Human Cytochrome P450 3A4 Research Articles

Lipid composition affects the thermal stability of cytochrome P450 3A4 in nanodiscs

Nanodiscs (NDs), self-assembled lipid bilayers encircled by membrane scaffold proteins (MSPs), offer a versatile platform for the reconstitution of membrane proteins for structural and biochemical investigations. Saturated, isoprenoid lipids are commonly found in thermophiles and have been associated with thermotolerance. To test whether these lipids confer additional stability on ND-incorporated membrane proteins, this study focuses on the thermal stability of human cytochrome P450 3A4 (CYP3A4) inside NDs composed of different phosphocholine lipids: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC). NDs were characterized using size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) and densitometric SDS-PAGE. CYP3A4-DPhPC-NDs were found to comprise three MSP copies instead of the canonical dimer, as reported before for the empty NDs. Rapid, thermally induced unfolding of CYP3A4 inside NDs measured using circular dichroism and differential scanning fluorimetry (nanoDSF) revealed that the CYP3A4 melting temperature was dependent on ND composition. In POPC and DMPC-CYP3A4-NDs the melting temperature was comparable to CYP3A4 without NDs (59 °C). CYP3A4 in DPhPC-NDs showed an increase in melting temperature of 4 °C. Decline in CYP3A4 integrity as well as ND aggregation and disintegration occur at similar rates for all membrane types when subjected to exposure at 37 °C for several hours. The POPC and DMPC- CYP3A4-NDs show significant lipid loss over time, which is not observed for DPhPC-NDs. The results demonstrate that thermally induced denaturation of protein-NDs is a complex, multifaceted process, which is not represented well by rapid thermal unfolding experiments.

Xin-yi-san contains potent human CYP1A2 inhibitors and its combined use with theophylline in treatment increases adverse risks in patients

BackgroundThe Xin-yi-san herbal decoction (XYS) is commonly used to treat patients with allergic rhinitis in Taiwan. Theophylline is primarily oxidized with high affinity by human cytochrome P450 (CYP)1A2, and has a narrow therapeutic index. PurposeThis study aimed to investigate the inhibition of human CYP1A2-catalyzed theophylline oxidation (THO) by XYS and its adverse effects in patients. MethodsHuman CYPs were studied in recombinant enzyme systems. The influence of concurrent XYS usage in theophylline-treated patients was retrospectively analyzed. ResultsAmong the major human hepatic and respiratory CYPs, XYS inhibitors preferentially inhibited CYP1A2 activity, which determined the elimination and side effects of theophylline. Among the herbal components of XYS decoction, Angelicae Dahuricae Radix contained potent THO inhibitors. Furanocoumarin imperatorin was abundant in XYS and Angelicae Dahuricae Radix decoctions, and non-competitively inhibited THO activity with Ki values of 77‒84 nM, higher than those (20‒52 nM) of fluvoxamine, which clinically interacted with theophylline. Compared with imperatorin, the intestinal bacterial metabolite xanthotoxol caused weaker THO inhibition. Consistent with the potency of the inhibitory effects, the docking analysis generated Gold fitness values in the order−fluvoxamine > imperatorin > xanthotoxol. During 2017‒2018, 2.6 % of 201,093 theophylline users consumed XYS. After inverse probability weighting, XYS users had a higher occurrence of undesired effects than non-XYS users; in particular, there was an approximately two-fold higher occurrence of headaches (odds ratio (OR), 2.14; 95 % confidence interval (CI), 1.99‒2.30; p < 0.001) and tachycardia (OR, 1.83; 95 % CI, 1.21‒2.77; p < 0.05). The incidence of irregular heartbeats increased (OR, 1.36; 95 % CI, 1.07‒1.72; p < 0.05) only in the theophylline users who took a high cumulative dose (≥ 24 g) of XYS. However, the mortality in theophylline users concurrently taking XYS was lower than that in non-XYS users (OR, 0.24; 95 % CI, 0.14‒0.40; p < 0.001). ConclusionXYS contains human CYP1A2 inhibitors, and undesirable effects were observed in patients receiving both theophylline and XYS. Further human studies are essential to reduce mortality and to adjust the dosage of theophylline in XYS users.

Induction of hepatic CYP3A4 expression by cholesterol and cholic acid: Alterations of gene expression, microsomal activity, and pharmacokinetics.

