Human Cytochrome P450 3A4 Research Articles

Cloning and in vivo metabolizing activity study of CYP3A4 on amiodarone drug residues: A possible probiotic and therapeutic option

BackgroundAmiodarone represents a principal antiarrhythmic pharmaceutical drug available in the market for the treatment of ventricular arrhythmias. However, despite its better efficacy, the usage of amiodarone is associated with extracardiac toxicity. The human body evolved a system of cytochrome P450 enzymes which play an essential role in the metabolism of harmful foreign substances. Therefore, CYP enzymes may either lead to the elimination or degradation of the leftover drug residues. ObjectiveThe present study focused on successful utilization of Saccharomyces cerevisiae strain OBS2 with probiotic- cum- therapeutic potential and expressing in silico enhanced human cytochrome P4503A4 for the degradation of leftover drug residues of amiodarone in vitro and in vivo. MethodologyIn this study, cytochrome P4503A4 (1W0E) was taken as a template and the predicted 3D model of mutant CYP3A4 was developed using different bioinformatics tools. Selected mutant (Glu165Asp) protein was reverse translated and transcribed into cDNA sequence. The cDNA of CYP3A4 was synthesized, cloned into p427TEF construct and transformed into Saccharomyces cerevisiae OBS2. The degradation of leftover drug residues of amiodarone in vitro and in vivo using recombinant Saccharomyces cerevisiae OBS2 expressing CYP3A4 was evaluated. ResultThe CYP3A4 activity in recombinant probiotic yeast was observed as 108 IU/mL and in vitro degradation of leftover drug residues of amiodarone was observed as 66.32 %. Whereas, in vivo degradation of leftover drug residues of amiodarone was observed as 72.61 % along with recovery of organ damage in histopathological studies of the animal model. ConclusionSaccharomyces cerevisiae OBS2 expressing CYP3A4 can be used for probiotic and therapeutic applications.

Characterization of the Inhibition of Allelic Variants of Cytochrome P450 2A6 with trans‐Cinnamic Aldehyde

Cigarette smoking and nicotine addiction continues to be a leading cause of preventable death. The enzyme cytochrome P450 2A6 (CYP2A6), which is highly polymorphic with over forty known alleles, is the primary enzyme that catalyzes the initial oxidation of nicotine in humans. Studies have shown that trans‐cinnamic aldehyde (tCA) is a mechanism‐based inactivator of this enzyme and is therefore a potential smoking cessation aid. However, the inhibitory effectiveness of tCA with the gene products of the different alleles is unknown. The goal of the work presented was to characterize inhibition of the CYP2A6 variant resulting from the *23 allele by tCA. This allele results in a point mutation (R203C). Site‐directed mutagenesis was used to engineer CYP2A6*23 from a truncated form of the human CYP2A6*1 gene. The recombinant protein was expressed in E. coli and purified. Using ligand‐binding titrations, we found that the Kd for tCA binding to CYP2A6*23 was 7.1 μM, whereas the Kd value for CYP2A6*1 was 10.6 μM. Consistent with previous reports, CYP2A6*23 had lower activity than that of CYP2A6*1; however the relative inhibition of both CYP2A6*23 and CYP2A6*1 by 80 μM tCA was similar. Therefore, while tCA has a slightly higher affinity for CYP2A6*23, the change to a cysteine at position 203 does not significantly affect the effectiveness of tCA as an inhibitor. So tCA could still be a potential smoking cessation aid for individuals with the CYP2A6*23 allele.Support or Funding InformationNational Institute on Drug Abuse Award Number 15DA042341; Pacific University Faculty Development Grant

Multistep Binding of the Non-Steroidal Inhibitors Orteronel and Seviteronel to Human Cytochrome P450 17A1 and Relevance to Inhibition of Enzyme Activity.

