Deciphering Structural Alterations Associated with Activity Reductions of Genetic Polymorphisms in Cytochrome P450 2A6 Using Molecular Dynamics Simulations.

Cytochrome P450 (CYP) 2A6 is a monooxygenase involved in the metabolism of various endogenous and exogenous chemicals, such as nicotine and therapeutic drugs. The genetic polymorphisms in CYP2A6 are a cause of individual variation in smoking behavior and drug toxicities. The enzymatic activities of the allelic variants of CYP2A6 were analyzed in previous studies. However, the three-dimensional structures of the mutants were not investigated, and the mechanisms underlying activity reduction remain unknown. In this study, to investigate the structural changes involved in the reduction in enzymatic activities, we performed molecular dynamics simulations for ten allelic mutants of CYP2A6. For the calculated wild type structure, no significant structural changes were observed in comparison with the experimental structure. On the other hand, the mutations affected the interaction with heme, substrates, and the redox partner. In CYP2A6.44, a structural change in the substrate access channel was also observed. Those structural effects could explain the alteration of enzymatic activity caused by the mutations. The results of simulations provide useful information regarding the relationship between genotype and phenotype.

Combined Linear Interaction Energy and Alchemical Solvation Free-Energy Approach for Protein-Binding Affinity Computation.

Calculating free energies of binding (ΔGbind) between ligands and their target protein is of major interest to drug discovery and safety, yet it is still associated with several challenges and difficulties. Linear interaction energy (LIE) is an efficient in silico method for ΔGbind computation. LIE models can be trained and used to directly calculate binding affinities from interaction energies involving ligands in the bound and unbound states only, and LIE can be combined with statistical weighting to calculate ΔGbind for flexible proteins that may bind their ligands in multiple orientations. Here, we investigate if LIE predictions can be effectively improved by explicitly including the entropy of (de)solvation into our free-energy calculations. For that purpose, we combine LIE calculations for the protein-ligand-bound state with explicit free-energy perturbation to rigorously compute the unbound ligand's solvation free energy. We show that for 28 Cytochrome P450 2A6 (CYP2A6) ligands, coupling LIE with alchemical solvation free-energy calculation helps to improve obtained correlation between computed and reference (experimental) binding data.

Cytochrome P450 2A6 and other human P450 enzymes in the oxidation of flavone and flavanone.

1. We previously reported that flavone and flavanone interact spectrally with cytochrome P450 (P450 or CYP) 2A6 and 2A13 and other human P450s and inhibit catalytic activities of these P450 enzymes. In this study, we studied abilities of CYP1A1, 1A2, 1B1, 2A6, 2A13, 2C9 and 3A4 to oxidize flavone and flavanone. 2. Human P450s oxidized flavone to 6- and 5-hydroxylated flavones, seven uncharacterized mono-hydroxylated flavones, and five di-hydroxylated flavones. CYP2A6 was most active in forming 6-hydroxy- and 5-hydroxyflavones and several mono- and di-hydroxylated products. 3. CYP2A6 was also very active in catalyzing flavanone to form 2'- and 6-hydroxyflavanones, the major products, at turnover rates of 4.8 min-1 and 1.3 min-1, respectively. Other flavanone metabolites were 4'-, 3'- and 7-hydroxyflavanone, three uncharacterized mono-hydroxylated flavanones and five mono-hydroxylated flavones, including 6-hydroxyflavone. CYP2A6 catalyzed flavanone to produce flavone at a turnover rate of 0.72 min-1 that was ∼3-fold higher than that catalyzed by CYP2A13 (0.29 min-1). 4. These results indicate that CYP2A6 and other human P450s have important roles in metabolizing flavone and flavanone, two unsubstituted flavonoids, present in dietary foods. Chemical mechanisms of P450-catalyzed desaturation of flavanone to form flavone are discussed.

Suitability of MMGBSA for the selection of correct ligand binding modes from docking results.

