Cytochrome P450 2A6 Inhibitors Research Articles

Systematic Review of Naturally Derived Substances That Act as Inhibitors of the Nicotine Metabolizing Enzyme Cytochrome P450 2A6.

Tobacco smoking has been highlighted as a major health challenge in modern societies. Despite not causing death directly, smoking has been associated with several health issues, such as cardiovascular diseases, respiratory disorders, and several cancer types. Moreover, exposure to nicotine during pregnancy has been associated with adverse neurological disorders in babies. Nicotine Replacement Therapy (NRT) is the most common strategy employed for smoking cessation, but despite its widespread use, NRT presents with low success and adherence rates. This is attributed partially to the rate of nicotine metabolism by cytochrome P450 2A6 (CYP2A6) in each individual. Nicotine addiction is correlated with the high rate of its metabolism, and thus, novel strategies need to be implemented in NRT protocols. Naturally derived products are a cost-efficient and rich source for potential inhibitors, with the main advantages being their abundance and ease of isolation. This systematic review aims to summarize the natural products that have been identified as CYP2A6 inhibitors, validated through in vitro and/or in vivo assays, and could be implemented as nicotine metabolism inhibitors. The scope is to present the different compounds and highlight their possible implementation in NRT strategies. Additionally, this information would provide valuable insight regarding CYP2A6 inhibitors, that can be utilized in drug development via the use of in silico methodologies and machine-learning models to identify new potential lead compounds for optimization and implementation in NRT regimes.

Synthesis and biological evaluation of coumarin derivatives as selective CYP2A6 inhibitors

Cytochrome P450 2A6 (CYP2A6) inhibitors are expected to be suitable as smoking cessation aids and for cancer prevention. Because the typical coumarin-based CYP2A6 inhibitor methoxsalen also inhibits CYP3A4, unintended drug-drug interactions are still a concern. Therefore, the development of selective CYP2A6 inhibitors is desirable. In this study, we synthesized coumarin-based molecules, determined the IC50 values for CYP2A6 inhibition, verified the possibility of mechanism-based inhibition, and compared the selectivity for CYP2A6 versus CYP3A4. The results demonstrated that we developed CYP2A6 inhibitors that were more potent and selective than methoxsalen.

CYP2C19 Plays a Major Role in the Hepatic N-Oxidation of Cotinine.

The primary mode of metabolism of nicotine is via the formation of cotinine by the enzyme CYP2A6. Cotinine undergoes further CYP2A6-mediated metabolism by hydroxylation to 3-hydroxycotinine and norcotinine, but can also form cotinine-N-glucuronide and cotinine-N-oxide (COX). The goal of this study was to investigate the enzymes that catalyze COX formation and determine whether genetic variation in these enzymes may affect this pathway. Specific inhibitors of major hepatic cytochrome P450 (P450) enzymes were used in cotinine-N-oxidation reactions using pooled human liver microsomes (HLMs). COX formation was monitored by ultrahigh-pressure liquid chromatography-tandem mass spectrometry and enzyme kinetic analysis was performed using microsomes from P450-overexpressing human embryonic kidney 293 (HEK293) cell lines. Genotype-phenotype analysis was performed in a panel of 113 human liver specimens. Inhibition of COX formation was only observed in HLMs when using inhibitors of CYP2A6, CYP2B6, CYP2C19, CYP2E1, and CYP3A4. Microsomes from cells overexpressing CYP2A6 or CYP2C19 exhibited similar N-oxidation activity against cotinine, with maximum reaction rate over Michaelis constant values (intrinsic clearance) of 4.4 and 4.2 nL/min/mg, respectively. CYP2B6-, CYP2E1-, and CYP3A4-overexpressing microsomes were also active in COX formation. Significant associations (P < 0.05) were observed between COX formation and genetic variants in CYP2C19 (*2 and *17 alleles) in HLMs. These results demonstrate that genetic variants in CYP2C19 are associated with decreased COX formation, potentially affecting the relative levels of cotinine in the plasma or urine of smokers and ultimately affecting recommended smoking cessation therapies. SIGNIFICANCE STATEMENT: This study is the first to elucidate the enzymes responsible for cotinine-N-oxide formation and genetic variants that affect this biological pathway. Genetic variants in CYP2C19 have the potential to modify nicotine metabolic ratio in smokers and could affect pharmacotherapeutic decisions for smoking cessation treatments.

