Human Cytochrome P450 Monooxygenase Research Articles

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.

Expanding the Diversity of Plant Monoterpenoid Indole Alkaloids Employing Human Cytochrome P450 3A4.

Human drug‐metabolizing cytochrome P450 monooxygenases (CYPs) have enormous substrate promiscuity; this makes them promising tools for the expansion of natural product diversity. Here, we used CYP3A4 for the targeted diversification of a plant biosynthetic route leading to monoterpenoid indole alkaloids. In silico, in vitro and in planta studies proved that CYP3A4 was able to convert the indole alkaloid vinorine into vomilenine, the former being one of the central intermediates in the ajmaline pathway in the medicinal plant Rauvolfia serpentina (L.) Benth. ex Kurz. However, to a much larger extent, the investigated conversion yielded vinorine (19R,20R)‐epoxide, a new metabolite with an epoxide functional group that is rare for indole alkaloids. The described work represents a successful example of combinatorial biosynthesis towards an increase in biodiversity of natural metabolites. Moreover, characterisation of the products of the in vitro and in planta transformation of potential pharmaceuticals with human CYPs might be indicative of the route of their conversion in the human organism.

Metabolic interactions of rosmarinic acid with human cytochrome P450 monooxygenases and uridine diphosphate glucuronosyltransferases

In light of the widespread use of herbal medicines containing rosmarinic acid (RA) and limited literature available thereon, we investigated the metabolic interactions of RA with human cytochrome P450 monooxygenases (CYPs) and uridine diphosphate glucuronosyltransferases (UGTs). The involvement of selected enzymes (CYP1A2, CYP2C19, CYP2E1, CYP3 A4, UGT1A1, UGT1A6, and UGT2B7) in the metabolism of RA and the inhibitory effect of RA on the enzyme activity were comprehensively evaluated using human recombinant isozyme system. Additionally, concentration-dependent RA metabolism mediated by phase I enzymes (including CYPs) or UGT was investigated in human liver microsome (HLM) system. A significant disappearance of RA was observed in the seven CYP and UGT isoforms studied, indicating their possible involvement in the metabolism of RA. Based on Michaelis-Menten kinetics, the metabolism study using HLM suggests that the UGT system may have a higher capacity and lower affinity for the metabolism of RA than phase I enzyme (including CYP) systems. Moreover, RA weakly inhibited CYP2C9 and 2E1 activities with IC50 values of 39.6 and 61.0 μM, respectively, while moderately inhibiting UGT1A1, 1A6, and 2B7 with IC50 values of 9.24, 19.1, and 23.4 μM, respectively. By constructing Line weaver–Burk plots, the type of inhibition exhibited by RA on CYP and UGT activities was determined as follows: CYP2C19, mixed inhibition; CYP2E1, UGT1A1, UGT1A6, and UGT2B7, competitive inhibition. Based on the comparison of the IC50 and Ki values obtained in the current study with the previously reported plasma concentrations of RA after oral dosing in humans, it is suggested that RA may significantly inhibit the activities of the tested UGTs, rather than CYPs, in clinical settings. Thus, the present study could provide a basis for further studies on clinically significant interactions between UGT substrate drugs and herbal medicines containing RA.

New Therapies of Neovascular AMD-Beyond Anti-VEGFs.

Neovascular age-related macular degeneration (nAMD) is one of the leading causes of blindness among the aging population. The current treatment options for nAMD include intravitreal injections of anti-vascular endothelial growth factor (anti-VEGF). However, standardized frequent administration of anti-VEGF injections only improves vision in approximately 30–40% of nAMD patients. Current therapies targeting nAMD pose a significant risk of retinal fibrosis and geographic atrophy (GA) development in nAMD patients. A need exists to develop new therapies to treat nAMD with effective and long-term anti-angiogenic effects. Recent research on nAMD has identified novel therapeutic targets and angiogenic signaling mechanisms involved in its pathogenesis. For example, tissue factor, human intravenous immune globulin, interferon-β signaling, cyclooxygenase-2 (COX-2) and cytochrome P450 monooxygenase lipid metabolites have been identified as key players in the development of angiogenesis in AMD disease models. Furthermore, novel therapies such as NACHT, LRR and PYD domains containing protein 3 (NLRP3) inflammasome inhibition, inhibitors of integrins and tissue factor are currently being tested at the level of clinical trials to treat nAMD. The aim of this review is to discuss the scope for alternative therapies proposed as anti-VEGFs for the treatment of nAMD.

