Electrochemically driven drug metabolism via cytochrome P450 2C9 isozyme microsomes with cytochrome P450 reductase and indium tin oxide nanoparticle composites

We describe herein an electrochemically driven drug metabolism strategy based on nanocomposites that integrate cyt P450 2C9 (CYP2C9) isozyme microsomes with cyt P450 reductase (CPR), indium tin oxide (ITO) nanoparticles and chitosan (CS). This novel bioelectronic system enables monitoring of the drug metabolism and enzyme inhibition.     

Electrochemical Activation of the Natural Catalytic Cycle of Cytochrome P450s in Human Liver Microsomes

The natural catalytic cycle of cytochrome (cyt) P450 enzymes in human liver microsome (HLM) films was activated electrochemically via the electron transfer sequence electrode→cyt P450 reductase (CPR)→cyt P450. Cyclic voltammograms for HLM films had midpoint potentials of ?0.50 V vs. SCE at pH 7.4 characteristic of CPR, not cyt P450s. HLM and CPR microsomes without cyt P450s did not electrocatalytically reduce H₂O₂, and did not shift midpoint potential when CO was added, also indicating that the peaks do not correspond to iron heme cyt P450 enzymes. Electrochemical activation of the natural cyt P450 cycle for substrate conversion via CPR in HLM films was confirmed by catalytic electrolysis in an electrochemical microfluidic array designed to generate and detect reactive metabolites by measuring their reactivity with DNA.     

Efficient bioelectronic actuation of the natural catalytic pathway of human metabolic cytochrome P450s

Cytochrome (cyt) P450s comprise the enzyme superfamily responsible for human oxidative metabolism of a majority of drugs and xenobiotics. Electronic delivery of electrons to cyt P450s could be used to drive the natural catalytic cycle for fundamental investigations, stereo- and regioselective synthesis, and biosensors. We describe herein 30 nm nanometer-thick films on electrodes featuring excess human cyt P450s and cyt P450 reductase (CPR) microsomes that efficiently mimic the natural catalytic pathway for the first time. Redox potentials, electron-transfer rates, CO-binding, and substrate conversion rates confirmed that electrons are delivered from the electrode to CPR, which transfers them to cyt P450. The film system enabled electrochemical probing of the interaction between cyt P450 and CPR for the first time. Agreement of film voltammetry data with theoretical simulations supports a pathway featuring a key equilibrium redox reaction in the natural catalytic pathway between reduced CPR and cyt P450 occurring within a CPR-cyt P450 complex uniquely poised for substrate conversion.     

A Minimal Functional Complex of Cytochrome P450 and FBD of Cytochrome P450 Reductase in Nanodiscs

Structural interactions that enable electron transfer to cytochrome‐P450 (CYP450) from its redox partner CYP450‐reductase (CPR) are a vital prerequisite for its catalytic mechanism. The first structural model for the membrane‐bound functional complex to reveal interactions between the full‐length CYP450 and a minimal domain of CPR is now reported. The results suggest that anchorage of the proteins in a lipid bilayer is a minimal requirement for CYP450 catalytic function. Akin to cytochrome‐b₅ (cyt‐b₅), Arg 125 on the C‐helix of CYP450s is found to be important for effective electron transfer, thus supporting the competitive behavior of redox partners for CYP450s. A general approach is presented to study protein–protein interactions combining the use of nanodiscs with NMR spectroscopy and SAXS. Linking structural details to the mechanism will help unravel the xenobiotic metabolism of diverse microsomal CYP450s in their native environment and facilitate the design of new drug entities.     

Bioelectrodes for evaluating molecular therapeutic and toxicity properties

Recent progress on material designs merged with nanotechnology and biotechnology strategies has advanced studies of complex biological samples on electrodes for cytochrome P450 (CYP)–driven biocatalytic reactions (e.g. liver membrane fractions, cells, and various organ-specific CYP extracts). In addition, protein engineering of CYP enzymes with their reductase partner in membranes (e.g. baculovirus- or Escherichia coli bacteria–expressed CYP microsomes) and other recombinant strategies (e.g. engineered CYP and reductase fusion domains and site-directed CYP mutagenesis) are promising sustainable approaches for offering abundant sources of CYP enzymes for electrocatalytic applications. The combination of in silico and experimental electroanalytical methods with hyphenated approaches and biological assays can offer early and rigorous profiling of new drugs and specialty chemicals for safe exposure and beneficial use.     

