Use of Phenoxyaniline Analogues To Generate Biochemical Insights into the Interactio n of Polybrominated Diphenyl Ether with CYP2B Enzymes.

Human hepatic cytochromes P450 (CYP) are integral to xenobiotic metabolism. CYP2B6 is a major catalyst of biotransformation of environmental toxicants, including polybrominated diphenyl ethers (PBDEs). CYP2B substrates tend to contain halogen atoms, but the biochemical basis for this selectivity and for species specific determinants of metabolism has not been identified. Spectral binding titrations and inhibition studies were performed to investigate interactions of rat CYP2B1, rabbit CYP2B4, and CYP2B6 with a series of phenoxyaniline (POA) congeners that are analogues of PBDEs. For most congeners, there was a <3-fold difference between the spectral binding constants (KS) and IC50 values. In contrast, large discrepancies between these values were observed for POA and 3-chloro-4-phenoxyaniline. CYP2B1 was the enzyme most sensitive to POA congeners, so the Val-363 residue from that enzyme was introduced into CYP2B4 or CYP2B6. This substitution partially altered the protein-ligand interaction profiles to make them more similar to that of CYP2B1. Addition of cytochrome P450 oxidoreductase (POR) to titrations of CYP2B6 with POA or 2'4'5'TCPOA decreased the affinity of both ligands for the enzyme. Addition of cytochrome b5 to a recombinant enzyme system containing POR and CYP2B6 increased the POA IC50 value and decreased the 2'4'5'TCPOA IC50 value. Overall, the inconsistency between KS and IC50 values for POA versus 2'4'5'TCPOA is largely due to the effects of redox partner binding. These results provide insight into the biochemical basis of binding of diphenyl ethers to human CYP2B6 and changes in CYP2B6-mediated metabolism that are dependent on POA congener and redox partner identity.

Structure-Function Analysis of Mammalian CYP2B Enzymes Using 7-Substituted Coumarin Derivatives as Probes: Utility of Crystal Structures and Molecular Modeling in Understanding Xenobiotic Metabolism.

Crystal structures of CYP2B35 and CYP2B37 from the desert woodrat were solved in complex with 4-(4-chlorophenyl)imidazole (4-CPI). The closed conformation of CYP2B35 contained two molecules of 4-CPI within the active site, whereas the CYP2B37 structure demonstrated an open conformation with three 4-CPI molecules, one within the active site and the other two in the substrate access channel. To probe structure-function relationships of CYP2B35, CYP2B37, and the related CYP2B36, we tested the O-dealkylation of three series of related substrates-namely, 7-alkoxycoumarins, 7-alkoxy-4-(trifluoromethyl)coumarins, and 7-alkoxy-4-methylcoumarins-with a C1-C7 side chain. CYP2B35 showed the highest catalytic efficiency (kcat/KM) with 7-heptoxycoumarin as a substrate, followed by 7-hexoxycoumarin. In contrast, CYP2B37 showed the highest catalytic efficiency with 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC), followed by 7-methoxy-4-(trifluoromethyl)coumarin (7-MFC). CYP2B35 had no dealkylation activity with 7-MFC or 7-EFC. Furthermore, the new CYP2B-4-CPI-bound structures were used as templates for docking the 7-substituted coumarin derivatives, which revealed orientations consistent with the functional studies. In addition, the observation of multiple -Cl and -NH-π interactions of 4-CPI with the aromatic side chains in the CYP2B35 and CYP2B37 structures provides insight into the influence of such functional groups on CYP2B ligand binding affinity and specificity. To conclude, structural, computational, and functional analysis revealed striking differences between the active sites of CYP2B35 and CYP2B37 that will aid in the elucidation of new structure-activity relationships.

Mechanism-Based Inactivation of Human Cytochrome P450 2B6 by Chlorpyrifos.

