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Structure-Activity Studies Reveal the Oxazinone Ring Is a Determinant of Cytochrome P450 2B6 Activity Toward Efavirenz.

ABSTRACT: Cytochrome P450 2B6 (CYP2B6) is primarily responsible for the metabolism of the anti-HIV drug efavirenz (EFV). We set out to explore the molecular basis for CYP2B6 activity toward EFV by examining the metabolism of eight EFV analogues. cDNA-expressed CYP2B6 formed monooxygenated metabolites from EFV analogues containing an intact oxazinone or oxazine ring, but not from analogues with a disrupted ring, suggesting this ring is important for metabolism of EFV by CYP2B6. Subsequent substrate depletion analysis of EFV and EFV analogues found to be CYP2B6 substrates revealed further differences between these CYP2B6 substrates. Compounds that were not found to be CYP2B6 substrates were still able to inhibit CYP2B6 activity toward a known substrate, bupropion, suggesting they do gain access to the CYP2B6 active site. Taken together, these data reveal structural characteristics of EFV, namely, the oxazinone ring, that are critical for CYP2B6 metabolism of compounds with the EFV chemical scaffold.

SUBMITTER: Cox PM

PROVIDER: S-EPMC4191608 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Structure-Activity Studies Reveal the Oxazinone Ring Is a Determinant of Cytochrome P450 2B6 Activity Toward Efavirenz.

Cox Philip M PM Bumpus Namandjé N NN

ACS medicinal chemistry letters 20140904 10

Cytochrome P450 2B6 (CYP2B6) is primarily responsible for the metabolism of the anti-HIV drug efavirenz (EFV). We set out to explore the molecular basis for CYP2B6 activity toward EFV by examining the metabolism of eight EFV analogues. cDNA-expressed CYP2B6 formed monooxygenated metabolites from EFV analogues containing an intact oxazinone or oxazine ring, but not from analogues with a disrupted ring, suggesting this ring is important for metabolism of EFV by CYP2B6. Subsequent substrate depleti ...[more]

PMID: 25309681

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Project description:The effects of genetic variation of cytochrome P450 2B6 (CYP2B6) and constitutive androstane receptor (CAR) on efavirenz (EFV) plasma concentration was evaluated among 312 HIV patients in Nairobi Kenya. The EFV plasma concentration at steady-state were determined using ultra-high-performance liquid chromatography with a tandem quadruple mass spectrometer (LC-MS/MS). Thirteen CYP2B6 (329G>T, 341T>C, 444 G>T/C, 15582C>T, 516G>T, 548T>G, 637T>C, 785A>G, 18492C>T, 835G>C, 1459C>T and 21563C>T) and one CAR (540C>T) single nucleotide polymorphisms (SNPs) were genotyped using real-time polymerase chain reaction. HIV drug resistance mutations were detected using an in-house genotypic assay. The EFV concentration of patients ranged from 4 ng/mL to 332697 ng/mL (median 2739.5 ng/mL, IQR 1878-4891.5 ng/mL). Overall, 22% patients had EFV concentrations beyond therapeutic range of 1000-4000 ng/mL (4.5%% < 1000 ng/mL and 31.7% > 4000 ng/mL). Five SNPs (15582C>T, 516G>T, 785A>G, 983T>C and 21563C>T) were associated with higher EFV plasma concentration while 18492C>T with lower EFV plasma concentration (p<0.05). Strong linkage disequilibrium (LD) was observed for 15582C>T, 516G>T, 785A>G, 18492C>T, 983T>C, 21563C>T, 1459C>T and CAR 540C>T. Sixteen haplotypes were observed and CTGCTTCC, CTGCTTCT, TTGCTTCT and CGACCCCT were associated with high EFV plasma concentration. In multivariate analysis, factors significantly associated with EFV plasma concentration included; the presence of skin rash (β = 1379, 95% confidence interval (CI) = 3216.9-3416.3; p < 0.039), T allele of CYP2B6 516G>T (β = 1868.9, 95% CI 3216.9-3416.3; p < 0.018), the C allele of CYP2B6 983T>C (β = 2638.3, 95% CI = 1348-3929; p < 0.0001), T allele of CYP2B6 21563C>T (β = 1737, 95% CI = 972.2-2681.9; p < 0.0001) and the presence of 5 to 7 numbers of SNPs per patient (β = 570, 95% CI = 362-778; p < 0.0001) and HIV viral load ≤1000 cells/mL (β = -4199.3, 95% CI = -7914.9 --483.6; p = 0.027). About 36.2% of the patients had EFV plasma concentrations beyond therapeutic window, posing high risk of treatment failure or toxicity. The SNPs of CYP2B6 516G>T, CYP2B6 983T>C, 21563C>T, presence of higher numbers of SNPs per patient and haplotypes CTGCTTCC, CTGCTTCT, TTGCTTCT and CGACCCCT could efficiently serves as genetic markers for EFV plasma concentration and could guide personalization of EFV based ART treatment in Kenya.

Project description:Cytochrome P450 2B6 (CYP2B6) belongs to the minor drug metabolizing P450s in human liver. Expression is highly variable both between individuals and within individuals, owing to non-genetic factors, genetic polymorphisms, inducibility, and irreversible inhibition by many compounds. Drugs metabolized mainly by CYP2B6 include artemisinin, bupropion, cyclophosphamide, efavirenz, ketamine, and methadone. CYP2B6 is one of the most polymorphic CYP genes in humans and variants have been shown to affect transcriptional regulation, splicing, mRNA and protein expression, and catalytic activity. Some variants appear to affect several functional levels simultaneously, thus, combined in haplotypes, leading to complex interactions between substrate-dependent and -independent mechanisms. The most common functionally deficient allele is CYP2B6*6 [Q172H, K262R], which occurs at frequencies of 15 to over 60% in different populations. The allele leads to lower expression in liver due to erroneous splicing. Recent investigations suggest that the amino acid changes contribute complex substrate-dependent effects at the activity level, although data from recombinant systems used by different researchers are not well in agreement with each other. Another important variant, CYP2B6*18 [I328T], occurs predominantly in Africans (4-12%) and does not express functional protein. A large number of uncharacterized variants are currently emerging from different ethnicities in the course of the 1000 Genomes Project. The CYP2B6 polymorphism is clinically relevant for HIV-infected patients treated with the reverse transcriptase inhibitor efavirenz, but it is increasingly being recognized for other drug substrates. This review summarizes recent advances on the functional and clinical significance of CYP2B6 and its genetic polymorphism, with particular emphasis on the comparison of kinetic data obtained with different substrates for variants expressed in different recombinant expression systems.

Dataset Information

Structure-Activity Studies Reveal the Oxazinone Ring Is a Determinant of Cytochrome P450 2B6 Activity Toward Efavirenz.

Publications

Structure-Activity Studies Reveal the Oxazinone Ring Is a Determinant of Cytochrome P450 2B6 Activity Toward Efavirenz.

Similar Datasets

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