Orteronel (TAK-700) is a substituted imidazole that was developed for the treatment of castration-resistant prostate cancer but was dropped in phase III clinical trials. Both enantiomers of this inhibitor of cytochrome P450 (P450) 17A1 show some selectivity in differentially blocking the 17α-hydroxylation and lyase activities of the enzyme. Although both enantiomers of this compound have sub-micromolar IC50 values and bind to the enzyme with a type II spectral change (indicative of nitrogen-iron bonding) and reported Kd values of 56 and 40 nM (R and S, respectively), the rates of binding to P450 17A1 were relatively slow. We considered the possibility that the drug is a slow, tight-binding inhibitor. Analysis of the kinetics of binding revealed rapid formation of an initial complex, presumably in the substrate binding site, followed by a slower change to the spectrum of a final iron complex. Similar kinetics were observed in the interaction of another inhibitor, the triazole (S)-seviteronel (VT-464), with P450 17A1. Kinetic tests and modeling indicate that the further change to the iron-complexed form of the orteronel- or seviteronel-P450 complex is not a prerequisite for enzyme inhibition. Accordingly, the inclusion of heme-binding heterocyclic nitrogen moieties in P450 17A1 inhibitors may not be necessary to achieve inhibition but may nevertheless augment the process.

Evaluation of the inhibition of chlorophenols towards human cytochrome P450 3A4 and differences among various species

Chlorophenols (CPs) are important pollutants detected frequently in the environment. This study intended to detect the inhibitory effects of fourteen CPs (2-CP, 3-CP, 4-CP, 4C2AP, 4C3MP, 2.4-DCP, 2.3.4-TCP, 2.4.5-TCP, 2.4.6-TCP, 3.4.5-TCP, 2.3.4.5-TECP, 2.3.4.6-TECP, 2.3.5.6-TECP and PCP) towards human liver cytochrome P450 3A4 (CYP3A4). Throughout the tests, testosterone was used as the probe substrate and CPs were used as inhibitors. A series of experiments (enzyme activity assays, preliminary screening tests, inhibition kinetics determination) were conducted to determine the inhibition of CPs towards human liver CYP3A4. CPs with the inhibitory effect >80% were selected for the inhibition evaluation in liver microsomes from different animal species (monkey, rat, dog, pig). The results showed that 2.3.4-TCP, 3.4.5-TCP, and 2.3.4.5-TECP inhibited the activities of CYP3A4 by 80.3%, 93.4%, 91.6%, respectively. Inhibition kinetics type were non-competitive and inhibition kinetics constant (Ki) values were 26.4 μM, 13.5 μM, and 8.8 μM for the inhibition of 2.3.4-TCP, 3.4.5-TCP, and 2.3.4.5-TECP towards human CYP3A4, respectively. Inhibition kinetics type was competitive and Ki value was 4.9 μM for the inhibition of 2.3.4-TCP towards CYP3A4 in Monkey liver microsomes (MyLMs). Inhibition kinetic types were non-competitive and Ki values were 8.1 μM and 28.7 μM for the inhibition of 3.4.5-TCP and 2.3.4.5-TECP towards CYP3A4 in MyLMs. Inhibition kinetic types were non-competitive and Ki values were 13.8 μM, 0.6 μM, and 6.1 μM for the inhibition of 2.3.4-TCP, 3.4.5-TCP, and 2.3.4.5-TECP towards CYP3A4 in Dog liver microsomes (DLMs), respectively. By comparing Ki values and inhibition kinetic types, the dog was the most suitable model to assess the inhibition of 2.3.4-TCP and 2.3.4.5-TECP towards CYP3A4, and monkey was the most suitable model to assess the inhibition of 3.4.5-TCP towards CYP3A4. In conclusion, our recent study on the inhibition of CPs towards CYP3A4 and species differences was important for further toxicological studies of CPs in human bodies.