Chlorpyrifos (CPS) is a commonly used pesticide which is metabolized by P450s into the toxic metabolite chlorpyrifos-oxon (CPO). Metabolism also results in the release of sulfur, which has been suggested to be involved in mechanism-based inactivation (MBI) of P450s. CYP2B6 was previously determined to have the greatest catalytic efficiency for CPO formation in vitro. Therefore, we characterized the MBI of CYP2B6 by CPS. CPS inactivated CYP2B6 in a time- and concentration-dependent manner with a kinact of 1.97 min(-1), a KI of 0.47 µM, and a partition ratio of 17.7. We further evaluated the ability of other organophosphate pesticides including chorpyrifos-methyl, diazinon, parathion-methyl, and azinophos-methyl to inactivate CYP2B6. These organophosphate pesticides were also potent MBIs of CYP2B6 characterized by similar kinact and KI values. The inactivation of CYP2B6 by CPS was accompanied by the loss of P450 detectable in the CO reduced spectrum and loss of detectable heme. High molecular weight aggregates were observed when inactivated CYP2B6 was run on SDS-PAGE gels indicating protein aggregation. Interestingly, we found that the rat homologue of CYP2B6, CYP2B1, was not inactivated by CPS despite forming CPO to a similar extent. On the basis of the locations of the Cys residues in the two proteins which could react with released sulfur during the metabolism of CPS, we investigated whether the C475 in CYP2B6, which is not conserved in CYP2B1, was the critical residue for inactivation by mutating it to a Ser. CYP2B6 C475S was inactivated to a similar extent as wild type CYP2B6 indicating that C475 is not likely the key difference between CYP2B1 and CYP2B6 with respect to inactivation. These results indicate that CPS and other organophosphate pesticides are potent MBIs of CYP2B6 which may have implications for the toxicity of these pesticides as well as the potential for pesticide-drug interactions.

Effects of Sublethal Exposure to a Glyphosate-Based Herbicide Formulation on Metabolic Activities of Different Xenobiotic-Metabolizing Enzymes in Rats.

The activities of different xenobiotic-metabolizing enzymes in liver subcellular fractions from Wistar rats exposed to a glyphosate (GLP)-based herbicide (Roundup full II) were evaluated in this work. Exposure to the herbicide triggered protective mechanisms against oxidative stress (increased glutathione peroxidase activity and total glutathione levels). Liver microsomes from both male and female rats exposed to the herbicide had lower (45%-54%, P < 0.01) hepatic cytochrome P450 (CYP) levels compared to their respective control animals. In female rats, the hepatic 7-ethoxycoumarin O-deethylase (a general CYP-dependent enzyme activity) was 57% higher (P < 0.05) in herbicide-exposed compared to control animals. Conversely, this enzyme activity was 58% lower (P < 0.05) in male rats receiving the herbicide. Lower (P < 0.05) 7-ethoxyresorufin O-deethlyase (EROD, CYP1A1/2 dependent) and oleandomycin triacetate (TAO) N-demethylase (CYP3A dependent) enzyme activities were observed in liver microsomes from exposed male rats. Conversely, in females receiving the herbicide, EROD increased (123%-168%, P < 0.05), whereas TAO N-demethylase did not change. A higher (158%-179%, P < 0.01) benzyloxyresorufin O-debenzylase (a CYP2B-dependent enzyme activity) activity was only observed in herbicide-exposed female rats. In herbicide-exposed rats, the hepatic S-oxidation of methimazole (flavin monooxygenase dependent) was 49% to 62% lower (P < 0.001), whereas the carbonyl reduction of menadione (a cytosolic carbonyl reductase-dependent activity) was higher (P < 0.05). Exposure to the herbicide had no effects on enzymatic activities dependent on carboxylesterases, glutathione transferases, and uridinediphospho-glucuronosyltransferases. This research demonstrated certain biochemical modifications after exposure to a GLP-based herbicide. Such modifications may affect the metabolic fate of different endobiotic and xenobiotic substances. The pharmacotoxicological significance of these findings remains to be clarified.

In vitro to in vivo extrapolation and physiologically based modeling of cytochrome P450 mediated metabolism in beagle dog gut wall and liver.

The beagle dog is a widely used in vivo model to guide clinical formulation development and to explore the potential for food effects. However, the results in dogs are often not directly translatable to humans. Consequently, a physiologically based modeling strategy has been proposed, using the dog as a validation step to verify model assumptions before making predictions in humans. One current weakness in this strategy is the lack of validated tools to incorporate gut wall metabolism into the dog model. In this study, in vitro to in vivo extrapolation factors for CYP2B11 and CYP3A12 mediated metabolism were established based on tissue enzyme abundance data reported earlier. Thereafter, physiologically based modeling of intestinal absorption in beagle dog was conducted in GastroPlus using V(max) and K(m) determined in recombinant enzymes as inputs for metabolic turnover. The predicted fraction of absorbed dose escaping the gut wall metabolism (F(g)) of all five reference compounds studied (domperidone, felodipine, nitrendipine, quinidine, and sildenafil) were within a two-fold range of the value estimated from in vivo data at single dose levels. However, further in vivo studies and analysis of the dose-dependent pharmacokinetics of felodipine and nitrendipine showed that more work is required for robust forecasting of nonlinearities. In conclusion, this study demonstrates an approach for prediction of the gut wall extraction of CYP substrates in the beagle dog, thus enhancing the value of dog studies as a component in a strategy for the prediction of human pharmacokinetics.