Engineering of new-to-nature halogenated indigo precursors in plants

Plants are versatile chemists producing a tremendous variety of specialized compounds. Here, we describe the engineering of entirely novel metabolic pathways in planta enabling generation of halogenated indigo precursors as non-natural plant products.Indican (indolyl-β-D-glucopyranoside) is a secondary metabolite characteristic of a number of dyers plants. Its deglucosylation and subsequent oxidative dimerization leads to the blue dye, indigo. Halogenated indican derivatives are commonly used as detection reagents in histochemical and molecular biology applications; their production, however, relies largely on chemical synthesis. To attain the de novo biosynthesis in a plant-based system devoid of indican, we employed a sequence of enzymes from diverse sources, including three microbial tryptophan halogenases substituting the amino acid at either C5, C6, or C7 of the indole moiety. Subsequent processing of the halotryptophan by bacterial tryptophanase TnaA in concert with a mutant of the human cytochrome P450 monooxygenase 2A6 and glycosylation of the resulting indoxyl derivatives by an endogenous tobacco glucosyltransferase yielded corresponding haloindican variants in transiently transformed Nicotiana benthamiana plants. Accumulation levels were highest when the 5-halogenase PyrH was utilized, reaching 0.93 ± 0.089 mg/g dry weight of 5-chloroindican. The identity of the latter was unambiguously confirmed by NMR analysis. Moreover, our combinatorial approach, facilitated by the modular assembly capabilities of the GoldenBraid cloning system and inspired by the unique compartmentation of plant cells, afforded testing a number of alternative subcellular localizations for pathway design. In consequence, chloroplasts were validated as functional biosynthetic venues for haloindican, with the requisite reducing augmentation of the halogenases as well as the cytochrome P450 monooxygenase fulfilled by catalytic systems native to the organelle.Thus, our study puts forward a viable alternative production platform for halogenated fine chemicals, eschewing reliance on fossil fuel resources and toxic chemicals. We further contend that in planta generation of halogenated indigoid precursors previously unknown to nature offers an extended view on and, indeed, pushes forward the established frontiers of biosynthetic capacity of plants.

Abstract 963: Ovarian-dependent SSM2ucd mouse mammary carcinoma cells depend on Cyp3a11 epoxygenase activity for proliferation