Comparison of steroid hormone hydroxylation mediated by cytochrome P450 3A subfamilies

Hydroxylation activity at the 6β-position of steroid hormones (testosterone, progesterone, and cortisol) by human cytochromes P450 (CYP) 3A4, polymorphic CYP3A5, and fetal CYP3A7 were compared to understand the catalytic properties of the major forms of human CYP3A subfamily. Testosterone, progesterone, and cortisol 6β-hydroxylation activities of recombinant CYP3A4, CYP3A5, and CYP3A7 were determined by liquid chromatography. Michaelis constants (Km) for CYP3A7-mediated 6β-hydroxylation of testosterone, progesterone, and cortisol were similar to those of CYP3A4 and CYP3A5. The maximal velocity (kcat) and kcat/Km values for CYP3A4 were the highest, followed by CYP3A5 and those for CYP3A7 were the lowest among three CYP3A subfamily members. A decrease in Km values for progesterone 6β-hydroxylation by CYP3A4, CYP3A5, and CYP3A7 in the presence of testosterone was observed, and the kcat values for CYP3A5 gradually increased with increasing testosterone. This indicated that testosterone stimulated progesterone 6β-hydroxylation by all three CYP3A subfamily members. However, progesterone inhibited testosterone 6β-hydroxylation mediated by CYP3A4, CYP3A5, and CYP3A7. In conclusion, the kcat values, rather than Km values, for 6β-hydroxylation of three steroid hormones mediated by CYP3A7 were different from those for CYP3A4 and CYP3A5. In addition, the inhibitory/stimulatory pattern of steroid-steroid interactions would be different among CYP3A subfamily members.

Clobetasol Propionate Is a Heme-Mediated Selective Inhibitor of Human Cytochrome P450 3A5.

The human cytochrome P450 (CYP) enzymes CYP3A4 and CYP3A5 metabolize most drugs and have high similarities in their structure and substrate preference. Whereas CYP3A4 is predominantly expressed in the liver, CYP3A5 is upregulated in cancer, contributing to drug resistance. Selective inhibitors of CYP3A5 are, therefore, critical to validating it as a therapeutic target. Here we report clobetasol propionate (clobetasol) as a potent and selective CYP3A5 inhibitor identified by high-throughput screening using enzymatic and cell-based assays. Molecular dynamics simulations suggest a close proximity of clobetasol to the heme in CYP3A5 but not in CYP3A4. UV-visible spectroscopy and electron paramagnetic resonance analyses confirmed the formation of an inhibitory type I heme-clobetasol complex in CYP3A5 but not in CYP3A4, thus explaining the CYP3A5 selectivity of clobetasol. Our results provide a structural basis for selective CYP3A5 inhibition, along with mechanistic insights, and highlight clobetasol as an important chemical tool for target validation.

Genetic Polymorphisms of the Human Cytochrome P450 1A1 (CYP1A1) and Cervical Cancer Susceptibility among Northeast Thai Women.

Background:CYP1A1 is an enzyme in phase I of the cytochrome P450 (CYP) superfamily, and plays a key role in detoxification of carcinogens. Host genetic predisposition in the CYP1A1 may be associated with an increased susceptibility to cervical cancer.The study aimed to evaluate four common polymorphisms of the CYP1A1 and cervical cancer susceptibility among Northeast Thai women. Methods:A case-control study was conducted involving 204 patients with squamous cell cervical cancer (SCCA) and 204 age-matched healthy controls. DNA was extracted from peripheral blood leucocytes. CYP1A1 m1, m3, and m4 genotypes were detected using PCR-RFLP, whereas the CYP1A1 m2 genotype was investigated using real-time PCR. Haplotype analysis was performed using PHASE algorithm version 2.1.1. Results: CYP1A1 m3 was monomorphic. Association between the common CYP1A1 polymorphisms, m1 and m2, and cervical cancer risk was not observed (p>0.05), nor was any association found between the m1–m2–m4 haplotype and cervical cancer risk (p>0.05). Interestingly, the CA genotype of CYP1A1 m4 was observed in 30.88% of the cervical cancer patients but was absent in healthy controls. Conclusion:Our results demonstrated a possible involvement of the CYP1A1 m4 polymorphism but no other common polymorphisms (viz., m1, m2, and m3) in the risk for cervical cancer.This finding may be useful when screening for risk of cervical cancer among Northeast Thai women.