The estimation of the correct binding mode and affinity of a ligand into a target protein using computational methods is challenging. However, docking can introduce poses from which the correct binding mode could be identified using other methods. Here, we analyzed the reliability of binding energy estimation using the molecular mechanics-generalized Born surface area (MMGBSA) method without and with energy minimization to identify the likely ligand binding modes within docking results. MMGBSA workflow (a) outperformed docking in recognizing the correct binding modes of androgen receptor ligands and (b) improved the correlation coefficient of computational and experimental results of rescored docking poses to phosphodiesterase 4B. Combined with stability and atomic distance analysis, MMGBSA helped to (c) identify the binding modes and sites of metabolism of cytochrome P450 2A6 substrates. The standard deviation of estimated binding energy within one simulation was lowered by minimization in all three example cases. Minimization improved the identification of the correct binding modes of androgen receptor ligands. Although only three case studies are shown, the results are analogous and indicate that these behaviors could be generalized. Such identified binding modes could be further used, for example, with free energy perturbation methods to understand binding energetics more accurately.

Exploring the structure characteristics and major channels of cytochrome P450 2A6, 2A13, and 2E1 with pilocarpine.

The majority of cytochromes P450 play a critical role in metabolism of endogenous and exogenous substrates, some of its products are carcinogens. Therefore, inhibition of P450 enzymes activity can promote the detoxification and elimination of chemical carcinogens. In this study, molecular dynamics (MD) simulations and adaptive steered molecular dynamics (ASMD) simulations were performed to explore the structure features and channel dynamics of three P450 isoforms 2A6, 2A13, and 2E1 bound with the common inhibitor pilocarpine. The binding free energy results combined with the PMF calculations give a reasonable ranking of binding affinity, which are consistent with the experimental data. Our results uncover how a sequence divergence of different CYP2 enzymes causes individual variations in major channel selections. On the basis of channel bottleneck and energy decomposition analysis, we propose a gating mechanism of their respective major channels in three enzymes, which may be attributed to a reversal of Phe209 in CYP2A6/2A13, as well as the rotation of Phe116 and Phe298 in CYP2E1. The hydrophobic residues not only make strong hydrophobic interactions with inhibitor, but also act as gatekeeper to regulate the opening of channel. The present study provides important insights into the structure-function relationships of three cytochrome P450s and the molecular basis for development of potent inhibitors.

Structural and functional effects of cytochrome b5 interactions with human cytochrome P450 enzymes.

The small heme-containing protein cytochrome b5 can facilitate, inhibit, or have no effect on cytochrome P450 catalysis, often in a P450-dependent and substrate-dependent manner that is not well understood. Herein, solution NMR was used to identify b5 residues interacting with different human drug-metabolizing P450 enzymes. NMR results revealed that P450 enzymes bound to either b5 α4-5 (CYP2A6 and CYP2E1) or this region and α2-3 (CYP2D6 and CYP3A4) and suggested variation in the affinity for b5 Mutations of key b5 residues suggest not only that different b5 surfaces are responsible for binding different P450 enzymes, but that these different complexes are relevant to the observed effects on P450 catalysis.

Functional Characterization of 34 CYP2A6 Allelic Variants by Assessment of Nicotine C-Oxidation and Coumarin 7-Hydroxylation Activities.

CYP2A6, a member of the cytochrome P450 (P450) family, is one of the enzymes responsible for the metabolism of therapeutic drugs and such tobacco components as nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and N-nitrosodiethylamine. Genetic polymorphisms in CYP2A6 are associated with individual variation in smoking behavior, drug toxicities, and the risk of developing several cancers. In this study, we conducted an in vitro analysis of 34 allelic variants of CYP2A6 using nicotine and coumarin as representative CYP2A6 substrates. These variant CYP2A6 proteins were heterologously expressed in 293FT cells, and their enzymatic activities were assessed on the basis of nicotine C-oxidation and coumarin 7-hydroxylation activities. Among the 34 CYP2A6 variants, CYP2A6.2, CYP2A6.5, CYP2A6.6, CYP2A6.10, CYP2A6.26, CYP2A6.36, and CYP2A6.37 exhibited no enzymatic activity, whereas 14 other variants exhibited markedly reduced activity toward both nicotine and coumarin. These comprehensive in vitro findings may provide useful insight into individual differences in smoking behavior, drug efficacy, and cancer susceptibility.

Study the influence of licorice and pomegranate drinks on nicotine metabolism in human urine by LC-orbitrap MS.