Cytochrome P450 reaction phenotyping and inhibition and induction studies of pinostrobin in human liver microsomes and hepatocytes

Pinostrobin (PI, 5‐hydroxy‐7‐methoxyflavanone) is a natural flavonoid known for its rich pharmacological activities. The objective of this study was to identify the human liver cytochrome P450 (CYP450) isoenzymes involved in the metabolism of PI. A single hydoxylated metabolite was obtained from PI after an incubation with pooled human liver microsomes (HLMs). The relative contributions of different CYP450s were evaluated using CYP450‐selective inhibitors in HLMs and recombinant human CYP450 enzymes, and the results revealed the major involvement of CYP1A2, CYP2C9 and CYP2E1 in PI metabolism. We also evaluated the ability of PI to inhibit and induce human cytochrome P450 enzymes in vitro . High‐performance liquid chromatography and liquid chromatography–tandem mass spectrometry analytical techniques were used to estimate the enzymatic activities of seven drug‐metabolizing CYP450 isozymes in vitro . In HLMs, PI did not inhibit CYP 1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 or CYP3A4 (IC₅₀ > 100 μm ). In the induction studies, PI had minimal effects on CYP1A2, CYP2B6and CYP3A4 activity. Based on these results, PI would not be expected to cause clinically significant CYP450 inhibition or induction.     

Enantioselective capillary electrophoresis for identification and characterization of human cytochrome P450 enzymes which metabolize ketamine and norketamine in vitro

Ketamine, a phencyclidine derivative, is used for induction of anesthesia, as an anesthetic drug for short term surgical interventions and in subanesthetic doses for postoperative pain relief. Ketamine undergoes extensive hepatic first-pass metabolism. Enantioselective capillary electrophoresis with multiple isomer sulfated β-cyclodextrin as chiral selector was used to identify cytochrome P450 enzymes involved in hepatic ketamine and norketamine biotransformation in vitro. The N-demethylation of ketamine to norketamine and subsequently the biotransformation of norketamine to other metabolites were studied via analysis of alkaline extracts of in vitro incubations of racemic ketamine and racemic norketamine with nine recombinantly expressed human cytochrome P450 enzymes and human liver microsomes. Norketamine was formed by CYP3A4, CYP2C19, CYP2B6, CYP2A6, CYP2D6 and CYP2C9, whereas CYP2B6 and CYP2A6 were identified to be the only enzymes which enable the hydroxylation of norketamine. The latter two enzymes produced metabolic patterns similar to those found in incubations with human liver microsomes. The kinetic data of ketamine N-demethylation with CYP3A4 and CYP2B6 were best described with the Michaelis–Menten model and the Hill equation, respectively. This is the first study elucidating the individual enzymes responsible for hydroxylation of norketamine. The obtained data suggest that in vitro biotransformation of ketamine and norketamine is stereoselective.     

In vitro metabolism of a new cardioprotective agent, KR-32570, in human liver microsomes

KR-32570 (5-(2-methoxy-5-chlorophenyl)furan-2-ylcarbonyl)guanidine) is a new reversible Na+/H+ exchanger inhibitor for preventing ischemia-reperfusion injury. This study was performed to identify the metabolic pathway of KR-32570 in human liver microsomes. Human liver microsomal incubation of KR-32570 in the presence of NADPH and UDPGA resulted in the formation of six metabolites, M1-M6. M1 was identified as O-desmethyl-KR-32570, on the basis of liquid chromatography/tandem mass spectrometric (LC/MS/MS) analysis with the synthesized authentic standard. M2 and M3 were suggested to be hydroxy-KR-32570 and hydroxy-O-desmethyl-KR-32570, respectively. M1, M2, and M3 were further metabolized to their glucuronide conjugates, M4, M5, and M6, respectively. In addition, the specific P450 isoforms responsible for KR-32570 oxidation to two major metabolites, O-desmethyl-KR-32570 and hydroxy-KR-32570, were identified using a combination of correlation analysis, chemical inhibition in human liver microsomes and metabolism by expressed recombinant P450 isoforms. The inhibitory potency of KR-32570 on clinically major P450s was investigated in human liver microsomes. The results show that CYP3A4 contributes to the oxidation of KR-32570 to hydroxy-KR-32570, and CYP1A2 play the predominant role in O-demethylation of KR-32570. KR-32570 was found to inhibit moderately the metabolism of CYP2C8 substrates.     