Assessment of CYP2A6 and CYP2C19 Genetic Variation on Cotinine‐N‐Oxide Formation and their Potential Effect on the Nicotine Metabolic Ratio (NMR)

SignificanceThe nicotine metabolic ratio (NMR) of 3‐hydroxycotinine (3HC)/cotinine in smokers is a determinant of CYP2A6 activity and is widely used in clinical practice to help select smoking cessation pharmacotherapy. The major mode of metabolism of nicotine is via the formation of cotinine and subsequently to 3HC by the enzyme CYP2A6. Cotinine is also metabolized to cotinine‐N‐glucuronide and to cotinine‐N‐oxide (COX), accounting for ~7 and 5% of urinary nicotine metabolites, respectively, in smokers. The goal of the present study was to investigate the enzymes that catalyze COX formation and determine whether genetic variation in these enzymes may affect this pathway.MethodsWe used specific inhibitors of the major hepatic cytochrome P450 (CYP) enzymes including CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 in cotinine‐N‐oxidation reactions using pooled human liver microsomes (HLM). COX formation was monitored by UPLC‐MS/MS, and enzyme kinetic analysis was performed using microsomal fractions from CYP‐overexpressing HEK293 cell lines. Additionally, genotype‐phenotype analysis was performed for a panel of 113 HLM specimens.ResultsInhibition of COX formation was only observed when using inhibitors of CYPs 2A6, 2B6, 2C19, 2E1, and 3A4. CYPs 2A6 and 2C19 exhibited similar oxidation activity against cotinine with Vmax/KM values of 4.4, and 4.2 nL•min−1•mg−1, respectively; the Vmax/KM value for the 2B6 isoform was of 2.2 nL•min−1•mg−1. The activity observed for both CYPs 2E1 and 3A4 were low, with KM’s greater than 10 mM. We measured the levels of COX formation in 113 HLM specimens and genotyped variants in CYPs 2A6 (*2, *9, and *14), 2B6 (*2, *5, *9, rs4803419 and rs8109525) and 2C19 (*2 and *17) using qPCR. An association between decreased levels of COX formation and CYP variants in the 2C19 (up to 35% reduction) and 2A6 (up to 38% reduction) isoforms was observed. No statistically significant correlation was observed with variants in the 2B6 isoform.ConclusionsThese results demonstrate that genetic variants in CYPs 2A6 and 2C19 are associated with decreased COX formation, potentially affecting the levels of free cotinine and consequently the calculated NMR in smokers and clinical practices surrounding smoking cessation therapies.Support or Funding InformationThis work is supported by grants from NIH, National Institutes of Environmental Health Sciences (grant R01‐ES025460; to P. Lazarus), the Fulbright‐Garcia Robles Program and CONACyT I2T2 (to Y. Perez‐Paramo), Health Sciences and Services Authority of Spokane, Washington (grant WSU002292 to College of Pharmacy and Pharmaceutical Sciences, Washington State University)

The Novel CYP2A6 Inhibitor, DLCI-1, Decreases Nicotine Self-Administration in Mice.

During tobacco and e-cigarette use, nicotine is mainly metabolized in the human liver by cytochrome P450 2A6 (CYP2A6). Given that a slower CYP2A6 metabolism has been associated with less vulnerability to develop nicotine dependence, the current studies sought to validate a novel CYP2A6 inhibitor, (5-(4-ethylpyridin-3-yl)thiophen-2-yl)methanamine (DLCI-1), for its effects on intravenous nicotine self-administration. Male and female mice were trained to self-administer nicotine across daily sessions. Once stable responding was achieved, DLCI-1 or vehicle control was administered prior to nicotine sessions. We found that the lower 25 mg/kg and moderate 50 mg/kg doses of DLCI-1 induced a significant decrease in nicotine intake for both males and females. DLCI-1 was further shown to be more effective than a moderate 1 mg/kg dose of bupropion on reducing nicotine intake and did not exert the adverse behavioral effects found with a high 75 mg/kg dose of bupropion. Although mice treated with DLCI-1 self-administered significantly less nicotine, similar nicotine-mediated behavioral effects on locomotion were observed. Together, along with the analysis of nicotine metabolites during self-administration, these findings support the contention that blocking hepatic nicotine metabolism would allow for similar activation of nicotinic acetylcholine receptors at lower nicotine doses. Moreover, these effects of DLCI-1 were specific to nicotine self-administration, as DLCI-1 did not result in any behavioral changes during food self-administration. Taken together, these studies validate DLCI-1 as a novel compound to decrease nicotine consumption, which may thereby promote tobacco and nicotine product cessation. SIGNIFICANCE STATEMENT: Current pharmacological approaches for nicotine and tobacco cessation have only been able to achieve limited efficaciousness in promoting long-term abstinence. In this work, we characterize the effects of a novel compound, (5-(4-ethylpyridin-3-yl)thiophen-2-yl)methanamine (DLCI-1), which inhibits the main enzyme that metabolizes nicotine, and we report a significant decrease in intravenous nicotine self-administration in male and female mice, supporting the potential of DLCI-1 as a novel tobacco cessation pharmacotherapeutic.

