the number of drugs that are metabolized by each CYP, the impact of CYP SNPs, as well as CYP expression patterns in different tissues. The most important polymorphic CYPs were found to be 1A2, 2D6, 2C9 and 2C19. Thirty-four common allele variants in Caucasians led to altered enzyme activity. To compare the relevant Caucasian SNPs with those of other ethnicities a search in 1,000 individual genomes was undertaken. We found 199 non-synonymous SNPs with frequencies over one percent in the 1,000 genomes, many of them not described so far. With knowledge of frequent mutations and their impact on CYP activities, it may be possible to predict patient response to certain drugs, as well as adverse side effects. With improved availability of genotyping, our data may provide a resource for an understanding of the effects of specific SNPs in CYPs, enabling the selection of a more personalized treatment regimen.     

ABCB1、CES1 和 CYP2C19 基因多态性与 氯吡格雷抵抗的相关性研究进展

氯吡格雷是目前全球临床使用最为广泛的血小板受体抑制剂,但其抗血小板效应存在明显个体差异,部分病人服用常规剂量氯吡格 雷后存在抵抗现象,甚至发生不良临床事件。多项研究表明,ABCB1、CES1 和 CYP2C19 基因多态性对氯吡格雷抵抗的产生发挥重要作用。 简介氯吡格雷体内吸收与代谢机制和氯吡格雷抵抗的定义,综述 ABCB1、CES1 和 CYP2C19 基因多态性对氯吡格雷抵抗的影响。     

Comparison between MassARRAY and pyrosequencing for CYP2C19 and ABCB1 gene variants of clopidogrel efficiency genotyping

Clopidogrel is one of the most frequently used drugs in patients to reduce cardiovascular events. Since patients with different genetic variations respond quite differently to clopidogrel therapy, the related genetic testing plays a vital role in its dosage and genetic testing related to clopidogrel therapy is currently considered as routine test worldwide. In this study, we aim to use two different methods MALDI-TOF mass spectrometry and pyrosequencing to detect gene variant of CYP2C19 and ABCB1. Six single nucleotides polymorphisms (SNP) within CYP2C19 (*2, *3, *4, *5, *17) and ABCB1 C3435T in 458 Chinese Han patients were determined using both MassARRAY and Pyrosequencing. Sanger sequencing was used for verification. Results of both methods were analyzed and compared. Allele frequencies of each SNP and distribution of different genotypes were calculated based on the MassARRAY and Sanger sequencing results. Both methods provided 100% call rates for gene variants, while results of six samples were different with two methods. With Sanger sequencing as the reference results, MassARRAY generated all the same results. The minor allele frequencies of the above six SNPs were 27.1% (CYP2C19*), 5.9% (CYP2C19*3), 0% (CYP2C19*4), 0% (CYP2C19*5), 1.1% (CYP2C19*17), 40.9% (ABCB1), respectively. MassARRAY provides accurate clopidogrel related genotyping with relatively high cost-efficiency, throughput and short time when compared with pyrosequencing.     