Abstract Introduction: Silencing of the human cytochrome P450 (CYP) monooxygenase enzyme CYP3A4 in the ER+ MCF-7 tumor cell line inhibits tumor growth in the mammary fat pad, but the relative roles of tumor cell intrinsic CYPs vs. vascular/microenvironment CYPs remain to be determined and a syngeneic animal model is needed to answer this question. The SSM2ucd ovarian dependent mouse mammary carcinoma cell line forms a transplantable estrogen-receptor positive (ER+) mouse mammary tumor in which growth dependence on cell intrinsic CYPs may be tested in vitro and in vivo. The SSM2ucd cell line is derived from 129SvJ: homozygous Stat1-null female mice, which develop ER+ mammary tumors (1,2). Expression profiling of spontaneously arising mouse mammary carcinomas in a TP53 KO model revealed that the CYP3A4 ortholog Cyp3a11 is up-regulated in these tumors. Methods: Cyp3a11 bactosomes were used to assay for NADPH dependent biosynthesis of EETs from arachidonic acid using an LC-MS/MS method. siRNA silencing and CRISPR/Cas9 knock out of the Cyp3a11 exons 2 and 3 was performed and the cell lines were characterized for EET dependence using an MTT assay. The highly potent inhibitor and chemical probe of CYP epoxygenase activity, hexyl-benzyl-biguanide (HBB), was used to test dependence of SSM2ucd cells on CYP epoxygenase activity. Total cellular EETs in SSM2ucd cells exposed to HBB at the IC50were measured by the LC-MS/MS method. Results: Cyp3a11 was shown to have robust NADPH dependent epoxygenase activity, while SSM2ucd cells were EET dependent for growth. Silencing of Cyp3a11 was demonstrated to inhibit proliferation of the SSM2ucd cell line, which was partly abrogated by EETs. CRISPR/Cas9 knock out of the Cyp3a11 in the SSM2ucd cell line was performed and exon 2 and exon 3 deleted cell lines were derived. SSM2ucd Cyp3a11 knock out cell lines were more sensitive to EET-induced proliferation under serum free condition than control lines. HBB potently inhibits the SSM2ucd cell line in vitro (IC50=25 uM) and reduces total cellular EET levels in this cell line at IC50. These results suggest that we may see inhibitory activity of HBB in a syngeneic 129/SvJ tumor transplantation model. Conclusions: The Cyp3a11 gene can be silenced in the SSM2ucd cell line, without disrupting cell viability, allowing further study of the roles of EETs in mouse mammary carcinoma proliferation, survival and clonogenicity. The Cyp3a11 gene has been knocked out in the SSM2 ucd cell line, allowing in vitro and in vivo studies of the roles of Cyp3a11 in the growth of ovarian dependent mouse mammary carcinoma. 1. Breast Cancer Res. 2012 Jan 20;14(1):R16 2. PLoS One. 2015 Jun 15;10(6):e0129895] Citation Format: Zhijun Guo, Patrick McGrarrah, Ted Bebi, Ashley Mooneyham, Alejandra Osorio, Cesar Herrera, Victor Arrieta, Sebastian Mohar, Irwin Hernandez, Robert Klink, Robert Cardiff, David A. Potter. Ovarian-dependent SSM2ucd mouse mammary carcinoma cells depend on Cyp3a11 epoxygenase activity for proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 963. doi:10.1158/1538-7445.AM2017-963

Advanced Approaches to Model Xenobiotic Metabolism in Bacterial Genotoxicology In Vitro.

During the past 30 years there has been considerable progress in the development of bacterial test systems for use in genotoxicity testing by the stable introduction of expression vectors (cDNAs) coding for xenobiotic-metabolizing enzymes into bacterial cells. The development not only provides insights into the mechanisms of bioactivation of xenobiotic compounds but also evaluates the roles of enzymes involved in metabolic activation or inactivation in chemical carcinogenesis. This review describes recent advances in bacterial genotoxicity assays and their future prospects, with a focus on the development and application of genetically engineering bacterial cells to incorporate some of the enzymatic activities involved in the bio-activation process of xenobiotics. Various genes have been introduced into bacterial umu tester strains encoding enzymes for genotoxic bioactivation, including bacterial nitroreductase and O-acetyltransferase, human cytochrome P450 monooxygenases, rat glutathione S-transferases, and human N-acetyltransferases and sulfotransferases. Their application has provided new tools for genotoxicity assays and for studying the role of biotransformation in chemical carcinogenesis in humans.

Metabolism of the14C-labeled herbicide clodinafop-propargyl in plant cell cultures of wheat and tobacco