Beta sheet 2-alpha helix C loop of cytochrome P450 reductase serves as a docking site for redox partners.

As a promiscuous redox partner, the biological role of cytochrome P450 reductase (CPR) depends significantly on protein-protein interactions. We tested a hypothesized CPR docking site by mutating D113, E115, and E116 to alanine and assaying activity toward various electron acceptors as a function of ionic strength. Steady-state cytochrome c studies demonstrated the mutations improved catalytic efficiency and decreased the impact of ionic strength on catalytic parameters when compared to wild type. Based on activity toward 7-ethoxy-4-trifluoro-methylcoumarin, CYP2B1 and CPR favored formation of an active CYP2B1*CPR complex and inactive (CYP2B1)(2)*CPR complex until higher ionic strength whereby only the binary complex was observed. The mutations increased dissociation constants only for the binary complex and suppressed the ionic strength effect. Studies with a non-binding substrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) suggest changes in activity toward cytochrome c and CYP2B1 reflect alterations in the route of electron transfer caused by the mutations. Electrostatic modeling of catalytic and binding parameters confirmed the importance of D113 and especially the double mutant E115 and E116 as mediators in forming charge-charge interactions between CPR and complex partners.

Effect of conformational dynamics on substrate recognition and specificity as probed by the introduction of a de novo disulfide bond into cytochrome P450 2B1.

The conformational dynamics of cytochrome P450 2B1 (CYP2B1) were investigated through the introduction of a disulfide bond to link the I- and K-helices by generation of a double Cys variant, Y309C/S360C. The consequences of the disulfide bonding were examined both experimentally and in silico by molecular dynamics simulations. Under high hydrostatic pressures, the partial inactivation volume for the Y309C/S360C variant was determined to be -21 cm3mol(-1), which is more than twice as much as those of the wild type (WT) and single Cys variants (Y309C, S360C). This result indicates that the engineered disulfide bond has substantially reduced the protein plasticity of the Y309C/S360C variant. Under steady-state turnover conditions, the S360C variant catalyzed the N-demethylation of benzphetamine and O-deethylation of 7-ethoxy-trifluoromethylcoumarin as the WT did, whereas the Y309C variant retained only 39% of the N-demethylation activity and 66% of the O-deethylation activity compared with the WT. Interestingly, the Y309C/S360C variant restored the N-demethylation activity to the same level as that of the WT but decreased the O-deethylation activity to only 19% of the WT. Furthermore, the Y309C/S360C variant showed increased substrate specificity for testosterone over androstenedione. Molecular dynamics simulations revealed that the engineered disulfide bond altered substrate access channels. Taken together, these results suggest that protein dynamics play an important role in regulating substrate entry and recognition.

Covalent modification of Thr302 in cytochrome P450 2B1 by the mechanism-based inactivator 4-tert-butylphenylacetylene.

The mechanism of inactivation of cytochrome P450 2B1 (CYP2B1) by 4-tert-butylphenylacetylene (BPA) has been characterized previously to be caused by the covalent binding of a reactive intermediate to the apoprotein rather than heme destruction (J Pharmacol Exp Ther 331:392-403, 2009). The identification of a BPA-glutathione conjugate and the increase in the mass of the BPA-adducted apoprotein have indicated that the mass of adduct is 174 Da, equivalent to the mass of BPA plus one oxygen atom. To identify the adducted residue, BPA-inactivated CYP2B1 was digested with trypsin, and the digest was then analyzed by using capillary liquid chromatography with a LTQ linear ion trap mass spectrometer as the detector. A mass shift of 174 Da was used for a SEQUEST database search. The tandem mass spectrometry fragmentation of the modified peptide and the identity of modified residue were determined. The results revealed a mass increase of 174 Da for the peptide sequence (296)FFAGTSSTTLR(308) in the I-helix of CYP2B1 and that the site of adduction formation is Thr302. Homology modeling and ligand docking studies showed that BPA binds in close proximity to both the heme iron and Thr302 with the distances being 2.96 and 3.42 A, respectively. The identification of Thr302 in the CYP2B1 active site as the site of covalent modification leading to inactivation by BPA supports previous hypotheses that this conserved Thr residue may play a crucial role for various functions in P450s.