CYP2C19 基因多态性与冠心病危险因素对氯吡格雷抵抗的影响

目的:观察细胞色素P450系统药物代谢酶CYP2C19基因多态性以及相关临床因素对氯吡格雷抵抗的影响。方法:选择2010年11月至2011年5月我科拟行PCI术治疗的冠心病患者共145例,均给予氯吡格雷300mg负荷剂量,75mg维持剂量。①通过流式细胞仪检测血管舒张因子刺激酸磷蛋白血小板反应性指数VASP PRI(以VASP PRI≥50%,定义为氯吡格雷抵抗)分为氯吡格雷抵抗组和氯吡格雷反应组。②检测入选患者的药物代谢酶CYP2C19的基因型;根据不同等位基因功能缺失,分为快代谢基因型(*1/*1)、中间代谢基因型(*1/*2、*1/*3)和慢代谢基因型(*2/*2、*2/*3、*3/*3)。③观察CYP2C19基因型及相关临床危险因素对氯吡格雷反应性的影响,④观察氯吡格雷抵抗与临床不良终点事件主要临床不良终点事件[心源性死亡、再发心肌梗死、靶病变再次血运重建术(TLR)]和次要临床终点事件(支架内血栓形成、脑血管意外、大出血)之间的相关性。结果:检测出氯吡格雷抵抗的患者31例,其发生率为20.67%;检测出CYP2C19慢代谢基因型携带患者19例,所占比例为12.67%。慢代谢基因型患者与(快代谢基因型+中间代谢基因型患者)之间VASP PRI比为(49.20±8.45)%VS(44.17±5.41)%,P<0.05,氯吡格雷抵抗发生率之比为35.49%(n=11)VS16.81%(n=20),P<0.05。多元回归分析提示CYP2C19慢代谢基因型(OR:4.43;95%CI:3.28-8.37,P<0.05)和2型糖尿病(OR:2.76;95%CI:2.13-6.14;P<0.05)是氯吡格雷抵抗的两种危险因素。临床随访结果显示氯吡格雷抵抗组与氯吡格雷反应组主要临床不良终点事件的发生率比为6.45%(n=2)vs2.63%(n=3),P<0.05。结论:携带CPY2C19慢代谢基因型和患有2型糖尿病是导致氯吡格雷抵抗的两种重要的危险因素,氯吡格雷抵抗的发生增加了临床不良终点事件的风险。     

Interplay between Genetic and Clinical Variables Affecting Platelet Reactivity and Cardiac Adverse Events in Patients Undergoing Percutaneous Coronary Intervention

Several clinical and genetic variables are associated with influencing high on treatment platelet reactivity (HTPR). The aim of the study was to propose a path model explaining a concurrent impact among variables influencing HTPR and ischemic events. In this prospective cohort study polymorphisms of CYP2C19*2, CYP2C19*17, ABCB1, PON1 alleles and platelet function assessed by Multiple Electrode Aggregometry were assessed in 416 patients undergoing percutaneous coronary intervention treated with clopidogrel and aspirin. The rates of major adverse cardiac events (MACE) were recorded during a 12-month follow up. The path model was calculated by a structural equation modelling. Paths from two clinical characteristics (diabetes mellitus and acute coronary syndrome (ACS)) and two genetic variants (CYP2C19*2 and CYP2C19*17) independently predicted HTPR (path coefficients: 0.11 0.10, 0.17, and -0.10, respectively; p<0.05 for all). By use of those four variables a novel score for prediction of HTPR was built: in a factor-weighted model the risk for HTPR was calculated with an OR of 3.8 (95%CI: 3.1–6.8, p<0.001) for a score level of ≥1 compared with a score of <1. While MACE was independently predicted by HTPR and age in the multivariate model (path coefficient: 0.14 and 0.13, respectively; p<0.05), the coexistence of HTPR and age ≥75 years emerged as the strongest predictor of MACE. Our study suggests a pathway, which might explain indirect and direct impact of variables on clinical outcome: ACS, diabetes mellitus, CYP2C19*2 and CYP2C19*17 genetic variants independently predicted HTPR. In turn, age ≥75 years and HTPR were the strongest predictors of MACE.     

The SPR-based biosensor test system for analysis of small compounds interaction with human cytochrome P450 51A1 (CYP51A1)

The SPR-based test for human cytochrome P450 51A1 (CYP51A1) ligand screening has been developed. Applicability of this system has been validated with known azole inhibitors of cytochromes P450. The studied azoles selectively interacted with human cytochrome P450 51A1, which showed the highest affinity towards ketoconazole. The efficiency of the SPR based assay has been tested using 19 steroid and triterpene compounds, which have not been investigated as potential ligands of CYP51A1.     