Effect of rifampin and itraconazole on the pharmacokinetics of zanubrutinib (a Bruton's tyrosine kinase inhibitor) in Asian and non-Asian healthy subjects

PurposeZanubrutinib (BGB-3111) is a potent Bruton’s tyrosine kinase inhibitor with promising clinical activity in B-cell malignancies. Zanubrutinib was shown to be mainly metabolized through cytochrome P450 3A (CYP3A) in vitro. We evaluated the effect of steady-state rifampin (a strong CYP3A inducer) and steady-state itraconazole (a strong CYP3A inhibitor) on the pharmacokinetics (PK), safety, and tolerability of zanubrutinib in healthy Asian and non-Asian subjects.MethodsIn this open-label, two-part clinical study, 20 participants received a single oral dose of zanubrutinib (320 mg) and oral rifampin (600 mg) in Part A, and 18 participants received a single oral dose of zanubrutinib (20 mg) and oral itraconazole (200 mg) in Part B. Serial blood samples were collected after administration of zanubrutinib alone and zanubrutinib in combination with rifampin or itraconazole for the measurement of PK parameters.ResultsCoadministration with rifampin decreased AUC0–∞ of zanubrutinib by 13.5-fold and Cmax by 12.6-fold. Coadministration with itraconazole increased the AUC0–∞ of zanubrutinib by 3.8-fold and Cmax by 2.6-fold. The PK of zanubrutinib was consistent between Asian and non-Asian subjects, and zanubrutinib was well tolerated in this study.ConclusionsThese results confirm that zanubrutinib is primarily metabolized by CYP3A in humans. The PK of zanubrutinib was comparable between Asian and non-Asian subjects and, therefore, no dose modifications are necessary for zanubrutinib in these ethnic populations.

Phylogenetic Analysis and In Silico Characterization of Cytochrome P450 1A (Cyp1A) Protein from the African Catfish, Clarias gariepinus (Burchell, 1822)

The CYP family enzymes are broadly used as biomarkers because of their pattern of expression. This study describes the application of in silico tools to predict the physico-chemical characters of CYP1A protein from the catfish, Clarias gariepinus. The nucleotide sequence analysis of C. gariepinus CYP1A gene showed higher similarity with C. batrachus and reflected in the phylogenetic tree. The comparative modelling results showed this CYP1A protein was highly similar with the 3-D crystal structure of human Cytochrome p450 1A1 (PDB: 1BE3). The prediction results depicted that most of the amino acids formed alpha helix. The predicted pI was 9.10, hydropathycity was -0.226, exposed and buried residues were 61.67, 38.33% respectively. Ramachandran plot analysis showed that most of the amino acids falling on the favoured region and exhibited right- handed alpha helices as the most stable secondary structure. Some amino acids were also found to form loops to interconnect different helices. The CYP1A protein was predicted to be localized in the mitochondrion of the eukaryotic cell.

Different Roles of Human Cytochrome P450 2C9 and 3A Enzymes in Diclofenac 4'- and 5-Hydroxylations Mediated by Metabolically Inactivated Human Hepatocytes in Previously Transplanted Chimeric Mice.