Cytochrome P450 Enzyme Contributions to the N‐oxide Detoxification Pathway of Tobacco‐Specific Nitrosamines; a Possible Role in Tobacco‐Related Cancer Risk

Tobacco‐specific nitrosamines (TSNAs) including 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) are among the most potent carcinogens found in tobacco products. The major mode of metabolism of NNK is by reduction to form 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol (NNAL), which, like other TSNAs including N′‐nitrosonornicotine (NNN), N′‐nitrosoanabasine (NAB), and N′‐nitrosoanabatine (NAT), is detoxified primarily by UDP‐glucuronosyltransferases (UGTs) to their glucuronide products, but also by N‐oxidation of the common pyridine ring. TSNA‐N‐oxides were identified previously in the urine of rodents exposed to TSNAs, and NNAL‐N‐oxide was found in the urine of smokers. The enzymes responsible for this conversion have not yet been determined. To identify enzymes that catalyze TSNA‐N‐oxide formation, we used specific inhibitors (furafylline, tranylcypromine, clopidrogrel, montelukast, sulfaphenazole, quinidine, chlomethiazole, and ketoconazole) of the major hepatic cytochrome P450 (CYP) enzymes including CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 in oxidation reactions using pooled human liver microsomes. Inhibition of NNK‐N‐oxide and NNAL‐N‐oxide formation was observed when using inhibitors of CYPs 2A6, 2C8, 2C19, 2E1 and 3A4; no detectable levels of NNN‐, NAT‐ or NAB‐N‐oxide formation were observed in human liver microsomes. To confirm results from these inhibition studies, NNK‐N‐oxide and NNAL‐N‐oxide formation were examined using microsomal fractions from CYP over‐expressing HEK293 cell lines. CYPs 2A13, 2C8 and 2E1 exhibited the highest oxidation activity against NNK, with Vmax/KM values of 675, 610 and 559 nL•min−1•mg−1, respectively, and against NNAL, with Vmax/KM values of 105, 67 and 86 nL•min−1•mg−1, respectively. These data suggest that several cytochrome P450 enzymes play an important role in the in situ detoxification of NNK and NNAL and that inter‐individual variability in the activity of these enzymes could potentially be important in TSNA‐induced cancer risk assessment.Support or Funding InformationThis work is supported by grants from NIH, National Institutes of Environmental Health Sciences (grant R01‐ES025460; to P. Lazarus), the Fulbright‐Garcia Robles Program (to Y. Perez‐Paramo), and the Health Sciences and Services Authority of Spokane, Washington (grant WSU002292 to College of Pharmacy and Pharmaceutical Sciences, Washington State University)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Vernonia cinerea Compounds on Drug-metabolizing Cytochrome P450s in Human Liver Microsomes.

Vernonia cinerea has been widely used in traditional medicines for various diseases and shown to aid in smoking abstinence and has anticancer properties. V.cinerea bioactive compounds, including flavonoids and hirsutinolide-type sesquiterpene lactones, have shown an inhibition effect on the nicotine-metabolizing cytochrome P450 2A6 (CYP2A6) enzyme and hirsutinolides reported suppressing cancer growth. In this study, V.cinerea ethanol extract and its bioactive compounds, including four flavonoids and four hirsutinolides, were investigated for an inhibitory effect on human liver microsomal CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 using cocktail inhibition assays combined with LC-MS/MS analysis. Among tested flavonoids, chrysoeriol was more potent in inhibition on CYP2A6 and CYP1A2 than other liver CYPs, with better binding efficiency toward CYP2A6 than CYP1A2 (Ki values in competitive mode of 1.93±0.05 versus 3.39±0.21μM, respectively). Hirsutinolides were prominent inhibitors of CYP2A6 and CYP2D6, with IC50 values of 12-23 and 15-41μM, respectively. These hirsutinolides demonstrated time-dependent inhibition, an indication of mechanism-based inactivation, toward CYP2A6. Quantitative prediction of microsomal metabolism of these flavonoids and hirsutinolides, including half-lives and hepatic clearance rate, was examined. These findings may have implications for further in vivo studies of V.cinerea. Copyright © 2017 John Wiley & Sons, Ltd.