Identification of cytochrome P450 2C2 protein complexes in mouse liver

Interactions of microsomal cytochromes P450 (CYPs) with other proteins in the microsomal membrane are important for their function. In addition to their redox partners, CYPs have been reported to interact with other proteins not directly involved in their enzymatic function. In this study, proteins were identified that interact with CYP2C2 in vivo in mouse liver. Flag-tagged CYP2C2 was expressed exogenously in mouse liver and was affinity purified, along with associated proteins which were identified by MS and confirmed by Western blotting. Over 20 proteins reproducibly copurified with CYP2C2. The heterogeneous sedimentation velocity of CYP2C2 and associated proteins by centrifugation in sucrose gradients and sequential immunoprecipitation analysis were consistent with multiple CYP2C2 complexes of differing composition. The abundance of CYPs and other drug metabolizing enzymes and NAD/NADP requiring enzymes associated with CYP2C2 suggest that complexes of these proteins may improve enzymatic efficiency or facilitate sequential metabolic steps. Chaperones, which may be important for maintaining CYP function, and reticulons, endoplasmic reticulum proteins that shape the morphology of the endoplasmic reticulum and are potential endoplasmic reticulum retention proteins for CYPs, were also associated with CYP2C2.     

Biocatalytic conversion of avermectin to 4'-oxo-avermectin: improvement of cytochrome p450 monooxygenase specificity by directed evolution

Discovery of the CYP107Z subfamily of cytochrome P450 oxidases (CYPs) led to an alternative biocatalytic synthesis of 4'-oxo-avermectin, a key intermediate for the commercial production of the semisynthetic insecticide emamectin. However, under industrial process conditions, these wild-type CYPs showed lower yields due to side product formation. Molecular evolution employing GeneReassembly was used to improve the regiospecificity of these enzymes by a combination of random mutagenesis, protein structure-guided site-directed mutagenesis, and recombination of multiple natural and synthetic CYP107Z gene fragments. To assess the specificity of CYP mutants, a miniaturized, whole-cell biocatalytic reaction system that allowed high-throughput screening of large numbers of variants was developed. In an iterative process consisting of four successive rounds of GeneReassembly evolution, enzyme variants with significantly improved specificity for the production of 4'-oxo-avermectin were identified; these variants could be employed for a more economical industrial biocatalytic process to manufacture emamectin.     

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.     

The cytochrome P450 complement (CYPome) of Streptomyces coelicolor A3(2)

In the present study we describe the complete cytochrome P450 complement, the "CYPome," of Streptomyces coelicolor A3(2). Eighteen cytochromes P450 (CYP) are described, in contrast to the absence of CYPs in Escherichia coli, and the twenty observed in Mycobacterium tuberculosis. Here we confirm protein identity as cytochromes P450 by heterologous expression in E. coli and measurement of reduced carbon monoxide difference spectra. We also report on their arrangement in the linear chromosome and relatedness to other CYPs in the superfamily. The future development of manipulation of antibiotic pathways and the use of streptomycetes in bioremediation and biotransformations will involve many of the new CYP forms identified here.     

Biocatalytic Conversion of Avermectin to 4″-Oxo-Avermectin: Improvement of Cytochrome P450 Monooxygenase Specificity by Directed Evolution

Discovery of the CYP107Z subfamily of cytochrome P450 oxidases (CYPs) led to an alternative biocatalytic synthesis of 4″-oxo-avermectin, a key intermediate for the commercial production of the semisynthetic insecticide emamectin. However, under industrial process conditions, these wild-type CYPs showed lower yields due to side product formation. Molecular evolution employing GeneReassembly was used to improve the regiospecificity of these enzymes by a combination of random mutagenesis, protein structure-guided site-directed mutagenesis, and recombination of multiple natural and synthetic CYP107Z gene fragments. To assess the specificity of CYP mutants, a miniaturized, whole-cell biocatalytic reaction system that allowed high-throughput screening of large numbers of variants was developed. In an iterative process consisting of four successive rounds of GeneReassembly evolution, enzyme variants with significantly improved specificity for the production of 4″-oxo-avermectin were identified; these variants could be employed for a more economical industrial biocatalytic process to manufacture emamectin.     