Human cytochrome P450 3A4 (CYP3A4) is a drug-metabolizing enzyme that is abundantly expressed in the liver and intestine. It is an important issue whether compounds of interest affect the expression of CYP3A4 because more than 30% of commercially available drugs are metabolized by CYP3A4. In this study, we examined the effects of cholesterol and cholic acid on the expression level and activity of CYP3A4 in hCYP3A mice that have a human CYP3A gene cluster and show human-like regulation of the coding genes. A normal diet (ND, CE-2), CE-2 with 1% cholesterol and 0.5% cholic acid (HCD) or CE-2 with 0.5% cholic acid was given to the mice. The plasma concentrations of cholesterol, cholic acid and its metabolites in HCD mice were higher than those in ND mice. In this condition, the expression levels of hepatic CYP3A4 and the hydroxylation activities of triazolam, a typical CYP3A4 substrate, in liver microsomes of HCD mice were higher than those in liver microsomes of ND mice. Furthermore, plasma concentrations of triazolam in HCD mice were lower than those in ND mice. In conclusion, our study suggested that hepatic CYP3A4 expression and activity are influenced by the combination of cholesterol and cholic acid invivo.

Induction of human hepatic cytochrome P-450 3A4 expression by antifungal succinate dehydrogenase inhibitors

Succinate dehydrogenase inhibitors (SDHIs) are widely-used fungicides, to which humans are exposed and for which putative health risks are of concern. In order to identify human molecular targets for these agrochemicals, the interactions of 15 SDHIs with expression and activity of human cytochrome P-450 3A4 (CYP3A4), a major hepatic drug metabolizing enzyme, were investigated in vitro. 12/15 SDHIs, i.e., bixafen, boscalid, fluopyram, flutolanil, fluxapyroxad, furametpyr, isofetamid, isopyrazam, penflufen, penthiopyrad, pydiflumetofen and sedaxane, were found to enhance CYP3A4 mRNA expression in human hepatic HepaRG cells and primary human hepatocytes exposed for 48 h to 10 µM SDHIs, whereas 3/15 SDHIs, i.e., benzovindiflupyr, carboxin and thifluzamide, were without effect. The inducing effects were concentrations-dependent for boscalid (EC50=22.5 µM), fluopyram (EC50=4.8 µM) and flutolanil (EC50=53.6 µM). They were fully prevented by SPA70, an antagonist of the Pregnane X Receptor, thus underlining the implication of this xenobiotic-sensing receptor. Increase in CYP3A4 mRNA in response to SDHIs paralleled enhanced CYP3A4 protein expression for most of SDHIs. With respect to CYP3A4 activity, it was directly inhibited by some SDHIs, including bixafen, fluopyram, fluxapyroxad, isofetamid, isopyrazam, penthiopyrad and sedaxane, which therefore appears as dual regulators of CYP3A4, being both inducer of its expression and inhibitor of its activity. The inducing effect nevertheless predominates for these SDHIs, except for isopyrazam and sedaxane, whereas boscalid and flutolanil were pure inducers of CYP3A4 expression and activity. Most of SDHIs appear therefore as in vitro inducers of CYP3A4 expression in cultured hepatic cells, when, however, used at concentrations rather higher than those expected in humans in response to environmental or dietary exposure to these agrochemicals.

Roles of human cytochrome P450 1A2, 2C9, and 3A4/5 enzymes in drug oxidations mediated by metabolically inactivated human hepatocytes in previously transplanted chimeric mice

Roles of human cytochrome P450 1A2, 2C9, and 3A4/5 enzymes in drug oxidations mediated by metabolically inactivated human hepatocytes in previously transplanted chimeric mice

Pathogenic Missense Mutations in Nicotine-metabolizing Cytochrome P450 2A6 Gene – An In silico Study

