Human Cytochrome P450 Research Articles

Melatonin Activation by Human Cytochrome P450 Enzymes: A Comparison between Different Isozymes.

Cytochrome P450 enzymes in the human body play a pivotal role in both the biosynthesis and the degradation of the hormone melatonin. Melatonin plays a key role in circadian rhythms in the body, but its concentration is also linked to mood fluctuations as well as emotional well-being. In the present study, we present a computational analysis of the binding and activation of melatonin by various P450 isozymes that are known to yield different products and product distributions. In particular, the P450 isozymes 1A1, 1A2, and 1B1 generally react with melatonin to provide dominant aromatic hydroxylation at the C6-position, whereas the P450 2C19 isozyme mostly provides O-demethylation products. To gain insight into the origin of these product distributions of the P450 isozymes, we performed a comprehensive computational study of P450 2C19 isozymes and compared our work with previous studies on alternative isozymes. The work covers molecular mechanics, molecular dynamics and quantum mechanics approaches. Our work highlights major differences in the size and shape of the substrate binding pocket amongst the different P450 isozymes. Consequently, substrate binding and positioning in the active site varies substantially within the P450 isozymes. Thus, in P450 2C19, the substrate is oriented with its methoxy group pointing towards the heme, and therefore reacts favorably through hydrogen atom abstraction, leading to the production of O-demethylation products. On the other hand, the substrate-binding pockets in P450 1A1, 1A2, and 1B1 are tighter, direct the methoxy group away from the heme, and consequently activate an alternative site and lead to aromatic hydroxylation instead.

Ni(II) complexes with in-situ generated N, O-bidentate aroylhydrazone Schiff base ligands: One-pot synthesis, characterization, crystal structure, and in-silico studies

A series of novel aroylhydrazone-based Ni(II) complexes were synthesized by using analogous Schiff base ligands prepared in-situ by reacting 4-[(4-methylbenzyl)oxy]benzoylhydrazine with different aldehydes in the presence of Ni(II) acetate tetrahydrate. The correspondent [NiL2] complexes, 4-7, were characterized by physicochemical techniques, viz., molar conductance, magnetic susceptibility measurement, IR, NMR, and mass spectroscopic techniques. The single-crystal X-ray structural analyses of complexes 4, 6 and 7 showed a distorted square-planar geometry for Ni atoms with deprotonated ligands in trans-configuration bound via azomethine-N and carbonylate-O atoms. Next, quantum chemical methods were incorporated to investigate their thermochemical, molecular orbital, and electrostatic potential properties. Computer-aided drug design methods were also implemented to assess their medicinal, pharmacological, and toxicological properties. Molecular docking and interaction analyses disclosed their potential binding affinity against human lung Cytochrome P450 2A13 receptor protein. Pharmacokinetic and toxicological studies suggest their relatively safe drug administrative properties.

In Silico ADME Prediction and Molecular Docking of 1,2,3-Triazole-based Compounds Against Human Aromatase Cytochrome P450

In Silico ADME Prediction and Molecular Docking of 1,2,3-Triazole-based Compounds Against Human Aromatase Cytochrome P450

Differences of Atomic-Level Interactions between Midazolam and Two CYP Isoforms 3A4 and 3A5.

CYP 3A4 and CYP 3A5 are two important members of the human cytochrome P450 family. Although their overall structures are similar, the local structures of the active site are different, which directly leads to obvious individual differences in drug metabolic efficacy and toxicity. In this work, midazolam (MDZ) was selected as the probe substrate, and its interaction with two proteins, CYP 3A4 and CYP 3A5, was studied by molecular dynamics simulation (MD) along with the calculation of the binding free energy. The results show that two protein-substrate complexes have some similarities in enzyme-substrate binding; that is, in both complexes, Ser119 forms a high occupancy hydrogen bond with MDZ, which plays a key role in the stability of the interaction between MDZ and the enzymes. However, the complex formed by CYP 3A4 and MDZ is more stable, which may be attributed to the sandwich structure formed by the fluorophenyl group of the substrate with Leu216 and Leu482. Our study interprets the binding differences between two isoform-substrate complexes and reveals a structure-function relationship from the atomic perspective, which is expected to provide a theoretical basis for accurately measuring the effectiveness and toxicity of drugs for individuals in the era of precision medicine.