ABSTRACTThe metabolism of 14C-clodinafop-propargyl (CfP) was examined in cell cultures of wheat (Triticum aestivum L. cv. ‘Heines Koga II’) and tobacco (Nicotiana tabacum L.). Besides the non-transgenic tobacco culture, cultures transformed separately with cDNA of human cytochrome P450-monooxygenases (P450s) CYP1A1, CYP1A2, CYP3A4, CYP2B6 and CYP2C19 were examined. Experiments with wheat were executed in the presence and absence of safener cloquintocet-mexyl (CqM). After 48 h of incubation, only about 10% of applied 14C was found in media (both tobacco and wheat). Non-extractable residues of 14C-CfP in wheat cells were 16.54% (without CqM) and 30.87% (with CqM). In all tobacco cultures, 82.41–92.46% of applied radioactivity was recovered in cell extracts. In contrast to wheat, non-extractable residues amounted only to 1.50–2.82%. As determined by radio-thin layer chromatography (TLC) and -high-performance liquid chromatography (HPLC), the parent CfP was not found in the cell extracts of wheat; in tobacco cell extracts, only traces of CfP were detected. After a hydrolysis of assumed carbohydrate conjugates of CfP derived polar 14C-labeled compounds, TLC and HPLC analysis showed that in wheat, a more complex pattern of metabolites of CfP were observed as compared to all tobacco cultures. In hydrolysates resulting from wheat, the identity of three primary products was confirmed by means of GC-EI-MS: free acid clodinafop (Cf), hydroxy-Cf hydroxylated at the pyridinyl moiety, and 4-(5-chloro-3-fluoropyridin-2-yloxy)phenol. In hydrolysates derived from all tobacco cultures, main metabolite was Cf besides only traces of further unidentified products. Differences among the different tobacco cultures (non-transgenic, transgenic) did not emerge. According to kinetics of disappearance of primary metabolite Cf as well as formation of polar soluble products and non-extractable residues, metabolization of CfP proceeded at a noticeably higher rate in wheat cells treated with safener CqM than in cells without CqM treatment. Thus, these results indicated a stimulation of CfP's metabolism by CqM, although metabolic profiles observed in CqM treated and non-treated cells (after hydrolysis) were qualitatively similar. The findings obtained from all tobacco cultures suggested that with the exception of ester cleavage to Cf, CfP cannot be metabolized by tobacco itself or by the human P450s examined.

Differences in metabolism of the marine biotoxin okadaic acid by human and rat cytochrome P450 monooxygenases.

The ingestion of seafood contaminated with the marine biotoxin okadaic acid (OA) can lead to diarrhetic shellfish poisoning with symptoms like nausea, vomiting and abdominal cramps. Both rat and the human hepatic cytochrome P450 monooxygenases (CYP) metabolize OA. However, liver cell toxicity of metabolized OA is mainly unclear. The aim of our study was to detect the cellular effects in HepG2 cells exposed to OA in the presence of recombinant CYP enzymes of both rat and human for the investigation of species differences. The results should be set in correlation with a CYP-specific metabolite pattern. Comparative metabolite profiles of OA after incubation in rat and human recombinant CYP enzymes were established by using LC-MS/MS technique. Results demonstrated that metabolism of OA to oxygenated metabolites correlates with detoxification which was mainly catalyzed by human CYP3A4 and CYP3A5. Detoxification by rat Cyp3a1 was lower compared to human CYP3A enzymes and activation of OA by Cyp3a2 was observed, coincident with minor overall conversion capacity of OA. By contrast human and rat CYP1A2 seem to activate OA into cytotoxic intermediates. In conclusion, different mechanisms of OA metabolism may occur in the liver. At low OA doses, the human liver is likely well protected against cytotoxic OA, but for high shellfish consumers a potential risk cannot be excluded.

Direct infusion-SIM as fast and robust method for absolute protein quantification in complex samples

Relative and absolute quantification of proteins in biological and clinical samples are common approaches in proteomics. Until now, targeted protein quantification is mainly performed using a combination of HPLC-based peptide separation and selected reaction monitoring on triple quadrupole mass spectrometers. Here, we show for the first time the potential of absolute quantification using a direct infusion strategy combined with single ion monitoring (SIM) on a Q Exactive mass spectrometer. By using complex membrane fractions of Escherichia coli, we absolutely quantified the recombinant expressed heterologous human cytochrome P450 monooxygenase 3A4 (CYP3A4) comparing direct infusion-SIM with conventional HPLC-SIM. Direct-infusion SIM revealed only 14.7% (±4.1 (s.e.m.)) deviation on average, compared to HPLC-SIM and a decreased processing and analysis time of 4.5min (that could be further decreased to 30s) for a single sample in contrast to 65min by the LC–MS method. Summarized, our simplified workflow using direct infusion-SIM provides a fast and robust method for quantification of proteins in complex protein mixtures.

Fungal unspecific peroxygenases: heme-thiolate proteins that combine peroxidase and cytochrome p450 properties.