Quantitative structure-activity relationships (QSARs) for inhibitors and substrates of CYP2B enzymes: importance of compound lipophilicity in explanation of potency differences.

The results of quantitative structure-activity relationship (QSAR) studies on inhibitors and substrates of cytochrome P450 2B (CYP2B) subfamily enzymes are reported. It was found that lipophilicity (in the form of log P) is the most important property for explaining the variations in inhibitory activity, and there are similarities between QSARs for both substrates and inhibitors for CYP2B6 (human), and also between those of other CYP2B enzymes, such as CYP2B1 (rat) and CYP2B4 (rabbit). Both linear and quadratic lipophilicity relationships are evidenced in human and other mammalian species, and the particular type of expression found is probably due to the nature of the compounds under investigation, as it is usually the homologous series which tend to show quadratic relationships in log P. The findings from QSAR studies can be rationalized by molecular modelling of the active site interactions with both P450 crystal structures and homology models of CYP2B subfamily enzymes.

Mechanism-based inactivation of CYP2B1 and its F-helix mutant by two tert-butyl acetylenic compounds: covalent modification of prosthetic heme versus apoprotein.

The mechanism-based inactivation of cytochrome CYP2B1 [wild type (WT)] and its Thr205 to Ala mutant (T205A) by tert-butylphenylacetylene (BPA) and tert-butyl 1-methyl-2-propynyl ether (BMP) in the reconstituted system was investigated. The inactivation of WT by BPA exhibited a k(inact)/K(I) value of 1343 min(-1)mM(-1) and a partition ratio of 1. The inactivation of WT by BMP exhibited a k(inact)/K(I) value of 33 min(-1)mM(-1) and a partition ratio of 10. Liquid chromatography/tandem mass spectrometry analysis (LC/MS/MS) of the WT revealed 1) inactivation by BPA resulted in the formation of a protein adduct with a mass increase equivalent to the mass of BPA plus one oxygen atom, and 2) inactivation by BMP resulted in the formation of multiple heme adducts that all exhibited a mass increase equivalent to BMP plus one oxygen atom. LC/MS/MS analysis indicated the formation of glutathione (GSH) conjugates by the reaction of GSH with the ethynyl moiety of BMP or BPA with the oxygen being added to the internal or terminal carbon. For the inactivation of T205A by BPA and BMP, the k(inact)/K(I) values were suppressed by 100- and 4-fold, respectively, and the partition ratios were increased 9- and 3.5-fold, respectively. Only one major heme adduct was detected following the inactivation of the T205A by BMP. These results show that the Thr205 in the F-helix plays an important role in the efficiency of the mechanism-based inactivation of CYP2B1 by BPA and BMP. Homology modeling and substrate docking studies were presented to facilitate the interpretation of the experimental results.

Modification of serine 360 by a reactive intermediate of 17-alpha-ethynylestradiol results in mechanism-based inactivation of cytochrome P450s 2B1 and 2B6.

17-alpha-Ethynylestradiol (17EE) is a mechanism-based inactivator of P450 2B1 and P450 2B6 in the reconstituted monooxygenase system. The loss in enzymatic activity was due to the binding of a reactive intermediate of 17EE to the apoprotein. P450 2B1 and P450 2B6 were inactivated by 17EE and digested with trypsin. The peptides obtained following digestion with trypsin of 17EE-inactivated P450 2B1 and P450 2B6 were separated by liquid chromatography and analyzed by ESI-MS. Adducted peptides exhibiting an increase in mass consistent with the addition of the mass of the reactive intermediate of 17EE were identified for each enzyme. Analysis of these modified peptides by ESI-MS/MS and precursor ion scanning facilitated the identification of the Ser360 in both enzymes as a site that had been adducted by a reactive intermediate of 17EE. A P450 2B1 mutant where Ser360 was replaced by alanine was constructed, expressed, and purified. Activity and inactivation studies indicated that mutation of the Ser360 residue to alanine did not prevent inactivation of the mutant enzyme by 17EE. These observations suggest that Ser360 is not critical for the catalytic function of these P450s. Spectral binding studies of the 17EE-inactivated P450 2B1 and P450 2B6 indicated that modification of the enzymes by the reactive intermediate of 17EE resulted in an enzyme that was no longer capable of binding substrates. These results suggest that the inactivation by 17EE may be due to modification of an amino acid residue in the substrate access channel near the point of entry into the active site.

