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Symposium: Cytochrome P450 –Multivalent Catalysts for Biotechnology and Pharmacology Nishith Gupta, Katrin Lehmann, Otmar Asperger P450non System from Acinetobacter sp. EB104: A Unique Bacterial Alkane Hydroxylase Maria Ludewig*, Carsten Schwarz+, Otmar Asperger*, Svante Pääbo+, Ulrich Hahn* Sequence Analysis of an Acinetobacter Plasmid Carrying the Genes of a P450-Dependent Alkane Monooxygenase Horst Honeck, Wolf-Hagen Schunck, Eduardo Barbosa Sicard Metabolism of eicosapentaenoic acid by CYP4A and CYP2C enzymes Rita Bernhardt Cytochrome P450 dependent steroid hydroxylases and their application in biotechnology and medicine Andreas Bichet, Frank Hannemann, Rita Bernhardt Using the yeast two-hybrid system as in vivo screening-system for interactions between adrenodoxin and its redoxpartners Calin-Aurel Dragan, Rita Bernhardt, Matthias Bureik Production of steroid hormones by fission yeast cells expressing human mitochondrial cytochromes P450 F. Peter Guengerich Mechanisms of Oxidations of Drugs and Carcinogens Catalyzed by Human P450s Annette Deul, Susanne Stevens, Uta Sander, Christian Kirchhoff, Joern Jungmann, Thomas Hesterkamp Use of cytochrome P450 isoenzyme assays in the hit-to-lead process of drug discovery Horst Honeck, Dominik N. Müller, Jürgen Theuer, Friedrich C. Luft, Wolf-H. Schunck, Eva Kärgel Cytochrome P450-Dependent Renal Metabolism of Arachidonic Acid in a Rat Model of Angiotensin II induced Hypertension and End-Organ Damage André Förster, Jana Gerber, Thomas Juretzek, Gerold Barth, Stephan Mauersberger FUNCTIONAL EXPRESSION OF HETEROLOGOUS CYTOCHROME P450 IN YARROWIA LIPOLYTICA AND ITS USE IN BIOTRANSFORMATION OF STEROIDS Kerstin Reichert, Matthias Rödel, Ralph Menzel Xenobiotically induced cytochrome P450 gene expression in Caenorhabditis elegans Dietmar Pfeifer, Martina Kaufmann, Bernd Fiebich, Olivia Spleiss Pharmacogenetic profiling with Biochips: an innovative concept to improve the efficiency of clinical trials Tanja Richter, Kathrin Klein, Thomas E. Mürdter, Michel Eichelbaum, Matthias Schwab, Ulrich M. Zanger ThioTEPA and Clopidogrel are specific mechanism-based inhibitors of human CYP2B6 Ivar Roots, Julia Kirchheiner, Jürgen Brockmöller, Christian Meisel, Kersti Oselin, Thomas Gerloff Pharmacogenetic studies on drug-metabolizing cytochromes P450 and possibilities for individual drug therapy Ilme Schlichting Crystallographic studies on the reaction mechanism of P450 Birgit Lauterbach, Eduardo Barbosa, Eva Kaergel, Horst Honeck, Jürgen Theuer, Hermann Haller, Friedrich C. Luft, Maik Gollasch, Wolf-Hagen Schunck Cytochrome P450-dependent eicosapentaenoic acid epoxygenation produces novel vasoactive metabolites Hannemann Frank, Bernhardt Rita, Zearo Silvia Activities of CYP11B isoforms from different species. Ulrich M. Zanger, Kathrin Klein, Tanja Richter, Thomas E. Mürdter, Ute Hofmann, Thomas Lang, Michel Eichelbaum, Matthias Schwab FUNCTIONAL SIGNIFICANCE OF GENETIC POLYMORPHISMS OF HUMAN CYP2B6 Nishith Gupta, Katrin Lehmann, Otmar Asperger P450non System from Acinetobacter sp. EB104: A Unique Bacterial Alkane Hydroxylase P450non system from Acinetobacter sp. EB104 represents the only bacterial alkane monooxygenase for which all components: P450, ferredoxin (Fd) and ferredoxin reductase (FdR) are available as isolated proteins. Thus, this system represents a preferable model to study the principles for cell-free oxidation of very hydrophobic and chemically inert substances. Recent cloning confirmed the isolated system identity and revealed, compared to other bacterial P450s, a prolonged N-terminus containing an amphipathic alpha-helix. Optical biosensor-based protein-protein interaction studies demonstrated the predominant formation of binary Fd/FdR and Fd/P450 complexes by electrostatic interactions. The P450 itself, oppositely to other bacterial P450s, tends to aggregate in the absence of detergents possibly attributing to the role of lipids for its functional or structural integrity. In accordance with optical spectral data, P450 preparations exhibit predominantly low-spin EPR spectra (g = 2.6, 2.26, 1.84) at 60°K with only weak high-spin signals (g = 4.0, 6.05). The equilibrium can be shifted by addition of lipids and potential