Lower overall survival in male patients with advanced disease undergoing allogeneic hematopoietic stem cell transplantation is associated with CYP1B1 Leu432Val polymorphism.

Human cytochrome P450 1B1 (CYP1B1) is an extrahepatic key enzyme involved in estrogen metabolism, steroid synthesis, and pro-carcinogen activation. In a single-center retrospective study, 382 patients who underwent allogeneic hematopoetic stem cell transplantation and their donors were genotyped for CYP1B1 C432G polymorphism by reverse transcription polymerase chain reaction. One hundred and sixty-nine patients (44%) were homozygous wild-type (wt) gene CC, 157 (41%) heterozygous CG and 56 (15%) homozygous gene mutated GG. Of interest, mutated CYP1B1 was more common in male (62%) than in female patients (48%) P=0.006, unlike in donors. Five-year estimate for overall survival (OS) was 58±4% (CC) versus 48±3% (CG and GG), P=0.048. Surprisingly, this difference was only evident in males (P=0.024): OS 58±6% versus 42±4%, whereas it was virtually absent in females. Importantly, this difference was only evident in male patients with advanced disease (AD) (n=118, P=0.002): OS 44±8% (CC) versus 32±6% (CG) versus 6±6% (GG), whereas it was virtually absent in male patients with early disease. One-year non-relapse mortality in male patients with AD was 8±4% (CC) versus 21±5% (CG) versus 50±12% (GG), P=0.002. Three-year relapse rate in male patients with AD was 31±7% (wt) versus 42±6% (mut), P=0.04. Multivariate analysis for OS in male patients with AD revealed CYP1B1 polymorphism as the only prognostic factor: RR 1.78, P=0.001. In conclusion, these results suggest that male patients with AD and mutant CYP1B1 polymorphism have lower OS after allogeneic hematopoetic stem cell transplantation due to a higher non-relapse mortality and a higher relapse rate.

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.

Comparative metabolism of fargesin in human, dog, monkey, mouse, and rat hepatocytes.

Fargesin, a bioactive lignan derived from Flos Magnoliae, possesses anti-inflammatory, anti-oxidative, anti-melanogenic, and anti-apoptotic effects. This study compared the metabolic profiles of fargesin in human, dog, monkey, mouse, and rat hepatocytes using liquid chromatography-high resolution mass spectrometry. In addition, we investigated the human cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT), and sulfotransferase (SULT) enzymes responsible for fargesin metabolism. The hepatic extraction ratio of fargesin among the five species ranged from 0.59 to 0.78, suggesting that it undergoes a moderate-to-extensive degree of hepatic metabolism. During metabolism, fargesin generates three phase 1 metabolites, including fargesin catechol (M1) and O-desmethylfargesin (M2 and M3), and 11 phase 2 metabolites, including O-methyl-M1 (M4 and M5) via catechol O-methyltransferase (COMT), glucuronides of M1, M2, M4, and M5, and sulfates of M1-M5. The production of M1 from fargesin via O-demethylenation is catalyzed by CYP2C9, CYP3A4, CYP2C19, and CYP2C8 enzymes, whereas the formation of M2 and M3 (O-desmethylfargesin) is catalyzed by CYP2C9, CYP2B6, CYP2C19, CYP3A4, CYP1A2, and CYP2D6 enzymes. M4 is metabolized to M4 glucuronide by UGT1A3, UGT1A8, UGT1A10, UGT2B15, and UGT2B17 enzymes, whereas M4 sulfate is generated by multiple SULT enzymes. Fargesin is extensively metabolized in human hepatocytes by CYP, COMT, UGT, and SULT enzymes. These findings help to elucidate the pharmacokinetics and drug interactions of fargesin.