Analysis of Species-Selectivity of Human, Mouse and Rat Cytochrome P450 1A and 2B Subfamily Enzymes using Molecular Modeling, Docking and Dynamics Simulations.

Cytochrome P450 (CYP) 1A and 2B subfamily enzymes are important drug metabolizing enzymes, and are highly conserved across species in terms of sequence homology. However, there are major to minor structural and macromolecular differences which provide for species-selectivity and substrate-selectivity. Therefore, species-selectivity of CYP1A and CYP2B subfamily proteins across human, mouse and rat was analyzed using molecular modeling, docking and dynamics simulations when the chiral molecules quinine and quinidine were used as ligands. The three-dimensional structures of 17 proteins belonging to CYP1A and CYP2B subfamilies of mouse and rat were predicted by adopting homology modeling using the available structures of human CYP1A and CYP2B proteins as templates. Molecular docking and dynamics simulations of quinine and quinidine with CYP1A subfamily proteins revealed the existence of species-selectivity across the three species. On the other hand, in the case of CYP2B subfamily proteins, no role for chirality of quinine and quinidine in forming complexes with CYP2B subfamily proteins of the three species was indicated. Our findings reveal the roles of active site amino acid residues of CYP1A and CYP2B subfamily proteins and provide insights into species-selectivity of these enzymes across human, mouse, and rat.

Lateral segregation of anionic phospholipids in model membranes induced by cytochrome P450 2B1: bi-directional coupling between CYP2B1 and anionic phospholipid.

The lateral segregation of anionic phospholipids phosphatidic acid (PA), phosphatidylinositol (PI), and phosphatidylserine (PS) was detected after addition of cytochrome P450 2B1 (CYP2B1). The tendency of lipid clustering was highly dependent on the type of anionic phospholipids examined. PA was the most highly clustered while PI and PS clustered to a lesser degree. Moreover, liposomes containing anionic phospholipids form anionic phospholipid-rich microdomains in the presence of CYP2B1. Anionic phospholipids (mostly notably PA) also increased the ability of CYP2B1 to bind to lipid monolayers. In addition to the ability of CYP2B1 to modulate the physical properties of the membrane, the membrane itself can have reciprocal effects on the activity and conformation of CYP2B1. The catalytic activity of CYP2B1 increased as a function of anionic phospholipid concentration and in the presence of 10 mol% PA, the activity increased by 85%. These results suggest a bi-directional coupling between the CYP2B1 and anionic phospholipids.

Peroxisome Proliferator-Activated Receptor α Activation Suppresses Cytochrome P450 Induction Potential in Mice Treated with Gemfibrozil.

Gemfibrozil, a peroxisome proliferator-activated receptor α (PPARα) agonist, is widely used for hypertriglyceridaemia and mixed hyperlipidaemia. Drug-drug interaction of gemfibrozil and other PPARα agonists has been reported. However, the role of PPARα in cytochrome P450 (CYP) induction by fibrates is not well known. In this study, wild-type mice were first fed gemfibrozil-containing diets (0.375%, 0.75% and 1.5%) for 14 days to establish a dose-response relationship for CYP induction. Then, wild-type mice and Pparα-null mice were treated with a 0.75% gemfibrozil-containing diet for 7 days. CYP3a, CYP2b and CYP2c were induced in a dose-dependent manner by gemfibrozil. In Pparα-null mice, their mRNA level, protein level and activity were induced more than those in wild-type mice. So, gemfibrozil induced CYP, and this action was inhibited by activated PPARα. These data suggested that the induction potential of CYPs was suppressed by activated PPARα, showing a potential role of this receptor in drug-drug interactions and metabolic diseases treated with fibrates.

Engineering mammalian cytochrome P450 2B1 by directed evolution for enhanced catalytic tolerance to temperature and dimethyl sulfoxide.