Convenient Gram-Scale Metabolite Synthesis by Engineered Fission Yeast Strains Expressing Functional Human P450 Systems

The growing need for the characterization of cytochrome P450 (P450) metabolites often necessitates their synthesis up to Gram-scale. This task may in principle be achieved by using various techniques including chemical synthesis, the use of laboratory animals, in vitro P450 systems or microbial biotransformation. However, these approaches are in many instances unfavorable due to low yields, laborious purification, costs of cofactors, or the formation of non-physiologic metabolites. The fission yeast Schizosaccharomyces pombe has previously been shown by others and us to be very well suited for the heterologous expression of human P450s. In this study, we demonstrate whole-cell biotransformation reactions carried out with fission yeast strains that coexpress human cytochrome P450 reductase (CPR) and one of the following P450 isoforms: CYP2B6, CYP2C9, CYP2C19, CYP2D6, or CYP3A4, respectively. These strains could successfully convert their respective standard substrates but showed different responses with respect to incubation pH, the presence of glucose, and temperature, respectively. In addition, the preparative of synthesis of 2.8?g of 4'-hydroxydiclofenac was achieved by whole-cell biotransformation of diclofenac using a CPR-CYP2C9 coexpressing fission yeast strain.     

A novel microplate-based HPLC-fluorescence assay for determination of NADPH-cytochrome P450 reductase activity

A 96‐well microplate‐based HPLC endpoint assay is described for the determination of NADPH‐cytochrome P450 reductase (CPR) activity. Novel sampling of NADPH into microplates was optimized. Separation was performed on a Zorbax Eclipse XDB‐C₁₈ analytical 4.6 × 150 mm, 5 µm column. To validate the method, recombinant human NADPH‐P450 reductase and microsomes with cytochrome P450 CYP1A1 were used. The mobile phase consisted of 80% acetonitrile and 20% water at a flow‐rate of 0.8 mL/min. The CPR activity was quantified using NADPH fluorescence at λ_Ex = 340 nm and λ_Em = 450 nm. Enzymatic activity was directly proportional to the decrease in NADPH fluorescence. This analytical process enables a highly sensitive endpoint determination for reductase activity in vitro and monitoring of the consumption of NADPH in enzymatic reactions. The method avoids the use of substrates and of organic solvents that may affect CPR and cytochrome P450 activity. In the reaction, molecular oxygen served as a proton source. The method can substitute spectrophotometric detection methods for its accuracy, high reproducibility (~100%) and sensitivity. The lower limit of detection, shown using the Agilent 1200 aparatus, is in the 250 nmol range. In addition, using this method it is possible to set up reactions in a high‐throughput format. Copyright © 2011 John Wiley & Sons, Ltd.     

UPLC-MS/MS结合多探针底物方法研究刺五加叶中黄酮苷类成分对CYP450活性的影响

利用超高效液相色谱-串联质谱联用(UPLC-MS/MS)的多反应监测(MRM)技术结合多探针底物方法, 研究了刺五加叶中的主要黄酮苷类化合物槲皮苷、金丝桃苷及芦丁对肝细胞色素P450酶(CYP450)亚型CYP1A2, CYP2C, CYP2E1, CYP2D和CYP3A活性的影响. 结果表明, 3种化合物对各CYP亚型酶均有抑制作用, 其中金丝桃苷和槲皮苷对CYP1A2催化的非那西丁的O-脱乙基反应抑制的IC₅₀值分别为46.53和49.75 μmol/L, 金丝桃苷和芦丁对CYP2E1催化的氯唑沙宗的6-羟基化反应抑制的IC₅₀值分别为99.87和86.36 μmol/L. 机理性抑制实验结果表明, 3种化合物对2种亚型酶的抑制作用是随着预孵时间延长而增强的机理性抑制.     