Nicotine-diet interactions have a particular importance on human health. Some food substances are subject to change hepatic CYP2A6 metabolism rate for nicotine and its levels in smokers consequently. This study investigates the effect of pomegranate and licorice drinks on nicotine metabolism, by a new developed and validated method for simultaneous determination of nicotine with its major metabolites (cotinine and nicotine N-oxide) in human urine, utilizing LC ESI-orbitrap-MS. Twenty-four Jordanian healthy and smoker volunteers were participated in two equal groups, crossover design for each of pomegranate and licorice test drink. In the study periods each group assigned either to drink test juice three times a day or to be avoided from test drink for 7 successive days, and then both groups switched their drink treatment in subsequent period. Early morning urine samples were collected from all volunteers after each period. Nicotine metabolism rate was evaluated from nicotine/cotinine and nicotine/nicotine N-oxide ratios in urine. A consistent trend of increase in metabolism rate for nicotine was observed from urine analysis under pomegranate or licorice drink conditions compared to control conditions. Pomegranate and licorice drinks are increasing the metabolism rate for nicotine in terms of induction effect for hepatic cytochrome p450 enzymes.

Structure-Function Studies of Naphthalene, Phenanthrene, Biphenyl, and Their Derivatives in Interaction with and Oxidation by Cytochromes P450 2A13 and 2A6.

Naphthalene, phenanthrene, biphenyl, and their derivatives having different ethynyl, propynyl, butynyl, and propargyl ether substitutions were examined for their interaction with and oxidation by cytochromes P450 (P450) 2A13 and 2A6. Spectral interaction studies suggested that most of these chemicals interacted with P450 2A13 to induce Type I binding spectra more readily than with P450 2A6. Among the various substituted derivatives examined, 2-ethynylnaphthalene, 2-naphthalene propargyl ether, 3-ethynylphenanthrene, and 4-biphenyl propargyl ether had larger ΔAmax/Ks values in inducing Type I binding spectra with P450 2A13 than their parent compounds. P450 2A13 was found to oxidize naphthalene, phenanthrene, and biphenyl to 1-naphthol, 9-hydroxyphenanthrene, and 2- and/or 4-hydroxybiphenyl, respectively, at much higher rates than P450 2A6. Other human P450 enzymes including P450s 1A1, 1A2, 1B1, 2C9, and 3A4 had lower rates of oxidation of naphthalene, phenanthrene, and biphenyl than P450s 2A13 and 2A6. Those alkynylated derivatives that strongly induced Type I binding spectra with P450s 2A13 and 2A6 were extensively oxidized by these enzymes upon analysis with HPLC. Molecular docking studies supported the hypothesis that ligand-interaction energies (U values) obtained with reported crystal structures of P450 2A13 and 2A6 bound to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, indole, pilocarpine, nicotine, and coumarin are of use in understanding the basis of possible molecular interactions of these xenobiotic chemicals with the active sites of P450 2A13 and 2A6 enzymes. In fact, the ligand-interaction energies with P450 2A13 4EJG bound to these chemicals were found to relate to their induction of Type I binding spectra.

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

BACKGROUND: 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. OBJECTIVE: 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. METHODS: We used our previous chemical inhibitor databases to construct improved 3-dimensional quantitative structureactivity relationship (3D-QSAR) models for CYP1A2, CYP2A6 and CYP2B6. RESULTS: 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. CONCLUSION: This in silico approach will provide a means for very rapid and high throughput prediction of cross-inhibition of these three CYP enzymes.

Synthesis, biological activity evaluation and molecular docking studies of novel coumarin substituted thiazolyl-3-aryl-pyrazole-4-carbaldehydes.

A novel series of coumarin substituted thiazolyl-3-aryl-pyrazole-4-carbaldehydes (4a-o) were synthesized via an efficient, one-pot multicomponent approach involving 3-(2-bromoacetyl)coumarins (1a-g), thiosemicarbazide (2) and substituted acetophenones (3a-c) utilizing Vilsmeier-Haack reaction condition with good yields. The title compounds structure was elucidated by spectroscopic data (IR, NMR and Mass) and elemental analysis. All the synthesized compounds were screened for their in vitro cytotoxic activity against MCF-7, DU-145 and HeLa cell lines and studied detailed about molecular interaction of probable target protein human microsomal cytochrome CYP450 2A6 using docking simulation. These coumarin derivatives were exhibiting moderate to appreciable cytotoxic activities. The compounds 4m and 4n exhibited significant cytotoxic activity with IC50 values having 5.75 and 6.25µM against HeLa cell line. Similarly compound 4n also exhibiting good anti cancer property and antibacterial activity against DU-145 cell line and Gram negative bacterial strains.