Reduced number of cardiovascular events and increased cost-effectiveness by genotype-guided antiplatelet therapy in patients undergoing percutaneous coronary interventions in the Netherlands

Aim

This study explores clinical outcome in cytochrome P450 2C19 (CYP2C19)-related poor metaboliser patients treated with either clopidogrel or prasugrel after percutaneous coronary intervention (PCI) and investigates whether this could be cost-effective.

Methods and results

This single-centre, observational study included 3260 patients scheduled for elective PCI between October 2010 and June 2013 and followed for adverse cardiovascular events until October 2014. Post PCI, CYP2C19 poor metaboliser patients were treated with clopidogrel or prasugrel, in addition to aspirin. In total, 32 poor metabolisers were treated with clopidogrel and 41 with prasugrel. The number of adverse cardiovascular events, defined as death from cardiovascular cause, myocardial infarction, stent thrombosis, every second visit to the catheterisation room for re-PCI in the same artery, or stroke, within 1.5 years of PCI, was significantly higher in the CYP2C19 poor metaboliser group treated with clopidogrel (n = 10, 31?%) compared with the poor metaboliser group treated with prasugrel (n = 2, 5?%) (p = 0.003). Costs per gained quality-adjusted life years (QALY) were estimated, showing that genotype-guided post-PCI treatment with prasugrel could be cost-effective with less than € 10,000 per gained QALY.

Conclusion

This study provides evidence that for CYP2C19-related poor metabolisers prasugrel may be more effective than clopidogrel to prevent major adverse cardiovascular events after PCI and this approach could be cost-effective.

     

Antitumor drug ellipticine inhibits the activities of rat hepatic cytochromes P450

Ellipticine is a potent antineoplastic agent, whose mode of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Recently, we found that ellipticine also forms the cytochrome P450 (CYP)-mediated covalent DNA adducts. Here, we study the effect of ellipticine on CYP enzymes in rat hepatic microsomes, studying its binding to the enzymes and its potential to inhibit the CYP activities measured with their selective substrates. Although ellipticine was reported to be a selective and strong inhibitor of CYP1A1/2, we found that its inhibitory potential is non-specific. Ellipticine is the most potent inhibitor for CYP3A-dependent 6beta-hydroxylation of progesterone, followed by CYP1A1/2-dependent ethoxyresorufin O-deethylation and CYP2B-mediated pentoxyresorufin O-depentylation. Lower inhibition was detected for 1'-hydroxylation of bufurarol, 21-hydroxylation of progesterone and 6-hydroxylation of chlorzoxazone catalyzed by CYP2D, CYP2C and CYP2E1, respectively. Ellipticine binds to several CYPs of rat hepatic microsomes. The binding titration of ellipticine typically give reverse type I spectrum with CYPs in rat hepatic microsomes. The results indicate that inhibition of CYPs by ellipticine cannot be explained only by its differential potency to bind to individual CYPs.     

Benzophenones as xanthone-open model CYP11B1 inhibitors potentially useful for promoting wound healing

The inhibition of steroidogenic cytochrome P450 enzymes has been shown to play a central role in the management of life-threatening diseases such as cancer, and indeed potent inhibitors of CYP19 (aromatase) and CYP17 (17α hydroxylase/17,20 lyase) are currently used for the treatment of breast, ovarian and prostate cancer. In the last few decades CYP11B1 (11-β-hydroxylase) and CYP11B2 (aldosterone synthase), key enzymes in the biosynthesis of cortisol and aldosterone, respectively, have been also investigated as targets for the identification of new potent and selective agents for the treatment of Cushing's syndrome, impaired wound healing and cardiovascular diseases.In an effort to improve activity and synthetic feasibility of our different series of xanthone-based CYP11B1 and CYP11B2 inhibitors, a small series of imidazolylmethylbenzophenone-based compounds, previously reported as CYP19 inhibitors, was also tested on these new targets, in order to explore the role of a more flexible scaffold for the inhibition of CYP11B1 and -B2 isoforms. Compound 3 proved to be very potent and selective towards CYP11B1, and was thus selected for further optimization via appropriate decoration of the scaffold, leading to new potent 4′-substituted derivatives. In this second series, 4 and 8, carrying a methoxy group and a phenyl ring, respectively, proved to be low-nanomolar inhibitors of CYP11B1, despite a slight decrease in selectivity against CYP11B2. Moreover, unlike the benzophenones of the first series, the 4′-substituted derivatives also proved to be selective for CYP11B enzymes, showing very weak inhibition of CYP19 and CYP17.Notably, the promising result of a preliminary scratch test performed on compound 8 confirmed the potential of this compound as a wound-healing promoter.     