To investigate the respective roles of cytochromes P450 2C9 and 3A in drug oxidation in human livers, the in vivo pharmacokinetics of S-warfarin and diclofenac were analyzed after intravenous administrations in chimeric mice that had been transplanted with human hepatocytes. P450 2C9 was metabolically inactivated in the humanized mice by orally pretreating them with tienilic acid. After intravenous administration of S-warfarin, a significant difference in the concentration-time profiles of the primary metabolite 7-hydroxywarfarin between untreated mice and mice treated with tienilic acid was observed. In contrast, there were no apparent differences in the profiles for S-warfarin between the treated and untreated groups. The mean values of the maximum concentrations (Cmax) and the areas under the plasma concentration versus time curves (AUCinfinity) for 7-hydroxywarfarin were significantly lower (22 and 16% of the untreated values, respectively) in the treated group. This presumably resulted from suppressed P450 2C9 activity in the primary oxidative metabolism in vivo in the treated group. After diclofenac administration, plasma levels of diclofenac, 5-hydroxydiclofenac, and diclofenac acylglucuronide were roughly similar in pretreated and untreated mice. However, the mean Cmax and AUCinfinity values for 4'-hydroxydiclofenac were significantly lower (38 and 53% of the untreated group, respectively) in the treated group. The reported value of ∼0.8 for the fraction of S-warfarin metabolized to 7-hydroxywarfarin mediated by P450 2C9 in in vitro systems was similar to the value implied by the present humanized-liver mouse model pretreated with tienilic acid in which the AUC of 7-hydroxywarfarin was reduced by 84%. In contrast, the fractions of diclofenac metabolized to 4'-hydroxydiclofenac in in vitro and in vivo experiments were inconsistent. These results suggested that humanized-liver mice orally treated with tienilic acid might constitute an in vivo model for metabolically inactivated P450 2C9 in human hepatocytes transplanted into chimeric mice. Moreover, diclofenac, a typical in vitro P450 2C9 probe substrate, was cleared differently in vitro and in humanized-liver mice in vivo.

Human Cytochrome P450 1A1 Adapts Active Site for Atypical Nonplanar Substrate.

The human cytochrome P450 1A1 (CYP1A1) is well known for chemical activation of procarcinogens and often has a substrate scope of small and highly planar compounds. Substrates deviating from these characteristics are certainly known, but how these larger and nonplanar substrates are accommodated and oriented within the CYP1A1 active site is not understood. Herein a new X-ray structure of CYP1A1 bound to the pan-Pim kinase inhibitor GDC-0339 reveals how the CYP1A1 active site cavity is reconfigured to bind larger and nonplanar compounds. The shape and size of the cavity are controlled by structural elements in the active site roof, with major changes in the conformation of the F helix break and relocation of Phe224 from the active site to the protein surface. This altered CYP1A1 active site architecture is consistent with the proposed mechanism for CYP1A1 generation of an unusual aminoazepane-rearranged metabolite for this substrate. SIGNIFICANCE STATEMENT: Cytochrome P450 1A1 metabolizes drugs, procarcinogens, and toxins and although previous structures have revealed how its stereotypical planar, aromatic compounds are accommodated in the CYP1A1 active site, this is not the case for flexible and nonplanar compounds. The current work determines the X-ray structure of CYP1A1 with such a flexible, nonplanar Pim kinase inhibitor, revealing significant modification of the CYP1A1 roof that accommodate this preclinical candidate and support an unusual intramolecular rearrangement reaction.

Human CYP1A1 inhibition by flavonoids

Cytochrome P4501A1 (CYP1A1) is involved in the metabolism of several genotoxic/carcinogenic environmental xenobiotics including polycyclic aromatic hydrocarbons (PAHs) like benzo[a]pyrene. Several authors had proposed CYP1A inhibition as a plausible strategy for cancer chemoprevention. Using ethoxyresorufin O-deethylase activity (EROD), we tested the inhibitory properties of nine flavonoids: quercetin, miricetin, luteolin, fisetin, morin, kaempferol, 5-hydroxyflavone (5-HF), 3-hydroxyflavone (3-HF), and flavone (F) against human recombinant CYP1A1. The last three compounds exerted the highest inhibitory effect with IC50 values of 0.07, 0.10 and 0.08 μM respectively; the more hydroxyl-groups were present, the lower the potency of inhibition was. Biochemical characterization leads to the conclusion that flavone and its hydroxy derivatives are mixed-type inhibitors. In silico studies have shown that, Phe224 and other aromatic residues in the human CYP1A1 active site play an important role in flavonoid-CYP interaction, through a π/π stacking between the aminoacid and the flavonoid C-ring. Outside the active site, the three flavonoids bind preferentially between A and K helices of the enzyme. Results from the Ames test using human S9 fraction revealed that none of the three compounds was mutagenic. We can consider 5-HF, 3-HF, and F as potential chemopreventive agents against genotoxic damage caused by metabolites resulting from CYP1A1 activity.