In vivo modulation of the behavioral effects of nicotine by the coumarins xanthotoxin, bergapten, and umbelliferone.

RationaleNicotine, a dominant alkaloid found in tobacco, is responsible for physical dependence, as well as addiction to cigarette smoking; consequently, smoking cessation is a very difficult process. Hepatic cytochrome P-450 2A6 (CYP2A6) is involved in the 70–80 % of the initial metabolism of nicotine and its co-metabolites. As this metabolism is slowed by inhibitors of CYP2A6, this kind of enzymatic inhibition has been proposed as a novel target for smoking cessation.ObjectivesNicotine administered alone improved memory acquisition and consolidation as well as exerted antidepressive activity in animal models. These effects persist for 24 h. However, they are completely extinguished 48 h after administration.MethodsTo investigate if the coumarins prolong the behavioral effects of nicotine, the forced swimming test (FST)—animal models of depression, and passive avoidance (PA) test—memory and learning paradigm were used.ResultsThis study revealed that three CYP2A6 inhibitors: two furanocoumarins, xanthotoxin (15 mg/kg) and bergapten (25 mg/kg), and the simple coumarin umbelliferone (25 mg/kg), prolonged the antidepressive and procognitive effects of nicotine.ConclusionsThese natural products may offer a new approach to the treatment of nicotinism as antidepressant and memory improvement actions are one of the main factors of nicotine dependence.

Common and Distinct Interactions of Chemical Inhibitors with Cytochrome P450 CYP1A2, CYP2A6 and CYP2B6 Enzymes

Tobacco smoking is a leading cause of preventable disease and death globally. Nicotine is the main addictive component in tobacco. Nicotine is eliminated from the body by biotransformation in the liver to inactive metabolites. This reaction is catalyzed by the cytochrome P450 2A6 (CYP2A6) enzyme. Administering chemical inhibitors of CYP2A6 has been shown to slow down the elimination of nicotine with consequent reduction in number of cigarettes smoked. We have systematically developed small molecule CYP2A6 inhibitors with good balance between potency and CYP selectivity. During this process we have noticed that many potent CYP2A6 inhibitors also inhibit other human liver CYP forms, most notably CYP1A2 and CYP2B6. This study aimed at defining common and distinct features of ligand binding to CYP1A2, CYP2A6 and CYP2B6 active sites. We used our previous chemical inhibitor databases to construct improved 3-dimensional quantitative structureactivity relationship (3D-QSAR) models for CYP1A2, CYP2A6 and CYP2B6. Combined 3D-QSAR and docking procedures yielded precise information about the common and distinct interactions of inhibitors and the enzyme active sites. Positioning of hydrogen bond donor/acceptor atoms and the shape and volume of the compound defined the potency and specificity of inhibition. A novel potent and selective CYP1A2 inhibitor was found. This in silico approach will provide a means for very rapid and high throughput prediction of cross-inhibition of these three CYP enzymes.

Binding free energies for nicotine analogs inhibiting cytochrome P450 2A6 by a combined use of molecular dynamics simulations and QM/MM-PBSA calculations