Identification of the effects of pathogenic genetic variations of human CYP2C9 and CYP2D6: an in silico approach

Genetic variations in the human cytochrome P450 family 2 subfamily C member 9 (CYP2C9) and cytochrome P450 family 2 subfamily D member 6 (CYP2D6) genes may affect drug metabolism and lead to alterations in phenotypes. Genetic variations are associated with toxicity, adverse drug reactions, inefficient treatment. Various in silico tools were combined to investigate the deleterious effects of missense non-synonymous single nucleotide polymorphisms (nsSNPs) of the human CYP2C9 and CYP2D6. The structural and functional effects of the high-risk non-synonymous SNPs in the human CYP2C9 and CYP2D6 were predicted by numerous computational mutation analysis methods. Out of 24 pathogenic missense SNPs in the CYP2C9, 22 nsSNPs had a decreasing effect on protein stability and 13 SNPs were showed to be located at conserved positions. Out of 27 high-risk deleterious non-synonymous SNPs in the human CYP2D6, 21 SNPs decreased protein stability and 16 nsSNPs were predicted to be positioned at conserved regions. Our present study suggests that the identified functional SNPs may affect drug metabolism associated with CYP2C9 and CYP2D6 enzymes.

A Combination of Novel HIV-1 Protease Inhibitor and Cytochrome P450 (CYP) Enzyme Inhibitor to Explore the Future Prospective of Antiviral Agents: Evotaz.

Viruses belong to the class of micro-organisms that are well known for causing infections in the human body. Antiviral medications are given out to prevent the spread of disease-causing viruses. When the viruses are actively reproducing, these agents have their greatest impact. It is particularly challenging to develop virus-specific medications since viruses share the majority of the metabolic functions of the host cell. In the continuous search for better antiviral agents, the United States Food and Drug Administration (USFDA) approved a new drug named Evotaz on January 29, 2015 for the treatment of human immunodeficiency virus (HIV). Evotaz is a combined once-daily fixed drug, containing Atazanavir, an HIV protease inhibitor, and cobicistat, an inhibitor of the human liver cytochrome P450 (CYP) enzyme. The medication is created such that it can kill viruses by concurrently inhibiting protease and CYP enzymes. The medicine is still being studied for a number of criteria, but its usefulness in children under the age of 12 is currently unknown. The preclinical and clinical characteristics of Evotaz, as well as its safety and efficacy profiles and a comparison of the novel drug with antiviral medications presently available in the market, are the main topics of this review paper.

Enhancing the invasive traits of breast cancers by CYP1B1 via regulation of p53 to promote uPAR expression

Human cytochrome P450 1B1 (CYP1B1) catalyzes estrogen metabolism to produce metabolites that promote the progression of breast cancer. Since the invasive properties of cancer cells cause cancer relapse, which dramatically reduces patient survival, we investigated the new pro-invasive mechanism involving CYP1B1 in breast cancer. Exploring clinical data from invasive breast cancer patients revealed that CYP1B1 exhibits a potential correlation with urokinase-type plasminogen activator receptor (uPAR). Interestingly, uPAR mRNA expression was elevated in invasive breast cancer patients carrying TP53 genes with driver mutations, and our results showed that CYP1B1 activates the uPAR pathway following regulation of p53 according to its mutant status. CYP1B1 suppressed wild-type (WT) p53 whereas it induced the oncogenic gain-of-function mutant p53R280K, not only via transcriptional regulation but also the protein stabilization and activation following phosphorylation on Ser15 residue of p53R280K. Intriguingly, results from CYP1B1 polymorphic gene study and 4-hydroxyestradiol (4-OHE2) treatment showed that CYP1B1 regulates p53s and uPAR through its enzymatic activity. Furthermore, effects of DMBA and TMS on uPAR expression disappeared in HCT116p53−/− cells, indicating that p53 is critical for uPAR induction by CYP1B1. Collectively, our results demonstrate that CYP1B1 may reduce the relapse-free survival rate of breast cancer patients by inducing invasive traits in cancer cells via p53 regulation based on the mutation status of TP53 genes and further activation of the uPAR pathway. The elucidation of the previously unknown molecular mechanism of CYP1B1 may provide evidence for the development of effective anti-cancer therapeutic strategies that target the progression of cancer invasion.