Modulating the coupling efficiency of human cytochrome P450 CYP3A4 at electrode surfaces through protein engineering

This study shows that regulating the electron flow to the heme of human cytochrome P450 CYP3A4, using artificial redox chains, can significantly enhance its coupling efficiency and catalytic activity at electrode surfaces. The human CYP3A4 was fused at the genetic level either to the reductase domain of CYP102A1 (BMR) to create the CYP3A4/BMR or to Desulfovibrio vulgaris flavodoxin (FLD) to create the CYP3A4/FLD. Direct electrochemistry of the CYP3A4, CYP3A4/BMR and CYP3A4/FLD on glassy carbon and gold electrodes showed that the BMR and FLD flavo-proteins reduced the electron transfer rate to the CYP3A4 heme. Electrocatalysis resulted in appreciably higher product formation with the immobilized CYP3A4/BMR and CYP3A4/FLD on both surfaces due to an increased coupling efficiency. Rotating disk electrode studies and quantification of hydrogen peroxide were consistent with the proposed mechanism of a longer lived iron-peroxy species in the immobilized CYP3A4/BMR and CYP3A4/FLD. The approaches in this study provide a better understanding of cytochrome P450 uncoupling at electrode surfaces and aids in the construction of improved cytochrome P450 biosensors and bioelectrocatalysts.     

Regioselective ω-hydroxylation of medium-chain n-alkanes and primary alcohols by CYP153 enzymes from Mycobacterium marinum and Polaromonas sp. strain JS666

The oxofunctionalization of saturated hydrocarbons is an important goal in basic and applied chemistry. Biocatalysts like cytochrome P450 enzymes can introduce oxygen into a wide variety of molecules in a very selective manner, which can be used for the synthesis of fine and bulk chemicals. Cytochrome P450 enzymes from the CYP153A subfamily have been described as alkane hydroxylases with high terminal regioselectivity. Here we report the product yields resulting from C(5)-C(12) alkane and alcohol oxidation catalyzed by CYP153A enzymes from Mycobacterium marinum (CYP153A16) and Polaromonas sp. (CYP153A P. sp.). For all reactions, byproduct formation is described in detail. Following cloning and expression in Escherichia coli, the activity of the purified monooxygenases was reconstituted with putidaredoxin (CamA) and putidaredoxin reductase (CamB). Although both enzyme systems yielded primary alcohols and α,ω-alkanediols, each one displayed a different oxidation pattern towards alkanes. For CYP153A P. sp. a predominant ω-hydroxylation activity was observed, while CYP153A16 possessed the ability to catalyze both ω-hydroxylation and α,ω-dihydroxylation reactions.     

Enhanced electron transfer and lauric acid hydroxylation by site-directed mutagenesis of CYP119

CYP119, a cytochrome P450 from a thermophilic organism for which a crystal structure is available, is shown here to hydroxylate lauric acid in a reaction supported by putidaredoxin and putidaredoxin reductase. This fatty acid hydroxylation activity is increased 15-fold by T214V and D77R mutations. The T214V mutation increases the rate by facilitating substrate binding and enhancing the associated spin state change, whereas the D77R mutation improves binding of the heterologous redox partner putidaredoxin to CYP119 and the rate of electron transfer from it to the heme group. A sequence alignment with P450(cam) can, therefore, be used to identify a part of the binding site for putidaredoxin on an unrelated P450 enzyme. This information can be used to engineer by mutagenesis an improved complementarity of the protein-protein interface that results in improved electron transfer from putidaredoxin to the P450 enzyme. As a result, the catalytic activity of the thermo- and barostable CYP119 has been incorporated into a catalytic system that hydroxylates fatty acids.     