ABSTRACT Introduction: Nicotine is an environmental agent that can have a significant impact on the emergence of nicotine dependence when it interacts with particular candidate nicotine genes. Pharmacogenetics may have a role in the interindividual difference in adolescent smoking cessation. Identification of missense mutations (which are deleterious) in advance will help to formulate medication targeting it, to prevent the consequences in the future. The current study aimed to identify more potential and functional mutations of the cytochrome P450 2A6 (CYP2A6) gene by employing computational tools. Materials and Methods: The Ensembl database was used to collect the data on missense mutations of the human CYP2A6 gene. Computational tools such as Sorting Intolerant From Tolerant, Polymorphism Phenotyping, and PROtein Variation Effect ANalyzer are used to identify and screen missense mutation. One hundred and thirty missense mutations were identified and screened. The stability of protein variants and their pathogenicity were identified using I-Mutant and MutPred, respectively. Results: One hundred and eighteen single-nucleotide polymorphisms were determined to be harmful among the 130 missense variants that were examined, as predicted by all three of the computational tools mentioned in the methodology section. I-Mutant Suite identified about 104 variants with decreased stability and the other 14 variants showed increased stability. MutPred tool identified that out of the 118 missense variants, 69 were found to be highly pathogenic, 33 were found to be pathogenic, and two of them were not pathogenic. Conclusions: Nearly a hundred alleles in the CYP2A6 gene were identified as potentially pathogenic variants using data mining.

Gene Polymorphisms and Drug-Drug Interactions Determine the Metabolic Profile of Blonanserin.

This study aimed to evaluate the effects of cytochrome P450 3A4 (CYP3A4) gene polymorphism and drug interaction on the metabolism of blonanserin. Human recombinant CYP3A4 was prepared using the Bac-to-Bac baculovirus expression system. A microsomal enzyme reaction system was established, and drug-drug interactions were evaluated using Sprague-Dawley rats. Ultra-performance liquid chromatography-tandem mass spectrometry was used to detect the concentrations of blonanserin and its metabolite. Compared with wild type CYP34A, the relative clearance of blonanserin by CYP3A4.29 significantly increased to 251.3%, while it decreased notably with CYP3A4.4, 5, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18, 23, 24, 28, 31, 33, and 34, ranging from 6.09% to 63.34%. Among 153 tested drugs, nimodipine, felodipine, and amlodipine were found to potently inhibit the metabolism of blonanserin. Moreover, the inhibitory potency of nimodipine, felodipine, and amlodipine varied with different CYP3A4 variants. The half-maximal inhibitory concentration and enzymatic kinetics assay demonstrated that the metabolism of blonanserin was noncompetitively inhibited by nimodipine in rat liver microsomes and was inhibited in a mixed manner by felodipine and amlodipine in both rat liver microsomes and human liver microsomes. When nimodipine and felodipine were coadministered with blonanserin, the area under the blood concentration-time curve (AUC)(0-t), AUC(0-∞), and C max of blonanserin increased. When amlodipine and blonanserin were combined, the C max of blonanserin C increased remarkably. The vast majority of CYP3A4 variants have a low ability to catalyze blonanserin. With combined administration of nimodipine, felodipine, and amlodipine, the elimination of blonanserin was inhibited. This study provides the basis for individualized clinical use of blonanserin. SIGNIFICANCE STATEMENT: The enzyme kinetics of novel CYP3A4 enzymes for metabolizing blonanserin were investigated. Clearance of blonanserin by CYP3A4.4, 5, 7-10, 12-14, 16-18, 23-24, 28, 31, 33, and 34 decreased notably, but increased with CYP3A4.29. Additionally, we established a drug interaction spectrum for blonanserin, in which nimodipine, felodipine, and amlodipine kinetics exhibited mixed inhibition. Moreover, their inhibitory potencies decreased with CYP3A4.4 and 5 compared to CYP3A4.1. This study provides essential data for personalized clinical use of blonanserin.

Differential Effects of Clotrimazole on X-Ray Crystal Structures of Human Cytochromes P450 3A5 and 3A4.