Stable Chinese Hamster Ovary Suspension Cell Lines Harboring Recombinant Human Cytochrome P450 Oxidoreductase and Human Cytochrome P450 Monooxygenases as Platform for In Vitro Biotransformation Studies.

In the liver, phase-1 biotransformation of drugs and other xenobiotics is largely facilitated by enzyme complexes consisting of cytochrome P450 oxidoreductase (CPR) and cytochrome P450 monooxygenases (CYPs). Generated from human liver-derived cell lines, recombinant in vitro cell systems with overexpression of defined phase-1 enzymes are widely used for pharmacological and toxicological drug assessment and laboratory-scale production of drug-specific reference metabolites. Most, if not all, of these cell lines, however, display some background activity of several CYPs, making it difficult to attribute effects to defined CYPs. The aim of this study was to generate cell lines with stable overexpression of human phase-1 enzymes based on Chinese hamster ovary (CHO) suspension cells. Cells were sequentially modified with cDNAs for human CPR in combination with CYP1A2, CYP2B6, or CYP3A4, using lentiviral gene transfer. In parallel, CYP-overexpressing cell lines without recombinant CPR were generated. Successful recombinant expression was demonstrated by mRNA and protein analyses. Using prototypical CYP-substrates, generated cell lines proved to display specific enzyme activities of each overexpressed CYP while we did not find any endogenous activity of those CYPs in parental CHO cells. Interestingly, cell lines revealed some evidence that the dependence of CYP activity on CPR could vary between CYPs. This needs to be confirmed in further studies. Recombinant expression of CPR was also shown to enhance CYP3A4-independent metabolisation of testosterone to androstenedione in CHO cells. We propose the novel serum-free CHO suspension cell lines with enhanced CPR and/or defined CYP activity as a promising "humanised" in vitro model to study the specific effects of those human CYPs. This could be relevant for toxicology and/or pharmacology studies in the pharmaceutical industry or medicine.

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.

Hepatic biotransformation of non-psychotropic phytocannabinoids and activity screening on cytochromes P450 and UDP-glucuronosyltransferases

This study examined the biotransformation of phytocannabinoids in human hepatocytes. The susceptibility of the tested compounds to transformations in hepatocytes exhibited the following hierarchy: cannabinol (CBN) > cannabigerol (CBG) > cannabichromene (CBC) > cannabidiol (CBD). Biotransformation included hydroxylation, oxidation to a carboxylic acid, dehydrogenation, hydrogenation, dehydration, loss/shortening of alkyl, glucuronidation and sulfation. CBN was primarily metabolized by oxidation of a methyl to a carboxylic acid group, while CBD, CBG and CBC were preferentially metabolized by direct glucuronidation. The study also screened for the activity of recombinant human cytochromes P450 (CYPs) and UDP-glucuronosyltransferases (UGTs), which could catalyze the hydroxylation and glucuronidation of the tested compounds, respectively. We found that CBD was hydroxylated mainly by CYPs 2C8, 2C19, 2D6; CBN by 1A2, 2C9, 2C19 and 2D6; and CBG by 2B6, 2C9, 2C19 and 2D6. CBC exhibited higher susceptibility to CYP-mediated transformation than the other tested compounds, mainly with CYPs 1A2, 2B6, 2C8, 2C19, 2D6 and 3A4 being involved. Further, CBD was primarily glucuronidated by UGTs 1A3, 1A7, 1A8, 1A9 and 2B7; CBN by 1A7, 1A8, 1A9 and 2B7; CBG by 1A3, 1A7, 1A8, 1A9, 2B4, 2B7 and 2B17; and the glucuronidation of CBC was catalyzed by UGTs 1A1, 1A8, 1A9 and 2B7.