Detection of electrophoretically separated cytochromes P450 by element-labelled monoclonal antibodies via laser ablation inductively coupled plasma mass spectrometry

Numerous structurally and enzymatically similar cytochromes P450 (CYPs) are involved in the metabolism of xenobiotics and are present in different amounts and with different enzyme profiles in human tissues and cells. Analysis of their adaptively regulated and individually variable patterns is a peculiar analytical challenge. We developed a laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) based method for concomitant detection and semiquantitative determination of electrophoretically separated and blotted CYPs. The first results are given here for the two enzymes CYP1A1 and CYP2E1. Specific monoclonal antibodies directed against the enzymes were differentially labelled with europium via a covalently linked chelator and with iodine, respectively. Analysis of the modified antibodies shows that both europium and iodine are coupled to the heavy and the light chains of the antibodies. Also, the antibodies maintained their antigen-binding properties after labelling as demonstrated by LA-ICP-MS-analysed immunoblots. The method allowed us to detect specifically and concomitantly both CYP enzymes in complex biological samples, i.e. microsomes of rat liver and minipig duodenum, which are characterized by different levels and proportions of the two CYP enzymes. A strong CYP1A1 signal is found in liver microsomes of 3-methylcholanthrene-treated rats, while it is (nearly) absent in liver microsomes of rats treated with isonocotinic acid hydrazide (isoniazid). The constitutively expressed CYP2E1 is found in microsomes of both treatment groups. Duodenal microsomes of minipigs orally exposed to polycyclic aromatic hydrocarbons show a clear CYP1A1 signal. Low levels of CYP2E1 can also be detected in these microsomes. The LA-ICP-MS method allows concomitant determination of CYPs, thereby exhibiting sensitivity similar to that of conventional chemoluminescence detection via peroxidase-labelled secondary antibodies. The latter method allows readout of a single CYP protein in a 1D separation. Although the results presented here are only for labelling by use of the elements iodine and europium, the same strategy can be applied also for other lanthanide elements in combination with chelating compounds, so LA-ICP-MS of western blots offers a new capability to be applied for highly multiplexed CYP determinations via labelled antibodies.     

UHPLC‐Q‐TOF‐MS/MS‐based screening and characterization of metabolites of cnidilin in human liver microsomes

Cnidilin is an active natural furocoumarin ingredient originating from well‐known traditional Chinese medicine Radix Angelicae Dahuricae . In the present study, an efficient approach was developed for the screening and identification of cnidilin metabolites using ultra‐high‐performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry. In this approach, an on‐line data acquisition method multiple mass defect filter combined with dynamic background subtraction was developed to trace all probable metabolites. Based on this analytical strategy, a total of 24 metabolites of cnidilin were detected in human liver microsomal incubation samples and the metabolic pathways were proposed. The results indicated that oxidation was the main biotransformation route for cnidilin in human liver microsomes. In addition, the specific cytochrome P450 (CYP) enzymes involved in the metabolism of cnidilin were identified using chemical inhibition and CYP recombinant enzymes. The results showed that CYP1A2 and CYP3A4 might be the major enzymes involved in the metabolism of cnidilin in human liver microsomes. The relationship between cnidilin and the CYP450 enzymes could provide us a theoretical basis of the pharmacological mechanism.     

Kinetic Characteristics of Curcumin and Germacrone in Rat and Human Liver Microsomes: Involvement of CYP Enzymes

Curcumin and germacrone, natural products present in the Zingiberaceae family of plants, have several biological properties. Among these properties, the anti-NSCLC cancer action is noteworthy. In this paper, kinetics of the two compounds in rat liver microsomes (RLMs), human liver microsomes (HLMs), and cytochrome P450 (CYP) enzymes (CYP3A4, 1A2, 2E1, and 2C19) in an NADPH-generating system in vitro were evaluated by UP-HPLC–MS/MS (ultrahigh-pressure liquid chromatography–tandem mass spectrometry). The contents of four cytochrome P450 (CYP) enzymes, adjusting by the compounds were detected using Western blotting in vitro and in vivo. The t_1/2 of curcumin was 22.35 min in RLMs and 173.28 min in HLMs, while 18.02 and 16.37 min were gained for germacrone. The V_max of curcumin in RLMs was about 4-fold in HLMs, meanwhile, the V_max of germacrone in RLMs was similar to that of HLMs. The single enzyme t1/2 of curcumin was 38.51 min in CYP3A4, 301.4 min in 1A2, 69.31 min in 2E1, 63.01 min in 2C19; besides, as to the same enzymes, t1/2 of germacrone was 36.48 min, 86.64 min, 69.31 min, and 57.76 min. The dynamic curves were obtained by reasonable experimental design and the metabolism of curcumin and germacrone were selected in RLMs/HLMs. The selectivities in the two liver microsomes differed in degradation performance. These results meant that we should pay more attention to drugs in clinical medication–drug and drug–enzyme interactions.     