Structural and biophysical characterization of human cytochromes P450 2B6 and 2A6 bound to volatile hydrocarbons: analysis and comparison.

X-ray crystal structures of complexes of cytochromes CYP2B6 and CYP2A6 with the monoterpene sabinene revealed two distinct binding modes in the active sites. In CYP2B6, sabinene positioned itself with the putative oxidation site located closer to the heme iron. In contrast, sabinene was found in an alternate conformation in the more compact CYP2A6, where the larger hydrophobic side chains resulted in a significantly reduced active-site cavity. Furthermore, results from isothermal titration calorimetry indicated a much more substantial contribution of favorable enthalpy to sabinene binding to CYP2B6 as opposed to CYP2A6, consistent with the previous observations with (+)-α-pinene. Structural analysis of CYP2B6 complexes with sabinene and the structurally similar (3)-carene and comparison with previously solved structures revealed how the movement of the F206 side chain influences the volume of the binding pocket. In addition, retrospective analysis of prior structures revealed that ligands containing -Cl and -NH functional groups adopted a distinct orientation in the CYP2B active site compared with other ligands. This binding mode may reflect the formation of Cl-π or NH-π bonds with aromatic rings in the active site, which serve as important contributors to protein-ligand binding affinity and specificity. Overall, the findings from multiple techniques illustrate how drugs metabolizing CYP2B6 and CYP2A6 handle a common hydrocarbon found in the environment. The study also provides insight into the role of specific functional groups of the ligand that may influence the binding to CYP2B6.

Effects of protein flexibility on the site of metabolism prediction for CYP2A6 substrates.

Structure-based prediction for the site of metabolism (SOM) of a compound metabolized by human cytochrome P450s (CYPs) is highly beneficial in drug discovery and development. However, the flexibility of the CYPs' active site remains a huge challenge for accurate SOM prediction. Compared with other CYPs, the active site of CYP2A6 is relatively small and rigid. To address the impact of the flexibility of CYP2A6 active site residues on the SOM prediction for substrates, in this work, molecular dynamics (MD) simulations and molecular docking were used to predict the SOM of 96 CYP2A6 substrates. Substrates with known SOM were docked into the snapshot structures from MD simulations and the crystal structures of CYP2A6. Compared to the crystal structures, the protein structures obtained from MD simulations showed more accurate prediction for SOM. Our results indicated that the flexibility of the active site of CYP2A6 significantly affects the SOM prediction results. Further analysis for the 40 substrates with definite Km values showed that the prediction accuracy for the low Km substrates is comparable to that by ligand-based methods.

Understanding a substrate's product regioselectivity in a family of enzymes: a case study of acetaminophen binding in cytochrome P450s.

Product regioselectivity as influenced by molecular recognition is a key aspect of enzyme catalysis. We applied large-scale two-dimensional (2D) umbrella sampling (USP) simulations to characterize acetaminophen (APAP) binding in the active sites of the family of Cytochrome P450 (CYP) enzymes as a case study to show the different regioselectivity exhibited by a single substrate in comparative enzymes. Our results successfully explain the experimentally observed product regioselectivity for all five human CYPs included in this study, demonstrating that binding events play an important role in determining regioselectivity. In CYP2C9 and CYP3A4, weak interactions in an overall large active site cavity result in a fairly small binding free energy difference between APAP reactive binding states, consistent with experimental results that show little preference for resulting metabolites. In contrast, in CYP1A2 and CYP2E1, APAP is strongly restrained by a compact binding pocket, leading to a preferred binding conformation. The calculated binding equilibrium of APAP within the compact active site of CYP2A6 is able to predict the experimentally documented product ratios and is also applied to explain APAP regioselectivity in CYP1A2 and CYP2C9. APAP regioselectivity seems to be related to the selectivity for one binding conformation over another binding conformation as dictated by the size and shape of the active site. Additionally, unlike docking and molecular dynamics (MD), our free energy calculations successfully reproduced a unique APAP pose in CYP3A4 that had been reported experimentally, suggesting this approach is well suited to find the realistic binding pose and the lowest-energy starting structure for studying the chemical reaction step in the future.