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.

In vitro inhibition of human cytochrome P450 enzymes by licoisoflavone B from Glycyrrhiza uralensis Fisch. ex DC.

Glycyrrhiza uralensis Fisch. ex DC, one of the 3 pharmacopeial species of licorice and widely used in dietary supplements, can inhibit certain cytochrome P450 (CYP) enzymes. Thereby, G. uralensis preparations have the potential to cause pharmacokinetic drug interactions when consumed along with prescription medicines. One compound (1.34 mg dry weight) responsible for inhibiting CYP2B6, CYP2C8, and CYP2C9 was isolated using bioactivity-guided fractionation from 250 g dried roots, stolons, and rhizomes. The enzyme kinetics and mechanisms of inhibition were determined using human liver microsomes, recombinant enzymes, and UHPLC-MS/MS-based assays. Identified as licoisoflavone B, this compound displayed reversible inhibition of CYP2C8 with an IC50 value of 7.4 ± 1.1 µM and reversible inhibition of CYP2C9 with an IC50 value of 4.9 ± 0.4 µM. The enzyme kinetics indicated that the mechanism of inhibition was competitive for recombinant CYP2C8, with a Ki value of 7.0 ± 0.7 μM, and mixed-type inhibition for recombinant CYP2C9, with a Ki value of 1.2 ± 0.2 μM. Licoisoflavone B moderately inhibited CYP2B6 through a combination of irreversible and reversible mechanisms with an IC50 value of 16.0 ± 3.9 µM.

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.

Allosteric modulation of cytochrome P450 enzymes by the NADPH cytochrome P450 reductase FMN-containing domain

NADPH-cytochrome P450 reductase delivers electrons required by heme oxygenase, squalene monooxygenase, fatty acid desaturase, and 48 human cytochrome P450 enzymes. While conformational changes supporting reductase intramolecular electron transfer are well defined, intermolecular interactions with these targets are poorly understood, in part because of their transient association. Herein the reductase FMN domain responsible for interacting with targets was fused to the N-terminus of three drug-metabolizing and two steroidogenic cytochrome P450 enzymes to increase the probability of interaction. These artificial fusion enzymes were profiled for their ability to bind their respective substrates and inhibitors and to perform catalysis supported by cumene hydroperoxide. Comparisons with the isolated P450 enzymes revealed that even the oxidized FMN domain causes substantial and diverse effects on P450 function. The FMN domain could increase, decrease, or not affect total ligand binding and/or dissociation constants depending on both P450 enzyme and ligand. As examples, FMN domain fusion has no effect on inhibitor ketoconazole binding to CYP17A1 but substantially altered CYP21A2 binding of the same compound. FMN domain fusion to CYP21A2 resulted in differential effects dependent on whether the ligand was 17α-hydroxyprogesterone versus ketoconazole. Similar enzyme-specific effects were observed on steady-state kinetics. These observations are most consistent with FMN domain interacting with the proximal P450 surface to allosterically impact P450 ligand binding and metabolism separate from electron delivery. The variety of effects on different P450 enzymes and on the same P450 with different ligands suggests intricate and differential allosteric communication between the P450 active site and its proximal reductase-binding surface.