Cytochromes P450 CYP3A5 and CYP3A4 exhibit differential plasticity that underlies differences in drug metabolism and drug-drug interactions. To extend previous studies, CYP3A4 and CYP3A5 were cocrystallized with clotrimazole, a compact ligand that binds to the heme iron in the catalytic center of the active site. Binding studies indicate that clotrimazole exhibits tight binding to CYP3A5 with a binding affinity (Kd) of <0.01 μM like that of CYP3A4. A single clotrimazole is bound to the heme iron in CYP3A4 that triggers expansion of active site cavity that reflects a loss of aromatic interactions between phenylalanine sidechains in the distal active site and increased conformational entropy for the F-F' connector due to reorientation of Phe-304 to accommodate clotrimazole. In contrast to CYP3A4, the CYP3A5 Phe-304 exhibits an induced fit along with Phe-213 to form edge-to-face aromatic interactions with heme-bound clotrimazole. These aromatic interactions between aromatic amino acids propagate by induced fits with a second clotrimazole residing in the distal active site and a third clotrimazole bound in an expanded entrance channel as well as between the three clotrimazoles. The large, expanded entrance channel surrounded by the C-terminal loop and the F' and A' helices in CYP3A5 suggests conformational selection for the binding of clotrimazole due to its large girth, which may also cause the entrance channel to remain open after the binding of the first clotrimazole to the heme iron. The additional binding sites suggest a path for sequential binding of one molecule to reach and bind to the heme iron. SIGNIFICANCE STATEMENT: Clotrimazole binds to the heme iron of CYP3A5 and CYP3A4. In CYP3A5, two clotrimazoles also bind in the distal active site and in an expanded entrance channel. Aromatic interactions between clotrimazoles and phenylalanine sidechains including Phe-304 indicate induced fits for each clotrimazole. In contrast to CYP3A5, displacement of the CYP3A4 Phe-304 rotamer by clotrimazole leads to extensive disruption of phenylalanine interactions that limit the space above the heme, to an expanded active site cavity, and to increased CYP3A4 conformational heterogeneity.

Synthesis of 6β-hydroxy androgens from a 3,5-diene steroid precursor to test for cytochrome P450 3A4-catalyzed hydroxylation of androstenedione

6β-Hydroxytestosterone is a biomarker for the activity of human cytochrome P450 3A4 (P450 3A4), the major drug metabolizing cytochrome P450 enzyme. Despite its significance, efficient routes for the chemical synthesis of 6β-hydroxytestosterone are rare. In this study, 6β-hydroxytestosterone was synthesized through the oxidation of a 3,5-diene precursor under the Uemura-Doyle reaction conditions using a dirhodium catalyst in the presence of tert-butylhydroperoxide. Mechanistic studies showed that some oxygen is incorporated from molecular oxygen and CH abstraction is partially rate-limiting. This reaction was used to synthesize 6β-hydroxyandrostenedione, which was used as a standard to test the hypothesis of whether P450 3A4 catalyzes the hydroxylation of androstenedione. Upon incubation of P450 3A4 with androstenedione, a hydroxylated product was formed, which matched the retention time of synthetic 6β-hydroxyandrostenedione. This reaction can be exploited to study other biochemical processes involving compounds with a 6 β -hydroxy-3-keto-Δ4 steroid backbone.

Design, synthesis and biological evaluation of chalcone derivatives as potent and orally active hCYP3A4 inhibitors

Human cytochrome P450 3A4 (hCYP3A4), one of the most important drug-metabolizing enzymes, catalyze the metabolic clearance of ∼50% therapeutic drugs. CYP3A4 inhibitors have been used for improving the in vivo efficacy of hCYP3A4-substrate drugs. However, most of existing hCYP3A4 inhibitors may trigger serious adverse effects or undesirable effects on endogenous metabolism. This study aimed to discover potent and orally active hCYP3A4 inhibitors from chalcone derivatives and to test their anti-hCYP3A4 effects both in vitro and in vivo. Following three rounds of screening and structural optimization, the isoquinoline chalcones were found with excellently anti-hCYP3A4 effects. SAR studies showed that introducing an isoquinoline ring on the A-ring significantly enhanced anti-CYP3A4 effect, generating A10 (IC50 = 102.10 nM) as a promising lead compound. The 2nd round of SAR studies showed that introducing a substituent group at the para position of the carbonyl group on B-ring strongly improved the anti-CYP3A4 effect. As a result, C6 was identified as the most potent hCYP3A4 inhibitor (IC50 = 43.93 nM) in human liver microsomes (HLMs). C6 also displayed potent anti-hCYP3A4 effect in living cells (IC50 = 153.00 nM), which was superior to the positive inhibitor ketoconazole (IC50 = 251.00 nM). Mechanistic studies revealed that C6 could potently inhibit CYP3A4-catalyzed N-ethyl-1,8-naphthalimide (NEN) hydroxylation in a competitive manner (Ki = 30.00 nM). Moreover, C6 exhibited suitable metabolic stability in HLMs and showed good safety profiles in mice. In vivo tests demonstrated that C6 (100 mg/kg, orally administration) significantly increased the AUC(0-inf) of midazolam by 3.63-fold, and strongly prolonged its half-life by 1.66-fold compared with the vehicle group in mice. Collectively, our findings revealed the SARs of chalcone derivatives as hCYP3A4 inhibitors and offered several potent chalcone-type hCYP3A4 inhibitors, while C6 could serve as a good lead compound for developing novel, orally active CYP3A4 inhibitors with improved druglikeness properties.