Structural Insights into the Interaction of Cytochrome P450 3A4 with Suicide Substrates: Mibefradil, Azamulin and 6',7'-Dihydroxybergamottin.

Human cytochrome P450 3A4 (CYP3A4) is the most important drug-metabolizing enzyme. Some drugs and natural compounds can act as suicide (mechanism-based) inactivators of CYP3A4, leading to unanticipated drug-drug interactions, toxicity and therapeutic failures. Despite significant clinical and toxicological implications, the mechanism-based inactivation remains incompletely understood. This study provides the first direct insights into the interaction of CYP3A4 with three suicide substrates: mibefradil, an antihypertensive drug quickly withdrawn from the market; a semi-synthetic antibiotic azamulin; and a natural furanocoumarin, 6′,7′-dihydroxybergamottin. Novel structural findings help better understand the suicide substrate binding and inhibitory mechanism, and can be used to improve the predictability of the binding ability, metabolic sites and inhibitory/inactivation potential of newly developed drugs and other chemicals relevant to public health.

Conformational Response of N-Terminally Truncated Cytochrome P450 3A4 to Ligand Binding in Solution.

Human cytochrome P450 3A4 (CYP3A4) is a membrane-associated monooxygenase that is responsible for metabolizing >50% of the pharmaceuticals in the current market, so studying its chemical mechanism and structural changes upon ligand binding will help provide deeper insights into drug metabolism and further drug development. The best-characterized cytochrome P450 is a bacterial form, P450cam, which undergoes significant conformational changes upon binding substrate and its redox partner, putidaredoxin. In contrast, most crystal structures of CYP3A4 with or without ligands have shown few changes, although allosteric effects and multiple-substrate binding in solution are well-documented. In this study, we use double electron-electron resonance (DEER) to measure distances between spatially separated spin-labels on CYP3A4 and molecular dynamics to interpret the DEER data. These methods were applied to a soluble N-terminally truncated CYP3A4 form, and the results show that there are few changes in the average structure upon binding ketoconazole, ritonavir, or midazolam. However, binding of midazolam, but not ketoconazole or ritonavir, resulted in a significant change in the motion and/or disorder in the F/G helix region near the substrate binding pocket. These results suggest that soluble CYP3A4 behaves in a unique way in response to inhibitor and substrate binding.

T-2 toxin upregulates the expression of human cytochrome P450 1A1 (CYP1A1) by enhancing NRF1 and Sp1 interaction

T-2 toxin is a major pollutant in crops and feedstuffs. Due to its high toxicity in a variety of organisms, T-2 toxin is of great concern as a threat to humans and to animal breeding. Overexpression of CYP1A1 may contribute to carcinogenesis, and CYP1A1 may be a promising target for the prevention and treatment of human malignancies. Therefore, it is essential to understand the regulatory mechanism by which T-2 toxin induces CYP1A1 expression in human cells. In this study, we confirmed that T-2 toxin (100 ng/mL) induced the expression of CYP1A1 in HepG2 cells through NRF1 and Sp1 bound to the promoter instead of through the well-recognized Aromatic hydrocarbon receptors (AhR). In cells treated with T-2 toxin, Sp1, but not NRF1, was significantly upregulated. However, T-2 toxin apparently promoted the interaction between NRF1 and Sp1 proteins, as revealed by IP analysis. Furthermore, in T-2 toxin-treated HepG2 cells, nuclear translocation of NRF1 was enhanced, while knockdown of Sp1 ablated NRF1 nuclear enrichment. Our results revealed that the upregulation of CYP1A1 by T-2 toxin in HepG2 cells depended on enhanced interaction between Sp1 and NRF1. This finding suggests the tumorigenic features of T-2 toxin might be related to the CYP1A1, which provides new insights to understand the toxicological effect of T-2 toxin.