Effects of acidic non-steroidal anti-inflammatory drugs on human cytochrome P450 4A11 activity: Roles of carboxylic acid and a sulfur atom in potent inhibition by sulindac sulfide

Cytochrome P450 4A11 (CYP4A11) has many endogenous and exogenous compounds containing a carboxyl group in their structure as substrates. If drugs with this characteristic potently attenuate the catalytic function of CYP4A11, drug-drug interactions may occur. Acidic non-steroidal anti-inflammatory drugs (NSAIDs) possess a carboxylic acid in their structure. However, it remains unclear whether these drugs inhibit CYP4A11 activity. The present study examined the inhibitory effects of acidic NSAIDs on CYP4A11 activity using human liver microsomes (HLMs) and recombinant CYP4A11. Sulindac sulfide, ibuprofen, and flurbiprofen effectively decreased the luciferin-4A O-demethylase activity of HLMs and recombinant CYP4A11 (inhibition rates of 30–96% at an inhibitor concentration of 100 μM), while salicylic acid, aspirin, diclofenac, mefenamic acid, indomethacin, etodolac, ketoprofen, loxoprofen, S-naproxen, pranoprofen, zaltoprofen, and oxaprozin exhibited weaker inhibitory activity (inhibition rates up to 23%). Among the drugs tested, sulindac sulfide was the most potent inhibitor of CYP4A11 activity. A kinetic analysis of the inhibition of CYP4A11 by sulindac sulfide revealed mixed-type inhibition for HLMs (Ki = 3.38 μM) and recombinant CYP4A11 (Ki = 4.19 μM). Sulindac sulfide is a pharmacologically active metabolite of sulindac (sulfoxide form), which is also oxidized to sulindac sulfone. To elucidate the role of a sulfur atom of sulindac sulfide in the inhibition of CYP4A11, the inhibitory effects of sulindac sulfide and its oxidized forms on CYP4A11 activity were examined. The potency of inhibition against HLMs was greater in the order of sulindac sulfide, sulindac, and sulindac sulfone; IC50 values were 6.16, 52.7, and 71.6 μM, respectively. The present results indicate that sulindac sulfide is a potent inhibitor of CYP4A11. These results and the molecular modeling of CYP4A11 with sulindac sulfide and its oxidized forms suggest that a sulfur atom of sulindac sulfide as well as its carboxylic acid play important roles in the inhibition of CYP4A11.

16R-HETE and 16S-HETE alter human cytochrome P450 1B1 enzyme activity probably through an allosteric mechanism.

Cytochrome P450 1B1 (CYP1B1) has been widely associated with the development of cardiac pathologies due to its ability to produce cardiotoxic metabolites like midchain hydroxyeicosatetraenoic acids (HETEs) from arachidonic acid (AA) through an allylic oxidation reaction. 16-HETE is a subterminal HETE that is also produced by CYP-mediated AA metabolism. 19-HETE is another subterminal HETE that was found to inhibit CYP1B1 activity, lower midchain HETEs, and have cardioprotective effects. However, the effect of 16-HETE enantiomers on CYP1B1 has not yet been investigated. We hypothesized that 16(R/S)-HETE could alter the activity of CYP1B1 and other CYP enzymes. Therefore, this study was carried out toinvestigate the modulatory effect of 16-HETE enantiomers on CYP1B1 enzyme activity, and to examine the mechanisms by which they exert these modulatory effects. To investigate whether these effects are specific to CYP1B1, we also investigated 16-HETE modulatory effects on CYP1A2. Our results showed that 16-HETE enantiomers significantly increased CYP1B1 activity in RL-14 cells, recombinant human CYP1B1, and human liver microsomes, as seen by the significant increase in 7-ethoxyresorufin deethylation rate. On the contrary, 16-HETE enantiomers significantly inhibited CYP1A2 catalytic activity mediated by the recombinant human CYP1A2 and human liver microsomes. 16R-HETE showed stronger effects than 16S-HETE. The sigmoidal binding mode of the enzyme kinetics data demonstrated that CYP1B1 activation and CYP1A2 inhibition occurred through allosteric regulation. In conclusion, our study provides the first evidence that 16R-HETE and 16S-HETE increase CYP1B1 catalytic activity through an allosteric mechanism.