A Fluorescent Chemosensor for Long‐Term Tracking of Cancer Cell Metastasis and Invasion via Enzyme‐Activated Anchoring

AbstractEvidence shows that the enzyme human cytochrome P450 1A1 (CYP1A1) is associated with cancer; indeed, it is shown to play a key role in the occurrence of many cancers. Therefore, the molecular imaging of CYP1A1 in cells is of great significance for revealing the process of cancer development. Herein, a chemosensor, DCBEM, is reported that is able to selectively recognize CYP1A1 and achieve long‐term labeling of the enzyme through an enzymolysis cascade reaction. The design of DCBEM relies on the reaction between the highly electrophilic intermediate (quinone methide) and the enzyme, which forms a fluorescent label with CYP1A1 via covalent bonding. The chemosensor reveals excellent specificity toward CYP1A1 and achieves high‐resolution monitoring of cell migration with a strong retention capacity in vivo (up to 4 days). Further, using DCBEM, the pathways of invasion of colon cancer and breast cancer cells are successfully visualized in living mice. This method of labeling enzymes provides a simple and efficient way to render ordinary fluorescent chemosensors suitable for the long‐term tracking of cancer cells, for which such molecular tools are currently lacking.

Interaction of CYP3A4 with caffeine: First insights into multiple substrate binding

Human cytochrome P450 3A4 (CYP3A4) is a major drug-metabolizing enzyme that shows extreme substrate promiscuity. Moreover, its large and malleable active site can simultaneously accommodate several substrate molecules of the same or different nature, which may lead to cooperative binding and allosteric behavior. Due to difficulty of crystallization of CYP3A4-substrate complexes, it remains unknown how multiple substrates can arrange in the active site. We determined crystal structures of CYP3A4 bound to three and six molecules of caffeine, a psychoactive alkaloid serving as a substrate and modulator of CYP3A4. In the ternary complex, one caffeine binds to the active site suitably for C8-hydroxylation, most preferable for CYP3A4. In the senary complex, three caffeine molecules stack parallel to the heme with the proximal ligand poised for 3-N-demethylation. However, the caffeine stack forms extensive hydrophobic interactions that could preclude product dissociation and multiple turnovers. In both complexes, caffeine is also bound in the substrate channel and on the outer surface known as a peripheral site. At all sites, aromatic stacking with the caffeine ring(s) is likely a dominant interaction, while direct and water-mediated polar contacts provide additional stabilization for the substrate-bound complexes. Protein-ligand interactions via the active site R212, intrachannel T224, and peripheral F219 were experimentally confirmed, and the latter two residues were identified as important for caffeine association. Collectively, the structural, spectral, and mutagenesis data provide valuable insights on the ligand binding mechanism and help better understand how purine-based pharmaceuticals and other aromatic compounds could interact with CYP3A4 and mediate drug-drug interactions.

Stimulatory and Inhibitory Effects of Steroid Hormones and Human Cytochrome P450 (CYP) 3A Inhibitors on Cortisol 6β-Hydroxylation Catalyzed by CYP3A Subfamilies.