The previously laboratory-evolved cytochrome P450 2B1 quadruple mutant V183L/F202L/L209A/S334P (QM), which showed enhanced H(2)O(2)-mediated substrate oxidation, has now been shown to exhibit a >3.0-fold decrease in K(m,HOOH) for 7-ethoxy-4-trifluoromethylcoumarin (7-EFC) O-deethylation compared with the parental enzyme L209A. Subsequently, a streamlined random mutagenesis and a high-throughput screening method were developed using QM to screen and select mutants with enhanced tolerance of catalytic activity to temperature and dimethyl sulfoxide (DMSO). Upon screening >3000 colonies, we identified QM/L295H and QM/K236I/D257N with enhanced catalytic tolerance to temperature and DMSO. QM/L295H exhibited higher activity than QM at a broad range of temperatures (35-55 degrees C) and maintained approximately 1.4-fold higher activity than QM at 45 degrees C for 6 h. In addition, QM/L295H showed a significant increase in T(m,app) compared with L209A. QM/L295H and QM/K236I/D257N exhibited higher activity than QM at a broad range of DMSO concentrations (2.5-15%). Furthermore, QM/K236I/D257N/L295H was constructed by combining QM/K236I/D257N with L295H using site-directed mutagenesis and exhibited a >2-fold higher activity than QM at nearly the entire range of DMSO concentrations. In conclusion, in addition to engineering mammalian cytochromes P450 for enhanced activity, directed evolution can also be used to optimize catalytic tolerance to temperature and organic solvent.

Functional interaction between amino-acid residues 242 and 290 in cytochromes P-450 2B1 and 2B11.

Previous studies have revealed the functional importance of the negatively charged amino-acid residue Asp-290 of the phenobarbital-inducible dog liver cytochrome P-450 (P-450) 2B11 (Harlow, G.R. and Halpert J.R. (1996) Arch. Biochem. Biophys. 326, 85-92). A search for P-450 2B11 residues capable of forming a charge pair with Asp-290 suggested the positively charged residue Lys-242 as a likely candidate. Replacement of Lys-242 with Asp in a P-450 2B11 fusion protein with rat NADPH-cytochrome P-450 reductase (reductase) resulted in very low holoenzyme expression levels in Escherichia coli, as did replacement of Asp-290 with Lys. Remarkably, however, expression levels of the double mutant Lys-242 --> Asp/Asp-290 --> Lys were dramatically increased above either single replacement alone. Similarly, the pair-wise substitutions Lys-242 --> Leu/Asp-290 --> Ile in P-450 2B11 and Leu-242 --> Lys/Ile-290 --> Asp in P-450 2B1 showed greater holoenzyme expression levels than the constituent single mutants, providing further evidence for the close proximity of these residues within the three-dimensional structure of these two enzymes. These results support the hypothesis that a functional interaction exists between residues 242 and 290, which may help to coordinate the relative positions of proposed helices G and I. All of the mutant combinations, including the additional P-450 2B11 double mutants Tyr-242/Asn-290 and Tyr-242/Ser-290, displayed altered stereoselectivity of androstenedione hydroxylation.

Comparison of substrate metabolism by cytochromes P450 2B1, 2B4, and 2B6: relationship of heme spin state, catalysis, and the effects of cytochrome b5.

The metabolism of selected substrates by cytochromes P450 (P450) 2B1, 2B4, and 2B6 was compared, and the effects of cytochrome b(5) (b(5)) on these reactions were assessed. There did not appear to be any trends regarding the effects of b(5) when the metabolism of a given substrate by the different P450 enzymes was compared. The changes in spin states of the P450 enzymes as a result of interactions with substrates and cytochrome b(5) were also determined. Only P450 2B4 demonstrated a relationship between spin state, reaction coupling and b(5) effects. The rates of benzphetamine and 7-ethoxy-4-trifluoromethylcoumarin metabolism by the three enzymes could be correlated with the proportions of high spin heme. Similarly, the proportion of reaction coupling during the metabolism of selected substrates was approximately equal to the proportion of high spin P450. The data are interpreted to indicate that a P450 conformational equilibrium coordinately regulates catalysis and spin state changes.

Homology modeling and molecular dynamics of CYP1A1 and CYP2B1 to explore the metabolism of aryl derivatives by docking and experimental assays.