Functional characterization of 27 CYP3A4 variants on macitentan metabolism in vitro.

Macitentan is a new choice for pulmonary hypertension treatment which is converted to active metabolite ACT132577 by human cytochrome P450 3A4. Human cytochrome P450 3A4 often occurred gene mutations. Gene polymorphism might cause a variety of changes of protein expression and thus give rise to metabolic difference. The aim of this study was to investigate the catalytic characteristics of 27 CYP3A4 protein variants on the metabolism of macitentan in vitro. The incubation mixtures (final volume of 200μl in 1m PBS) consisted of 1pmol wild-type CYP3A4.1 or other CYP3A4 protein variants, 2.38pmol CYP b5 and macitentan (10-600μm) with 1mm NADPH. All specimens were processed using same approach with acetonitrile precipitation. The metabolite of macitentan was analysed by ultraperformance liquid chromatography-tandem mass spectrometry. Most CYP3A4 protein variants (CYP3A4.9, .11, .12, .13, .17, .20, .23, .24, .28, .29, .33, .34) exhibited a sharp decrease, meanwhile nearly one in five variants (CYP3A4.3, .4, .5, .10, .15, .16) showed a significant rise in intrinsic clearance. The relative clearance of CYP3A4 protein variants was ranged from 5.53 to 501.00%. Twenty-seven CYP3A4 protein variants displayed different catalytic characteristics towards macitentan in vitro, especially CYP3A4.5, .17, .20, .23. It is important to pay more attention to the dosage of macitentan in order to get better treatment for pulmonary arterial hypertension.

Interspecies Variation in NCMN-O-Demethylation in Liver Microsomes from Various Species.

NCMN (N-(3-carboxy propyl)-4-methoxy-1,8-naphthalimide), a newly developed ratiometric two-photon fluorescent probe for human Cytochrome P450 1A (CYP1A), shows the best combination of specificity and reactivity for real-time detection of the enzymatic activities of CYP1A in complex biological systems. This study aimed to investigate the interspecies variation in NCMN-O-demethylation in commercially available liver microsomes from human, mouse, rat, beagle dog, minipig and cynomolgus monkey. Metabolite profiling demonstrated that NCMN could be O-demethylated in liver microsomes from all species but the reaction rate varied considerably. CYP1A was the major isoform involved in NCMN-O-demethylation in all examined liver microsomes based on the chemical inhibition assays. Furafylline, a specific inhibitor of mammalian CYP1A, displayed differential inhibitory effects on NCMN-O-demethylation in all tested species. Kinetic analyses demonstrated that NCMN-O-demethylation in liver microsomes form rat, minipig and cynomolgus monkey followed biphasic kinetics, while in liver microsomes form human, mouse and beagle dog obeyed Michaelis-Menten kinetics, the kinetic parameters from various species are much varied, while NCMN-O-demethylation in MLM exhibited the highest similarity of specificity, kinetic behavior and intrinsic clearance as that in HLM. These findings will be very helpful for the rational use of NCMN as a practical tool to decipher the functions of mammalian CYP1A or to study CYP1A associated drug-drug interactions in vivo.

AhR regulates the expression of human cytochrome P450 1A1 (CYP1A1) by recruiting Sp1.