The inhibitory and stimulatory effects of several compounds, including steroid hormones and azole antifungal agents, on cortisol 6β-hydroxylation activity by cytochrome P450 (CYP) 3A4, polymorphically expressed CYP3A5, and fetal CYP3A7 were compared with those on testosterone 6β-hydroxylation to clarify the catalytic properties of the predominant forms of the human CYP3A subfamily. 6β-Hydroxylation activities of cortisol and testosterone by CYP3A4, CYP3A5, and CYP3A7 in the absence or presence of dehydroepiandrosterone (DHEA), α-naphthoflavone (ANF), ketoconazole, itraconazole, and voriconazole were measured using high-performance liquid chromatography. Lower concentrations of DHEA and ANF increased cortisol 6β-hydroxylation activities catalyzed by CYP3A4 but not those catalyzed by CYP3A5 and CYP3A7. The inhibition strength of azole antifungal agents against cortisol 6β-hydroxylation catalyzed by all CYP3A subfamilies was similar to that of testosterone 6β-hydroxylation. Although the Michaelis constant (Km) increased 2-fold in the presence of 20 μM DHEA compared to that of the control, the maximal velocity (Vmax) values gradually increased with increasing DHEA. For ANF, both Km and Vmax values increased, although the Km value decreased at 2.5 μM concentrations. Ketoconazole and itraconazole competitively inhibited cortisol 6β-hydroxylation mediated by CYP3A4 with similar inhibition constants. The inhibitory/stimulatory pattern among CYP3A subfamily members differed between cortisol and testosterone, and CYP3A4 was found to be the most sensitive in terms of inhibition by azole antifungals among the CYP3A subfamily members investigated.

Enantioselective metabolism of novel chiral insecticide Paichongding by human cytochrome P450 3A4: A computational insight

As a novel chiral neonicotinoid insecticide, Paichongding (IPP) has been widely applied in agriculture due to its excellent insecticidal activity. However, the enantioselective metabolism of IPP stereoisomers (5R7R-IPP, 5S7S-IPP, 5R7S-IPP, and 5S7R-IPP) mediated by enzymes in non-target organisms, especially the cytochrome P450s (CYPs), remains unknown. To address this knowledge gap, we developed an integrated computational framework to elucidate the binding interactions and enantioselective metabolism of IPP stereoisomers in human CYP3A4. The results reveal that 5R7R-IPP shows much stronger binding affinity to CYP3A4 than 5S7S-IPP, while enantiomers 5R7S-IPP and 5S7R-IPP have no essential difference in their binding potential, owing to their specific interactions with key CYP3A4 residues. Although enantiomers 5R7R-IPP and 5S7S-IPP feature distinct binding modes resulting from the chiral differences, their transformation activities are slightly different, with C5 and C13 being the primary metabolic sites, respectively. In contrast, CYP3A4 preferably metabolizes 5R7S-IPP over 5S7R-IPP. The metabolism of epimers 5R7R-IPP and 5R7S-IPP share C5-hydroxylation routes due to the conserved 5R-conformaitons, but differ with the transformation routes at C11/C13 and C3 sites. The 7R-chirality of 5S7R-IPP significantly reduces the metabolic potency compared to 5S7S-IPP. CYP3A4-catalyzed hydroxylation and desaturation of IPP stereoisomers generate various chiral metabolites, with C5- and C13-hydroxyIPPs further transforming into depropylated products. Furthermore, the toxicity assessment reveals that IPP, along with the majority of its hydroxylated, desaturated, and depropylated metabolites, can potentially induce adverse effects on human health, specifically hepatotoxicity, respiratory toxicity, and carcinogenicity. This study provides valuable insights into the enantioselective fate of chiral IPP metabolism by CYP3A4, and the identified metabolites can serve as potential biomarkers for monitoring IPP exposure and associated health risk in human body.

CYP1A inhibitors: Recent progress, current challenges, and future perspectives.

Mammalian cytochrome P450 1A (CYP1A) are key phase I xenobiotic-metabolizing enzymes that play a distinctive role in metabolic activation or metabolic clearance of a variety of procarcinogens, drugs, and endogenous substances. Human CYP1A subfamily contains two members (hCYP1A1 and hCYP1A2), which are known to catalyze the oxidative activation of some environmental procarcinogens into carcinogenic species. Increasing evidence has demonstrated that CYP1A inhibitor therapies are promising strategies for cancer chemoprevention or overcoming CYP1A-associated drug toxicity and resistance. Herein, we reviewed recent advances in the discovery and characterization of hCYP1A inhibitors, from the discovery approaches to structural features and biomedical applications of hCYP1A inhibitors. The inhibition potentials, inhibition modes, and inhibition constants of all reported hCYP1A inhibitors are comprehensively summarized. Meanwhile, the structural features and structure-activity relationships of different classes of hCYP1A1 and hCYP1A2 inhibitors are analyzed and discussed in depth. Furthermore, the major challenges and future directions for this field are presented and highlighted. Collectively, the information and knowledge presented here will strongly facilitate the researchers to discover and develop more efficacious CYP1A inhibitors for specific purposes, such as chemo-preventive agents or as tool molecules in hCYP1A-related fundamental studies.