Effect of genetic and coexisting polymorphisms on platelet response to clopidogrel in Chinese Han patients with acute coronary syndrome

Polymorphisms of CYP2C19 are associated with platelet response to clopidogrel. This study was conducted to evaluate the contribution of the previously identified polymorphisms to the response of clopidogrel in a cohort of Chinese Han patients. A total of 222 acute coronary syndrome patients undergoing percutaneous coronary intervention treated with clopidogrel were enrolled from September 2012 to June 2013. Residual platelet aggregations for all patients were measured by the VerifyNow P2Y12 system. Sixteen single-nucleotide polymorphisms among nine genes were genotyped including CYP2C19, ABCB1 and PON1. In this study, CYP2C19*2 and CYP2C19*17 were strongly associated with higher platelet aggregation and lower platelet aggregation to clopidogrel treatment, respectively (P<0.001). Patients with CYP2C19*2 allele had a higher risk of high on-treatment platelet reactivity than non carriers (adjusted OR, 5.434; 95% CI, 1.918–15.399, P=0.01). The coexistence of CYP2B6*9 (rs8192719) and P2Y12 (rs2046934) and the coexistence of CYP2B6*1B (rs7254579) and P2Y12 (rs2046934) were also associated with poor response to clopidogrel. No significant relation of CYP2C19*3 and other polymorphisms to the platelet aggregation was found. In conclusion, CYP2C19*2, CYP2C19*17 coexistence of CYP2B6*9 (rs8192719) and P2Y12 (rs2046934) and coexistence of CYP2B6*1B (rs7254579) and P2Y12 (rs2046934) were identified to be associated with response to clopidogrel treatment in Chinese Han patients.     

Comparison of the 1.85 A structure of CYP154A1 from Streptomyces coelicolor A3(2) with the closely related CYP154C1 and CYPs from antibiotic biosynthetic pathways

The genus Streptomyces produces two-thirds of microbially derived antibiotics. Polyketides form the largest and most diverse group of these natural products. Antibiotic diversity of polyketides is generated during their biosynthesis by several means, including postpolyketide modification performed by oxidoreductases, a broad group of enzymes including cytochrome P450 monooxygenases (CYPs). CYPs catalyze site-specific oxidation of macrolide antibiotic precursors significantly affecting antibiotic activity. Efficient manipulation of Streptomyces CYPs in generating new antibiotics will require identification and/or engineering of monooxygenases with activities toward a diverse array of chemical substrates. To begin to link structure to function of CYPs involved in secondary metabolic pathways of industrially important species, we determined the X-ray structure of Streptomyces coelicolor A3(2) CYP154A1 at 1.85 A and analyzed it in the context of the closely related CYP154C1 and more distant CYPs from polyketide synthase (EryF) and nonribosomal peptide synthetase (OxyB) biosynthetic pathways. In contrast to CYP154C1, CYP154A1 reveals an active site inaccessible from the molecular surface, and an absence of catalytic activities observed for CYP154C1. Systematic variations in the amino acid patterns and length of the surface HI loop correlate with degree of rotation of the F and G helices relative to the active site in CYP154A1-related CYPs, presumably regulating the degree of active site accessibility and its dimensions. Heme in CYP154A1 is in a 180 degrees flipped orientation compared with most other structurally determined CYPs.