Molecular dynamics (MD) simulations and hybrid quantum mechanical/molecular mechanical (QM/MM) calculations have been performed to explore the dynamic behaviors of cytochrome P450 2A6 (CYP2A6) binding with nicotine analogs (that are typical inhibitors) and to calculate their binding free energies in combination with Poisson–Boltzmann surface area (PBSA) calculations. The combined MD simulations and QM/MM-PBSA calculations reveal that the most important structural parameters affecting the CYP2A6-inhibitor binding affinity are two crucial internuclear distances, that is, the distance between the heme iron atom of CYP2A6 and the coordinating atom of the inhibitor, and the hydrogen-bonding distance between the N297 side chain of CYP2A6 and the pyridine nitrogen of the inhibitor. The combined MD simulations and QM/MM-PBSA calculations have led to dynamic CYP2A6-inhibitor binding structures that are consistent with the observed dynamic behaviors and structural features of CYP2A6-inhibitor binding, and led to the binding free energies that are in good agreement with the experimentally-derived binding free energies. The agreement between the calculated binding free energies and the experimentally-derived binding free energies suggests that the combined MD and QM/MM-PBSA approach may be used as a valuable tool to accurately predict the CYP2A6-inhibitor binding affinities in future computational design of new, potent and selective CYP2A6 inhibitors.

Inhibitory Potency of 8-Methoxypsoralen on Cytochrome P450 2A6 (CYP2A6) Allelic Variants CYP2A6*15, CYP2A6*16, CYP2A6*21 and CYP2A6*22: Differential Susceptibility Due to Different Sequence Locations of the Mutations

Human cytochrome P450 2A6 (CYP2A6) is a highly polymorphic isoform of CYP2A subfamily. Our previous kinetic study on four CYP2A6 allelic variants (CYP2A6*15, CYP2A6*16, CYP2A6*21 and CYP2A6*22) have unveiled the functional significance of sequence mutations in these variants on coumarin 7-hydroxylation activity. In the present study, we further explored the ability of a typical CYP2A6 inhibitor, 8-methoxypsoralen (8-MOP), in inhibition of these alleles and we hypothesized that translational mutations in these variants are likely to give impact on 8-MOP inhibitory potency. The CYP2A6 variant and the wild type proteins were subjected to 8-MOP inhibition to yield IC50 values. In general, a similar trend of change in the IC50 and Km values was noted among the four mutants towards coumarin oxidation. With the exception of CYP2A6*16, differences in IC50 values were highly significant which implied compromised interaction of the mutants with 8-MOP. Molecular models of CYP2A6 were subsequently constructed and ligand-docking experiments were performed to rationalize experimental data. Our docking study has shown that mutations have induced enlargement of the active site volume in all mutants with the exception of CYP2A6*16. Furthermore, loss of hydrogen bond between 8-MOP and active site residue Asn297 was evidenced in all mutants. Our data indicate that the structural changes elicited by the sequence mutations could affect 8-MOP binding to yield differential enzymatic activities in the mutant CYP2A6 proteins.

Nicotine and 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone Binding and Access Channel in Human Cytochrome P450 2A6 and 2A13 Enzymes

Cytochromes P450 (CYP) from the 2A subfamily are known for their roles in the metabolism of nicotine, the addictive agent in tobacco, and activation of the tobacco procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Although both the hepatic CYP2A6 and respiratory CYP2A13 enzymes metabolize these compounds, CYP2A13 does so with much higher catalytic efficiency, but the structural basis for this has been unclear. X-ray structures of nicotine complexes with CYP2A13 (2.5 Å) and CYP2A6 (2.3 Å) yield a structural rationale for the preferential binding of nicotine to CYP2A13. Additional structures of CYP2A13 with NNK reveal either a single NNK molecule in the active site with orientations corresponding to metabolites known to form DNA adducts and initiate lung cancer (2.35 Å) or with two molecules of NNK bound (2.1 Å): one in the active site and one in a more distal staging site. Finally, in contrast to prior CYP2A structures with enclosed active sites, CYP2A13 conformations were solved that adopt both open and intermediate conformations resulting from an ∼2.5 Å movement of the F to G helices. This channel occurs in the same region where the second, distal NNK molecule is bound, suggesting that the channel may be used for ligand entry and/or exit from the active site. Altogether these structures provide multiple new snapshots of CYP2A13 conformations that assist in understanding the binding and activation of an important human carcinogen, as well as critical comparisons in the binding of nicotine, one of the most widely used and highly addictive drugs in human use.