Inhibition Effect of Okanin Toward Human Cytochrome P450 3A4 and 2D6 with Multi-spectroscopic Studies and Molecular Docking.

Okanin, a major flavonoid of a popular herb tea, Coreopsis tinctoria Nutt., showed strong inhibition on CYP3A4 and CYP2D6. The strong interaction between okanin and CYPs were determined by enzyme kinetics, multispectral technique and molecular docking. The inhibition type of two enzymes, CYP3A4 and CYP2D6, by okanin are mixed and non-competitive inhibition type, respectively. The IC50 values and the binding constant of okanin to CYP3A4 can be deduced that the interaction was stronger than that of CYP2D6. The Conformations of CYP3A4 and CYP2D6 were changed by okanin. The evidence from fluorescence measurement along with molecular docking verified that these two CYPs were bound with okanin by hydrogen bonds and hydrophobic forces. Our investigation suggested that okanin may lead to interactions between herb and drug by inhibiting CYP3A4 and CYP2D6 activities, thus its consumption should be taken with caution.

TET3-mediated DNA demethylation and chromatin remodeling regulate T-2 toxin-induced human CYP1A1 expression and cytotoxicity in HepG2 cells

T-2 toxin is a hazardous environmental pollutant that poses a risk to both farm animals and humans. Our previous research has reported that T-2 toxin highly induced the expression of human cytochrome P450 1A1 (CYP1A1), which may be a representative inducible marker of T-2 toxin and mediate the toxicity of T-2 toxin. In this study, we found that T-2 toxin decreased the DNA methylation levels of the CpG islands on the CYP1A1 promoter by inducing the expression of eleven translocation family protein 3 (TET3) and facilitating its binding to the promoter. These DNA methylation changes then generated an activated chromatin structure on the CYP1A1 promoter by releasing the repressor complex methyl-binding protein 2 (MeCP2) and histone deacetylase 2 (HDAC2), increasing the active histone modification markers, including H3K4ac, H3K9ac and H3K14ac, and facilitating RNA pol II and NRF1/Sp1 recruitment, which ultimately led to the transcriptional activation of CYP1A1. Interestingly, TET3-mediated CYP1A1 induction enhanced the cytotoxicity of T-2 toxin through inhibiting cell proliferation. Our results demonstrate that T-2 toxin-induced CYP1A1 expression is detrimental to cells and clearly show how T-2 toxin inhibits cell proliferation through regulating CYP1A1 expression from an epigenetic perspective. The findings broaden our current knowledge of the epigenetic mechanisms regulating environmental factors-induced CYP1A1 expression and cytotoxicity. TET3 may serve as a potential new target for toxicogenic detoxification.

Human cytochrome P450 17A1 structures with metabolites of prostate cancer drug abiraterone reveal substrate-binding plasticity and a second binding site

Abiraterone acetate is a first-line therapy for castration-resistant prostate cancer. This prodrug is deacetylated invivo to abiraterone, which is a potent and specific inhibitor of cytochrome P450 17A1 (CYP17A1). CYP17A1 performs two sequential steps that are required for the biosynthesis of androgens that drive prostate cancer proliferation, analogous to estrogens in breast cancer. Abiraterone can be further metabolized invivo on the steroid A ring to multiple metabolites that also inhibit CYP17A1. Despite its design as an active-site-directed substrate analog, abiraterone and its metabolites demonstrate mixed competitive/noncompetitive inhibition. To understand their binding, we solved the X-ray structures of CYP17A1 with three primary abiraterone metabolites. Despite different conformations of the steroid A ring and substituents, all three bound in the CYP17A1 active site with the steroid core packed against the I helix and the A ring C3 keto or hydroxyl oxygen forming a hydrogen bond with N202 similar to abiraterone itself. The structure of CYP17A1 with 3-keto, 5α-abiraterone was solved to 2.0Å, the highest resolution to date for a CYP17A1 complex. This structure had additional electron density near the F/G loop, which is likely a second molecule of the inhibitor and which may explain the noncompetitive inhibition. Mutation of the adjacent Asn52 to Tyr positions its side chain in this space, maintains enzyme activity, and prevents binding of the peripheral ligand. Collectively, our findings provide further insight into abiraterone metabolite binding and CYP17A1 function.