Enzymatic generation of alloxan radicals in rat liver microsomes: possible participation of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P-450 reductase.

Electron spin resonance studies showed that addition of rat liver microsomes to the reaction system of alloxan with reduced nicotinamide adenine dinucleotide phosphate (NADPH) resulted in a marked increase in the generation of alloxan radicals (AH.), whereas heat-denatured microsomes were without such effect. Oxidation of NADPH by alloxan was also stimulated by microsomes. The microsomes from rats treated with phenobarbital, an inducer of cytochrome P-450 reductase, greatly stimulated both the AH.generation and the NADPH oxidation. However, the microsomes from rats treated with 3-methylcholanthrene, an inducer of DT-diaphorase, did not have stimulative effect greater than the control microsomes. These results suggest the possibility that NADPH-linked AH.generations in microsomal membranes is catalyzed by NADPH-cytochrome P-450 reductase.     

Capillary electrophoresis to assess drug metabolism induced in vitro using single CYP450 enzymes (Supersomes): application to the chiral metabolism of mephenytoin and methadone

Capillary electrophoresis (CE) with multiwavelength absorbance detection is demonstrated to be an effective tool for the assessment of in vitro drug metabolism studies using microsomes containing single human cytochrome P450 enzymes (CYPs) expressed in baculovirus-infected insect cells (Supersomes). Mephenytoin (MEPH), dextromethorphan, diclofenac, caffeine, and methadone (MET) were successfully applied as test substrates for CYP2C19, CYP2D6*1, CYP2C9*1, CYP1A2, and CYP3A4, respectively. For each system, the CE-based assay could be shown to permit the simultaneous analysis of the parent drug and its targeted metabolite. Using a chiral micellar electrokinetic capillary chromatography assay, the aromatic hydroxylation of MEPH catalyzed by CYP2C19 could thereby be confirmed to be highly stereoselective, an aspect that is in agreement with data obtained via urinary analysis after intake of racemic MEPH by extensive metabolizer phenotypes. The MET to 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) conversion was investigated with a chiral zone electrophoresis assay. Incubation of racemic and nonracemic MET with CYP3A4 revealed no stereoselectivity for the transformation to EDDP, whereas no EDDP formation was observed with CYP1A2. CYP2C9 and CYP2C19 provided enhanced formation of R-EDDP and CYP2D6 incubation resulted in the preferential conversion to S-EDDP. Investigations using racemic MET and human liver microsomes revealed a modest stereoselectivity with an R/S EDDP ratio < 1 which is similar to the in vivo findings in urine.     

Preparation, characterization, and substrate metabolism of gold-immobilized cytochrome P450 2C9

The cytochrome P450 enzymes represent an important class of heme-containing enzymes. There is considerable interest in immobilizing these enzymes on a surface so that interactions between a single enzyme and other species can be studied with respect to electron transfer, homodimer or heterodimer interactions, or for construction of biological-based chips for standardizing cytochrome P450 metabolism or for high-throughput screening of pharmaceutical agents. Previous studies have generally immobilized P450 enzymes in a matrix or on a surface. Here, we have attached CYP2C9 to gold substrates such that the resulting construct maintains the ability to bind and metabolize substrates in the presence of NADPH and cytochrome P450 reductase. The activity of these chips is directly dependent upon the linkers used to attach CYP2C9 and to the presence of key molecules in the active site during enzyme attachment. A novel method to detect substrate-enzyme binding, namely, superconducting quantum interference device (SQUID) magnetometry, was used to monitor the binding of substrates. Most significantly, conditions that allow measurable CYP2C9 metabolism to occur have been developed.