Docking, molecular dynamics simulation studies, and structure-based QSAR model on cytochrome P450 2A6 inhibitors

In this study, we used molecular docking and molecular dynamics (MD) simulation studies to analyze the interactions of a series of naphthalene and non-naphthalene derivatives (n = 38) as potent inhibitors of cytochrome P450 2A6 (CYP2A6) and to construct a structure-based quantitative structure activity relationship (QSAR) model. A 6000 ps MD simulation in a cubic water box were employed to build 3D structure of the CYP2A6 in a water environment. After reaching the equilibrium, the most potent inhibitor (2-bromonaphthalene) was docked into the CYP2A6 to realize the binding site of the enzyme. The docking analysis showed that π–π interaction of the inhibitors with four phenylalanine residues at positions of 107, 111, 118, and 480 plays an important role in the activities of the inhibitors. Then, an additional 6000 ps MD simulation was performed on the complex of the CYP2A6–2-bromonaphthalene to explore the effect of the inhibitor on the stability of the protein–inhibitor complex. Radius of gyration values for CYP2A6 and CYP2A6–2-bromonaphthalene complex were 2.23 ± 0.01 and 2.21 ± 0.01 nm, respectively, which revealed that the structure of the CYP2A6 in the presence of 2-bromonaphthalene has not changed. Finally, all the inhibitors docked into the enzyme and the docked configuration of the inhibitors with the lowest free energy was used to calculate the most feasible descriptors. The selected descriptors were related to the inhibitory activities using multiple linear regression (MLR) and least squares support vector regression (LS-SVR) models. The Q2 values for MLR and LS-SVR were 0.695 and 0.728, respectively.

8-Methoxypsoralen, a potent human CYP2A6 inhibitor, inhibits lung adenocarcinoma development induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in female A/J mice

Previously, we demonstrated that 8-methoxypsoralen (methoxsalen), a potent human cytochrome P450 2A6 (CYP2A6) inhibitor, strongly suppresses lung adenoma induction by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in female A/J mice. In the present study, we examined the inhibitory effects of methoxsalen on the development of lung adenocarcinomas, as well as on adenomas and alveolar hyperplasia. Female A/J mice were treated with methoxsalen at doses of 12.5 or 1.25 mg/kg body weight, administered by stomach tube once daily for 3 days. One hour after the final treatment, NNK was injected i.p. at a dose of 2 mg/mouse. The experiments were terminated 52 weeks after the first methoxsalen treatment, and lung adenomas and adenocarcinomas were analyzed histopathologically. Pretreatment with methoxsalen significantly reduced the incidence of adenocarcinomas from 94.7 to 46.7% (12.5 mg/kg) and 44.4% (1.25 mg/kg), and their tumor multiplicity from 4.68 to 0.87 (12.5 mg/kg) and 0.61 (1.25 mg/kg) tumors/mouse. The tumor multiplicity of adenomas and adenocarcinomas in the methoxsalen-treated groups was significantly reduced from 12.47 to 5.67 (12.5 mg/kg) and 4.28 (1.25 mg/kg) tumors/mouse. Approximately 60% of the adenocarcinomas arose within adenomas. In comparing the methoxsalen + NNK and NNK alone groups, there was no significant difference in the frequency of such compound lesions, indicating that pretreatment with methoxsalen did not suppress the eventual progression of adenomas to adenocarcinomas. These results clearly demonstrate that methoxsalen, a potent human CYP2A6 inhibitor, inhibits not only lung adenoma but also adenocarcinoma development.

Exploring QSAR and QAAR for inhibitors of cytochrome P450 2A6 and 2A5 enzymes using GFA and G/PLS techniques

A series of naphthalene and non-naphthalene derivatives ( n = 42) having cytochrome P450 2A6 and 2A5 inhibitory activities, reported by Rahnasto et al., were subjected to QSAR and QAAR studies. The analyses were performed using electronic, spatial, shape and thermodynamic descriptors to develop quantitative models for prediction of the inhibitory activities and to explore importance of different descriptors for the responses. The data set was divided into training and test sets (with test set size being approximately 25% of the full data set size) based on K-means clustering applied on the standardized descriptor matrix. Genetic function approximation (GFA) and genetic partial least-squares (G/PLS) were used as chemometric tools for modeling, and the derived equations were of acceptable statistical and prediction (both internal and external) qualities although different equations varied in quality in a wide range ( R 2: 0.561–0.898, R a 2: 0.508–0.870, Q 2: 0.495–0.814, R pred 2: 0.615–0.914, r 2: 0.679–0.905, r 0 2: 0.639–0.904, r m 2: 0.494–0.876). In the case of CYP2A5 inhibition, the GFA derived QSAR model is better than the G/PLS derived model considering both internal and external validations. In the case of CYP2A6 inhibitory potency data, the GFA derived QSAR model is better than the G/PLS model considering internal validation whereas the latter is better in external validation (which is more important) than the former. The model development process was subjected to randomization test at 90% confidence level by taking into account the whole pool of descriptors, while the developed models were also subjected to randomization test (99% confidence level) for validation. Based on the randomization test results, GFA models are found to be superior to the G/PLS models. Among the parameters, which were found important in modeling both the responses, were different Jurs descriptors, electronic descriptors (like Sr, Apol), steric descriptors (like shadow indices, Molref), shape descriptors (like COSV, Fo) and lipophilicity descriptors. This indicates that the CYP2A5 and CYP2A6 inhibition of these compounds is related to charge distribution, surface area, electronic, hydrophobic and spatial properties of the molecules.