Newly Identified Tree Shrew Cytochrome P450 2A13 is Expressed in Liver and Lung and Encodes a Functional Drug-Metabolizing Enzyme Similar to Dog Cytochrome P450 2A13 and Pig Cytochrome P450 2A19.

The tree shrew, a non-rodent primate-like species, is used in various fields of biomedical research, including hepatitis virus infection, myopia, depression, and toxicology. Recent genome analysis found that the numbers of cytochrome P450 (P450 or CYP) genes are similar in tree shrews and humans and their sequence identities are high. Although the P450s are a family of important drug-metabolizing enzymes, they have not yet been fully investigated in tree shrews. In the current study, tree shrew CYP2A13 cDNA was isolated from liver, and its characteristics were compared with those of pig, dog, and human CYP2As. Tree shrew CYP2A13 amino acid sequences were highly identical (87-92%) to the human CYP2As and contained sequence motifs characteristic of P450s. Phylogenetic analysis revealed that tree shrew CYP2A13 was more closely related to human CYP2As than to rat CYP2As, similar to dog and pig CYP2As. Among the tissue types analyzed, tree shrew CYP2A13 mRNA was preferentially expressed in liver and lung, similar to dog CYP2A13 mRNA, whereas dog CYP2A25 and pig CYP2A19 mRNAs were predominantly expressed in liver. Tree shrew liver microsomes and tree shrew CYP2A13 proteins heterologously expressed in Escherichia coli catalyzed coumarin 7-hydroxylation and phenacetin O-deethylation, just as human, dog, and pig CYP2A proteins and liver microsomes do. These results demonstrate that tree shrew CYP2A13 is expressed in liver and lung and encodes a functional drug-metabolizing enzyme. SIGNIFICANCE STATEMENT: Novel tree shrew cytochrome P450 2A13 (CYP2A13) was identified and characterized in comparison with human, dog, and pig CYP2As. Tree shrew CYP2A13 isolated from liver had high sequence identities and close phylogenetic relationships to its human homologs and was abundantly expressed in liver and lung at the mRNA level. Tree shrew CYP2A13 metabolized coumarin and phenacetin, human selective CYP2A6 and CYP2A13 substrates, respectively, similar to dog and pig CYP2As, and is a functional drug-metabolizing enzyme likely responsible for drug clearances.

Hydroxylation and lyase reactions of steroids catalyzed by mouse cytochrome P450 17A1 (Cyp17a1)

Cytochrome P450 17A1 (CYP17A1) catalyzes 17α-hydroxylation and 17,20-lyase reactions with steroid hormones. Mice contain an orthologous Cyp17a1 enzyme in the genome, and its amino acid sequence has high similarity with human CYP17A1. We purified recombinant mouse Cyp17a1 and characterized its oxidation reactions with progesterone and pregnenolone. The open reading frame of the mouse Cyp17a1 gene was inserted and successfully expressed in Escherichia coli and then purified using Ni2+-nitrilotriacetic acid (NTA) affinity column chromatography. Purified mouse Cyp17a1 displayed typical Type I binding titration spectral changes upon the addition of progesterone, 17α-OH progesterone, pregnenolone, and 17α-OH pregnenolone, with similar binding affinities to those of human CYP17A1. Catalytic activities for 17α-hydroxylation and 17,20-lyase reactions were studied using ultra-performance liquid chromatography (UPLC)-mass spectrometry analysis. Mouse Cyp17a1 showed cytochrome b5 stimulation in catalysis. In comparison to human enzyme, much higher specificity constants (kcat/Km) were observed with mouse Cyp17a1. In the reactions of Δ4-steroids (progesterone and 17α-OH progesterone), the specificity constants were 2100 times higher than the human enzyme. The addition of cytochrome b5 produced significant stimulation of 17,20-lyase activities of mouse Cyp17a1. Two Arg mutants of mouse Cyp17a1 (R347H and R358Q) displayed a larger decrease in 17,20-lyase reaction (from 17α-OH pregnenolone to dehydroepiandrosterone, DHEA) than 17α-hydroxylation, indicating that –as in human CYP17A1–these basic residues in mouse Cyp17a1 are important in interacting with the cytochrome b5 protein in the lyase reactions.