Eleven years ago, a secreted heme-thiolate peroxidase with promiscuity for oxygen transfer reactions was discovered in the basidiomycetous fungus, Agrocybe aegerita. The enzyme turned out to be a functional mono-peroxygenase that transferred an oxygen atom from hydrogen peroxide to diverse organic substrates (aromatics, heterocycles, linear and cyclic alkanes/alkenes, fatty acids, etc.). Later similar enzymes were found in other mushroom genera such as Coprinellus and Marasmius. Approximately one thousand putative peroxygenase sequences that form two large clusters can be found in genetic databases and fungal genomes, indicating the widespread occurrence of such enzymes in the whole fungal kingdom including all phyla of true fungi (Eumycota) and certain fungus-like heterokonts (Oomycota). This new enzyme type was classified as unspecific peroxygenase (UPO, EC 1.11.2.1) and placed in a separate peroxidase subclass. Furthermore, UPOs and related heme-thiolate peroxidases such as well-studied chloroperoxidase (CPO) represent a separate superfamily of heme proteins on the phylogenetic level. The reactions catalyzed by UPOs include hydroxylation, epoxidation, O- and N-dealkylation, aromatization, sulfoxidation, N-oxygenation, dechlorination and halide oxidation. In many cases, the product patterns of UPOs resemble those of human cytochrome P450 (P450) monooxygenases and, in fact, combine the catalytic cycle of heme peroxidases with the "peroxide shunt" of P450s. Here, an overview on UPOs is provided with focus on their molecular and catalytic properties.

Regioselective oxidation of phospho‐NSAIDs by human cytochrome P450 and flavin monooxygenase isoforms: implications for their pharmacokinetic properties and safety

Phospho-ibuprofen (MDC-917) and phospho-sulindac (OXT-328) are highly effective in cancer and arthritis treatment in preclinical models. Here, we investigated their metabolism by major human cytochrome P450s (CYPs) and flavin monooxygenases (FMOs). The CYP/FMO-catalysed metabolism of phospho-ibuprofen and phospho-sulindac was studied by using in silico prediction modelling and a direct experimental approach. The CYP isoforms catalyse the oxidation of non-steroidal anti-inflammatory drugs (NSAIDs) and phospho-NSAIDs, with distinct activity and regioselectivity. CYP1A2, 2C19, 2D6 and 3A4 oxidize phospho-ibuprofen, but not ibuprofen; whereas CYP2C9 oxidizes ibuprofen, but not phospho-ibuprofen. All CYPs tested oxidize phospho-sulindac, but not sulindac. Among the five CYPs evaluated, CYP3A4 and 2D6 are the most active in the oxidation of phospho-ibuprofen and phospho-sulindac respectively. FMOs oxidized phospho-sulindac and sulindac, but not phospho-ibuprofen or ibuprofen. FMOs were more active towards phospho-sulindac than sulindac, indicating that phospho-sulindac is a preferred substrate of FMOs. The susceptibility of phospho-NSAIDs to CYP/FMO-mediated metabolism was also reflected in their rapid oxidation by human and mouse liver microsomes, which contain a full complement of CYPs and FMOs. Compared with conventional NSAIDs, the higher activity of CYPs towards phospho-ibuprofen and phospho-sulindac may be due to their greater lipophilicity, a key parameter for CYP binding. CYPs and FMOs play an important role in the metabolism of phospho-NSAIDs, resulting in differential pharmacokinetic profiles between phospho-NSAIDs and NSAIDs in vivo. The consequently more rapid detoxification of phospho-NSAIDs is likely to contribute to their greater safety.

Identification of Human Cytochrome P450 and Flavin-Containing Monooxygenase Enzymes Involved in the Metabolism of Lorcaserin, a Novel Selective Human 5-Hydroxytryptamine 2C Agonist