Identification of inhibitors of the nicotine metabolising CYP2A6 enzyme—an in silico approach

The compulsive nature of tobacco use is attributable to nicotine addiction. Nicotine is eliminated by metabolism through the cytochrome P450 2A6 (CYP2A6) enzyme in liver. Inhibition of CYP2A6 by chemical compounds may represent a potential supplement to anti-smoking therapy. The purpose of this study was to rationally design potent inhibitors of CYP2A6. 3D-QSAR models were constructed to find out which structural characteristics are important for inhibition potency. Specifically located hydrophobic and hydrogen donor features were found to affect inhibition potency. These features were used in virtual screening of over 60,000 compounds in the Maybridge chemical database. A total of 22 candidate molecules were selected and tested for inhibition potency. Four of these were potent and selective CYP2A6 inhibitors with IC(50) values lower than 1 muM. They represent novel structures of CYP2A6 inhibitors, especially N1-(4-fluorophenyl)cyclopropane-1-carboxamide. This compound can be used as a lead in the design of CYP2A6 inhibitor drugs to combat nicotine addiction.

Synthetic Inhibitors of Cytochrome P-450 2A6: Inhibitory Activity, Difference Spectra, Mechanism of Inhibition, and Protein Cocrystallization

A series of 3-heteroaromatic analogues of nicotine were synthesized to delineate structural and mechanistic requirements for selectively inhibiting human cytochrome P450 (CYP) 2A6. Thiophene, substituted thiophene, furan, substituted furan, acetylene, imidazole, substituted imidazole, thiazole, pyrazole, substituted pyrazole, and aliphatic and isoxazol moieties were used to replace the N-methylpyrrolidine ring of nicotine. A number of potent inhibitors were identified, and several exhibited high selectivity for CYP2A6 relative to CYP2E1, -3A4, -2B6, -2C9, -2C19, and -2D6. The majority of these inhibitors elicited type II difference spectra indicating the formation of a coordinate covalent bond to the heme iron. The majority of inhibitors were reversible inhibitors although several mechanism-based inactivators were identified. Most of the inhibitors were also relatively metabolically stable. X-ray crystal structures of CYP2A6 cocrystallized with three furan analogues bearing methanamino side chains indicated that the amine side chain coordinated to the heme iron. The pyridyl moiety was positioned to accept a hydrogen bond from Asn297, and all three inhibitors exhibited orthogonal aromatic-aromatic interactions with protein side chains. For comparison, the cocrystal structure of 4,4'-dipyridyl disulfide was also obtained and showed that the pyridine moiety could assume a different orientation than that observed for the 3-heteroaromatic pyridines examined. For the 3-heteroromatic pyridines, N-methyl and N,N-dimethyl amino groups increased the apparent Ki and distorted helix I of the protein. Substitution of a phenyl ring for the pyridyl ring also increased the apparent Ki, which is likely to reflect the loss of the hydrogen bonding interaction with Asn297. In contrast, inhibitory potency for other P450s was increased, and the selectivity of the phenyl analogues for CYP2A6 was decreased relative to the pyridyl compounds. The results suggest that inhibitors that compliment the active site features of CYP2A6 can exhibit significant selectivity for CYP2A6 relative to other human liver drug-metabolizing P450s.