Cytochrome P450 1A1 (CYP1A1) is abundant in the kidney, liver, and intestine and is involved in the phase I metabolism of numerous endogenous and exogenous compounds. Therefore, exploring the regulatory mechanism of its basal expression in humans is particularly important to understand the bioactivation of several procarcinogens to their carcinogenic derivatives. Site-specific mutagenesis and deletion of the transcription factor binding site determined the core cis-acting elements in the human CYP1A1 proximal and distal promoter regions. The proximal promoter region [overlapping xenobiotic-responsive element (XRE) and GC box sequences] determined the basal expression of CYP1A1. In human hepatocellular carcinoma cells (HepG2) with aryl hydrocarbon receptor (AhR) or specificity protein 1 (Sp1) knockdown, we confirmed that AhR and Sp1 are involved in basal CYP1A1 expression. In HepG2 cells overexpressing either AhR or Sp1, AhR determined the proximal transactivation of basal CYP1A1 expression. Via DNA affinity precipitation assays and ChIP, we found that AhR bound to the promoter and recruited Sp1 to transactivate CYP1A1 expression. The coordinated interaction between Sp1 and AhR was identified to be DNA mediated. Our work revealed a basal regulatory mechanism of an interesting human gene by which AhR interacts with Sp1 through DNA and recruits Sp1 to regulate basal CYP1A1 expression.

Effects of catalpol on the activity of human liver cytochrome P450 enzymes

1. Catalpol possesses numerous pharmacological activities, and however, little data available for the effects of catalpol on the activity of human liver cytochrome P450 (CYP) enzymes.2. This study investigates the inhibitory effects of catalpol on the main human liver CYP isoforms. In this study, the inhibitory effects of catalpol on the eight human liver CYP isoforms 1A2, 2A6, 2E1, 2D6, 2C9, 2C19, 2C8 and 3A4 were investigated in human liver microsomes.3. The results indicated that catalpol could inhibit the activity of CYP3A4, CYP2E1 and CYP2C9, with IC50 values of 14.27, 22.4 and 14.69 μM, respectively, but those other CYP isoforms were not affected. Enzyme kinetic studies showed that catalpol was not only a noncompetitive inhibitor of CYP3A4, but also a competitive inhibitor of CYP2E1 and CYP2C9, with Ki values of 7.40, 10.75 and 7.37 μM, respectively. In addition, catalpol is a time-dependent inhibitor for CYP3A4, with maximum inactivation (kinact) and 50% maximum inactivation (KI) values of 0.02 min−1 and 1.86 μM, respectively.4. The in vitro studies of catalpol with CYP isoforms suggest that catalpol has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP3A4, CYP2E1 and CYP2C9. Further in vivo studies are needed in order to evaluate the significance of this interaction.

Inhibition of human cytochrome P450 2A6 by 7-hydroxycoumarin analogues: Analysis of the structure-activity relationship and isoform selectivity

Compared with coumarin, 7-hydroxycoumarin could serve as a better hit for developing CYP2A6 inhibitors. In this study, a series of 7-hydroxycoumarin and its structural analogues were collected to study their structure-activity relationship (SAR) and isoform selectivity for inhibiting CYP2A6. All tested coumarins except a C4 phenyl derivative (11) showed higher inhibitory activities for CYP2A6 over the other CYP isoforms, including CYP1A2, CYP2D6, CYP2E1, CYP3A4, CYP2C8, and CYP2C9. Of these coumarins, 6,7-dihydroxycoumarin (1) and 7,8-dihydroxycoumarin (9) were found to be potent inhibitors of CYP2A6 with IC50/Ki value of 0.39/0.25 and 4.61/3.02 μM, respectively, compared to methoxalen as positive control (IC50/Ki = 0.43/0.26 μM). In contrast, other coumarins showed low or decreased CYP2A6-inhibiting activities. SAR analysis showed that hydroxy groups might be important for CYP2A6 inhibition, and the rank order of sites for hydroxy substitution was C6 > C7 > C8. In addition, either hydrophobic or hydrophilic substituents introduced into C4, C6 and C8 led to a reduction in CYP2A6-inhibiting activity, and the degree of influence was dependent on the size and electrical charge of substituents. Furthermore, inhibition kinetic analysis and docking simulations demonstrated that the 8-O-glucosylated coumarin derivative (17) exhibited noncompetitive inhibition against CYP2A6, while competitive inhibition patterns were noted for the other tested coumarins. The mechanisms underlying the inhibitors binding to CYP2A6 were further investigated by molecular docking study. The findings presented herein are very helpful for developing highly selective and more potent CYP2A6 inhibitors.