Lorcaserin, a selective serotonin 5-hydroxytryptamine 2C receptor agonist, is being developed for weight management. The oxidative metabolism of lorcaserin, mediated by recombinant human cytochrome P450 (P450) and flavin-containing monooxygenase (FMO) enzymes, was examined in vitro to identify the enzymes involved in the generation of its primary oxidative metabolites, N-hydroxylorcaserin, 7-hydroxylorcaserin, 5-hydroxylorcaserin, and 1-hydroxylorcaserin. Human CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, CYP3A4, and FMO1 are major enzymes involved in N-hydroxylorcaserin; CYP2D6 and CYP3A4 are enzymes involved in 7-hydroxylorcaserin; CYP1A1, CYP1A2, CYP2D6, and CYP3A4 are enzymes involved in 5-hydroxylorcaserin; and CYP3A4 is an enzyme involved in 1-hydroxylorcaserin formation. In 16 individual human liver microsomal preparations (HLM), formation of N-hydroxylorcaserin was correlated with CYP2B6, 7-hydroxylorcaserin was correlated with CYP2D6, 5-hydroxylorcaserin was correlated with CYP1A2 and CYP3A4, and 1-hydroxylorcaserin was correlated with CYP3A4 activity at 10.0 μM lorcaserin. No correlation was observed for N-hydroxylorcaserin with any P450 marker substrate activity at 1.0 μM lorcaserin. N-Hydroxylorcaserin formation was not inhibited by CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, and CYP3A4 inhibitors at the highest concentration tested. Furafylline, quinidine, and ketoconazole, selective inhibitors of CYP1A2, CYP2D6, and CYP3A4, respectively, inhibited 5-hydroxylorcaserin (IC(50) = 1.914 μM), 7-hydroxylorcaserin (IC(50) = 0.213 μM), and 1-hydroxylorcaserin formation (IC(50) = 0.281 μM), respectively. N-Hydroxylorcaserin showed low and high K(m) components in HLM and 7-hydroxylorcaserin showed lower K(m) than 5-hydroxylorcaserin and 1-hydroxylorcaserin in HLM. The highest intrinsic clearance was observed for N-hydroxylorcaserin, followed by 7-hydroxylorcaserin, 5-hydroxylorcaserin, and 1-hydroxylorcaserin in HLM. Multiple human P450 and FMO enzymes catalyze the formation of four primary oxidative metabolites of lorcaserin, suggesting that lorcaserin has a low probability of drug-drug interactions by concomitant medications.

Enzyme and protein engineering: engineering a self-sufficient human cytochrome P450 monooxygenase system

Enzyme and protein engineering: engineering a self-sufficient human cytochrome P450 monooxygenase system

Generation of novel medicinal plants by metabolic engineering

Plants produce a plethora of valuable natural products, many of which are used by humans as nutrients, colorants, flavors, fragrances, and pharmaceutics. For numerous natural products the dependency on natural occurring resources strongly limits their availability due to their occurance in eventually endangered species, due to the minute amounts of metabolites present in plant tissue, and due to the reluctance of many medicinal plants to propagation and cultivation in sufficient quantities. During the last decades an enormous amount of knowledge has been generated regarding the biochemistry and genetics of numerous complex natural product biosynthesis pathways in plants, like those leading to morphinans, terpenoid indole alkaloids, or lignans. This steadily increasing knowledge know opens up opportunities to engineer these pathways in heterologous hosts but also for metabolic engineering of those pathways to significantly enhance the yield of the desired products in planta. Additionally, novel catalysts like the human cytochrome P450 monooxygenases can be engineered into plants, presenting unique options to manipulate plant metabolism (1,2). Current achievements as well as future perspectives of metabolic engineering of medicinal plants will be evaluated in this presentation.

Recombinant human cytochrome P450 monooxygenases for drug metabolite synthesis

Cytochrome P450 monooxygenases (CYPs) are important enzymes in the metabolism of xenobiotics. Therefore, several approaches to clone and overexpress the human isoforms have been made. In addition to microsomes or S9 preparations, these recombinant human isoforms have found diverse application in drug development. We discuss and give examples of the use of bacterial whole cell systems with rec. human CYPs for the preparative scale synthesis of drug metabolites.

In Vitro Profiling and Mass Balance of the Anti-Cancer Agent Laromustine [14C]-VNP40101M by Rat, Dog, Monkey and Human Liver Microsomes