Genetic and Environmental Influences on Therapeutic and Toxicity Outcomes: Studies with CYP2A6

The idea that the liver enzyme cytochrome P450 2A6 (CYP2A6), known also as nicotine C-oxidase, is one of the determinants of smoking addiction and smoking behavior is primarily based on its role in nicotine metabolism and disposition. The results of studies linking the CYP2A6 genetic polymorphism with smoking dependence and smoking behavior however remain controversial. The most likely causes of the controversies appeared to be consideration given to a few allelic variants coupled with the uses of the CYP2A6 alleles lacking in vivo phenotypic validation. In the present review, we summarize research findings on biological significance of CYP2A6 and gene polymorphisms together with a discussion on CYP2A6 inhibitors that hold the promise of uses in smoking cessation. In addition, we provide the phenotype/genotype information derived from our systematic investigation on the relationship between CYP2A6 genotypes, smoking habits and coumarin metabolism phenotypes in a group of 393 normal adults (197 women and 196 men), 16 to 60 years of age, whose exposure to cadmium and lead were also determined, enabling us to assess the CYP2A6 phenotypic variability associated with CYP2A6 genotypes and environmental exposure. The results indicate that the phenotype of CYP2A6 enzyme in liver is an outcome of interactions between the CYP2A6 gene, cadmium, nicotine and possibly its metabolites.

5-Substituted, 6-Substituted, and Unsubstituted 3-Heteroaromatic Pyridine Analogues of Nicotine as Selective Inhibitors of Cytochrome P-450 2A6

A series of 5- and 6-substituted and unsubstituted 3-heteroaromatic analogues of nicotine were synthesized in an effort to delineate the structural requirements for selectively inhibiting human cytochrome P-450 (CYP) 2A6, the major nicotine metabolizing enzyme. Thiophene, substituted thiophene, furan, substituted furan, imidazole, substituted imidazole, pyridine, substituted pyridine, thiazole, and quinoline moieties were used to replace the N-methylpyrrolidine ring of nicotine. Bromo and methyl groups were introduced at the 5-position of the pyridine ring and fluoro, chloro, and methoxy groups were placed at the 6-position of the pyridine ring in order to explore the structure-activity relationship (SAR) of inhibition of CYP2A6. The inhibitory activity of the most potent CYP2A6 inhibitors on the functional activity of human cytochrome P450s 3A4, 2E1, 2B6, 2C9, 2C19, and 2D6 was also examined to determine inhibitor selectivity. We identified 36 compounds that were more potent than nicotine at inhibition of coumarin 7-hydroxylase (CYP2A6) activity. We also found a number of compounds to be highly selective for the inhibition of human CYP2A6 versus the other human CYPs examined.

Halothane-dependent lipid peroxidation in human liver microsomes is catalyzed by cytochrome P4502A6 (CYP2A6).

Halothane is extensively (approximately 50%) metabolized in humans and undergoes both oxidative and reductive cytochrome P450-catalyzed hepatic biotransformation. Halothane is reduced under low oxygen tensions by CYP2A6 and CYP3A4 in human liver microsome to an unstable free radical, and then to the volatile metabolites chlorodifluoroethene (CDE) and chlorotrifluoroethane (CTE). The free radical is also thought to initiate lipid peroxidation. Halothane-dependent lipid peroxidation has been shown in animals in vitro and in vivo but has not been evaluated in humans. This investigation tested the hypothesis that halothane causes lipid peroxidation in human liver microsomes, identified P450 isoforms responsible for halothane-dependent lipid peroxidation, and tested the hypothesis that lipid peroxidation is prevented by inhibiting halothane reduction. Halothane metabolism was determined using human liver microsomes or cDNA-expressed P450. Lipid peroxidation was quantified by malondialdehyde (MDA) formation using high-pressure liquid chromatography-ultraviolet analysis of the thiobarbituric acid-MDA adduct. CTE and CDE were determined by gas chromatography-mass spectrometry. Halothane caused MDA formation in human liver microsomes at rates much lower than in rat liver microsomes. Human liver microsomal MDA production exhibited biphasic enzyme kinetics, similar to CDE and CTE production. MDA production was inhibited by the CYP2A6 inhibitor methoxsalen but not by the CYP3A4 inhibitor troleandomycin. Halothane-dependent MDA production was catalyzed by cDNA-expressed CYP2A6 but not CYP3A4 or P450 reductase alone. CYP2A6-catalyzed MDA production was inhibited by methoxsalen or anti-CYP2A6 antibody. Halothane causes lipid peroxidation in human liver microsomes, which is catalyzed by CYP2A6, and inhibition of halothane reduction prevents halothane-dependent lipid peroxidation in vitro.