Since many drugs are metabolized by cytochrome P450 (CYP450), biotransformation studies using these enzymes are valuable in drug development. In this work, the biotransformation by CYP1A1 and CYP2B1 of two acetylcholinesterase (AChE) inhibitors, 4-(4'-hydroxy-phenylamino)-4-oxo propanoic acid (A) and 1H-pyrrolidine-1-(4'-hydroxy-phenyl)-2,5-dione (B), was investigated through docking and molecular dynamics (MD) simulations and by experimental methods using rat liver microsomes pretreated with ß-naphthoflavone and phenobarbital (CYP1A1 and CYP2B1 inducers, respectively). The target proteins were initially built by homology modeling, and the resulting three-dimensional structures were refined by MD to obtain fifteen snapshots of each P450 isoform. These snapshots were used to dock compounds A and B as well as the reference compound acetaminophen (APAP). We confirmed that APAP produces a toxic intermediate (N-acetyl-p-benzoquinone imine) upon interaction of its amide group with the heme iron of CYP1A1. However, neither A nor B presented this kind of interaction within any snapshot with CYP1A1. On the other hand, when APAP, A and B were docked on CYP2B1, their hydroxyl group was located near the heme iron on the snapshot at 3.5 ns. Furthermore, B maintained the same position on all snapshots of this isoform. Therefore, theoretical results suggests that A and B do not generate toxic metabolites. These data were supported by HPLC analysis showing only one metabolite from A and B, which was identified by GC-MS as the hydroxylated product. Altogether, our results suggest that neither test compound is toxic.

Exploring QSARs for binding affinity of azoles with CYP2B and CYP3A enzymes using GFA and G/PLS techniques.

Eighteen azole compounds, including some commercial fungicides, reported by Daisuke Itokawa et al. have been subjected to quantitative structure-activity relationship (QSAR) analysis for their binding affinity with cytochrome enzymes CYP3A and CYP2B. The analyses were performed using electronic (Charge, FCharge, Apol, Dipole, HOMO, LUMO, and Sr), spatial (Radius of gyration, Jurs descriptors, Shadow indices, Area, PMI-mag, Density, Vm), different topological parameters (E-state index, kappa shape index, molecular connectivity index, subgraph cont, information content indices), and thermodynamic (Alog P, Alog P98, Molref) descriptors calculated using CERIUS2 version 10 software. Genetic function approximation and genetic partial least squares were used as chemometric tools for modeling. The derived binding affinity models are of high statistical quality (leave-one-out Q(2) ranging from 0.946 to 0.977). The selectivity models also satisfy the statistical significance (Q(2) ranging from 0.680 to 0.761). The models indicate that the binding affinity of these compounds is related to topological, steric, electronic, and spatial properties of the molecules. The spline-based genetic models indicate optimum range of different parameters. The models have also been validated by leave-25%-out cross-validation.

Identification of 17-alpha-ethynylestradiol-modified active site peptides and glutathione conjugates formed during metabolism and inactivation of P450s 2B1 and 2B6.

The oral contraceptive 17-alpha-ethynylestradiol (17EE) is a mechanism-based inactivator of cytochrome P450s (P450s) 2B1 and 2B6. Inactivation of P450s 2B1 and 2B6 in the reconstituted system by [3H]17EE resulted in labeling of the P450 apoprotein. Mass spectral analysis of 17EE-inactivated P450 2B1 showed an increase in the mass of the apoprotein by 313 Da, consistent with the mass of 17EE plus one oxygen atom. P450s 2B1 and 2B6 were inactivated with [3H]17EE and digested with CNBr. Separation of these peptides resulted in the identification of one major labeled peptide for each enzyme. N-Terminal sequencing of these peptides yielded the amino acid sequences PYTDAVIHEI (for P450 2B1) and PYTEAV (for P450 2B6) that corresponded to amino acids P347-M376 and P347-M365 in P450s 2B1 and 2B6, respectively. Electrospray ionization (ESI)-liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption ionization (MALDI)-MS analysis of the P450 2B1-derived peptide resulted in a mass of 3654 Da consistent with the mass of the P347-M376 peptide (3385 Da) plus a 268 Da 17EE adduct. Chemically reactive intermediates of 17EE that were generated during the metabolism of 17EE by P450s 2B1 and 2B6 were trapped with gluthathione (GSH). ESI-LC-MS/MS analysis of 17EE-GSH conjugates from the incubation mixtures indicated that P450s 2B1 and 2B6 generated different reactive 17EE intermediates that were responsible for the inactivation and protein modification or the formation of GSH conjugates by these two enzymes.