Laromustine (VNP40101M; 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-(methylamino) carbonylhydrazine) is a novel sulfonylhydrazine alkylating agent. For the first time In vitro profiling and mass balance of [14C] VNP40101M in rat, dog, monkey and human liver microsomes was investigated. Also, the role of human cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) enzymes in the conversion of [14C] VNP40101M by NADPH-fortified human liver microsomes was determined. In this study, [14C]-VNP40101M was converted to five radioactive components (C-1, C-2, C-3, C-4 and C-7) after 60 min of incubation with dog, monkey and human liver microsomes. With the exception of C-3, the same components were detected with rat liver microsomes. In the presence of NADPH, after 60 min of incubation, the loss of substrate for rat, dog, monkey and human was 63, 82, 76 and 64%, respectively and mass balance ranged from 91.0 – 99.3%. In the absence of NADPH, after 60 min of incubation with [14C] VNP40101M (100 µM), the loss of substrate for rat, dog, monkey and human liver microsomes was 59, 53, 61 and 59%, respectively and mass balance ranged from 100.6 – 116.4%. The profiles of metabolites were similar. The relative abundance of individual metabolites was not species dependent. The formation of C-7 was not observed in zero cofactor (no NADPH) or zero-protein samples, suggesting that its formation was enzymatic. The formation of C-1, C-2, C-3, and C-4 increased with respect to incubation time. Using a panel of CYP enzymes including rCYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4, it was shown that C-7 formation was catalyzed by CYP2B6 and CYP3A4/5. The results of this study suggest that (1) P450 plays a role in C-7 formation but plays little or no role in the conversion of [14C] VNP40101M to C-1 through C-4, and (2) the relative abundance of individual degradation/metabolite products were not species dependent. These findings provide a comprehensive understanding of the metabolism of this new agent.

Bioconversion of the antihistaminc drug loratadine by tobacco cell suspension cultures expressing human cytochrome P450 3A4

In this study we have expanded the metabolic potential of plant cell suspension cultures by introducing active human cytochrome P450 monooxygenase 3A4 into tobacco cells. Exogenously supplied loratadine was metabolized in a 3A4-specific manner, showing the capacity of this system for the generation of metabolites.

Bioconversion of Mono- and Sesquiterpenoids by Recombinant Human Cytochrome P450 Monooxygenases

Cytochrome P450 monooxygenases play an important role in the biosynthesis and metabolism of terpenoids. We explored the potential of recombinant human liver cytochrome P450 monooxygenases CYP1A2, CYP2C9, and CYP3A4, heterologously expressed in Escherichia coli, to convert mono- and sesquiterpenoids to human metabolites. This natural product group is a diverse class of secondary metabolites and includes several industrially and pharmaceutically interesting compounds. Incubation of cedrol with CYP3A4 resulted in a bioconversion of 74% (±8.9%) after 1 h of the unknown metabolites 2-hydroxycedrol and 4-hydroxycedrol, which have been structurally elucidated by 1H and 13C NMR and GC-MS. We conclude that recombinant human cytochrome P450 enzymes can be useful tools in a combinatorial biosynthesis strategy for the production of new natural products and for in vitro metabolization studies.

Hepatic Metabolism of MK-0457, a Potent Aurora Kinase Inhibitor: Interspecies Comparison and Role of Human Cytochrome P450 and Flavin-Containing Monooxygenase

MK-0457 (N-[4([4-(4-methylpiperazin-1-yl)-6-[(3-methyl-1H-pyrazol-5 -yl)amino]pyrimidin-2-yl]thio)phenyl]cyclopropanecarboxamide), an Aurora kinase inhibitor in development for the treatment of cancer, was evaluated for its in vitro metabolism in different species. This compound primarily underwent N-oxidation and N-demethylation in human, monkey, dog, and rat liver preparations. However, N-demethylation was less significant in dogs. The formation of minor metabolites varied with species, but all metabolites generated in human hepatocytes were observed in animals. Results of immunoinhibition, selective chemical inhibition, thermal inactivation, and metabolism by recombinant cytochromes P450 and flavin-containing monoogygenases (FMOs) strongly suggest that CYP3A4 and FMO3 comparably contributed to MK-0457 N-oxidation in human liver microsomes, where the reaction conformed to Michaelis-Menten kinetics. These studies indicate a major role of CYP2C8 in the N-demethylation reaction, whereas CYP3A4 only made a minor contribution. However, significant substrate inhibition was observed with MK-0457 N-demethylation at high substrate concentrations (>10 microM) in human liver microsomes relative to the anticipated therapeutic exposure. A multienzyme metabolic pathway such as this may mitigate the potential of drug interactions in clinical treatment with MK-0457.