substrates. Functional activity of reconstituted systems is distinctively increased by Triton X-100 or rhamnolipids. P450non shows high regio-selectivity for terminal C-atoms independently of chain-length. Oxidation of alkyl chains results in corresponding carboxylic acids indicating a multi-functional enzyme. contact: PD Dr Otmar Asperger Universität Leipzig Institut für Biochemie [email protected] Talstraße 33 04103 Leipzig (D) Maria Ludewig*, Carsten Schwarz+, Otmar Asperger*, Svante Pääbo+, Ulrich Hahn* Sequence Analysis of an Acinetobacter Plasmid Carrying the Genes of a P450-Dependent Alkane Monooxygenase In addition to a nonheme iron alkane hydroxylase Acinetobacter sp. EB104 contains a P450-dependent alkane monooxygenase that consists of the haem protein P450non (CYP153) as the terminal oxygenase, a ferredoxin (Fd) and a NADH-dependent ferredoxin reductase (FdR). It was shown by southern hybridisation that the three genes of the P450 system are localised on the native plasmid pAC450 of A. sp. EB104. They are organised in a single operon together with a putative transcription regulator of the AraC/XylS family. To exploit this system for the biotransformation of chemically inert and highly hydrophobic compounds genes of enzymes involved in further steps of alkane oxidation should be identified. Therefore we sequenced the entire plasmid by shot-gun cloning. The draft sequence implies that the P450 operon is part of a 17-kb composite transposon. In the non-transposon region an open reading frame for a protein putatively involved in conjugation was detected suggesting the acquirement of the P450 system by horizontal gene transfer. ORF's with similarities to those of DNA-interacting proteins indicate that the plasmid encodes its own replication assembly. Furthermore, an ORF with similarity to a transport protein for long-chain fatty acids was found, but sequences typical for alcohol or aldehyde dehydrogenases were not detected. contact: PD Dr. Otmar Asperger Universität Leipzig Institut für Biochemie [email protected] Talstr. 33 04103 Leipzig (D) additional information * University of Leipzig, Institute of Biochemistry + Max-Planck-Institute for Evolutionary Anthropology, Leipzig Horst Honeck, Wolf-Hagen Schunck, Eduardo Barbosa Sicard Metabolism of eicosapentaenoic acid by CYP4A and CYP2C enzymes CYP4A and CYP2C enzymes hydroxylate and/or epoxygenate arachidonic acid (AA; 20:4 n-6) to metabolites involved in the regulation of vascular tone and kidney function. Eicosapentaenoic acid (EPA; 20:5 n-3) is structurally closely related to AA and represents a major (n-3) polyunsaturated fatty acid in fish oil. In the present study, we addressed the question whether or not P450 enzymes with known function in AA metabolism are able to accept EPA as an alternative substrate. We examined the rat AA hydroxylases CYP4A1, 4A2 and 4A3, a corresponding mouse P450 (Cyp4a12) and the AA epoxygenases CYP2C11 (from rat) and CYP2C8 (from human). Active microsomal P450 systems were obtained by co-expression with NAPDH-P450 reductase in Spodoptera frugiperda cells (SF9). All six P450 isoforms selected converted both AA and EPA. The catalytic efficiencies were almost equal or even higher with EPA as substrate. The CYP4As produced 20- and 19-OH-EPA. Cyp 4a12 showed a high regioselectivity in hydroxylating almost exclusively the C-20 position. CYP4A1 and Cyp4a12 also produced significant amounts of 17,18-epoxyeicosatetraenoic acid (17,18-EETeTr). CYP2C11 and 2C8 generated a series of regioisomeric EPA epoxides, among them, 17,18-EETeTr was the main product. Chiral-phase HPLC showed that the R,S- and S,R-enantiomers of 17,18-EETeTr occurred in a ratio of 64:36 for 4A1, 76:24 for 4a12, 28:72 for 2C8 and 72:28 for 2C11. In conclusion, EPA represents an efficient substrate for members of the CYP4A and CYP2C subfamilies. Competition between EPA and AA for conversion by these P450 enzymes and the resulting formation of alternative metabolites may contribute to the beneficial cardiovascular effects attributed to diets rich in EPA. contact: Dipl. Biol. Eduardo Barbosa Sicard Max Delbrueck Centre for Molecular Medicine [email protected] Robert-Roessle-Str. 10 13092 Berlin (Germany) Rita Bernhardt Cytochrome P450 dependent steroid hydroxylases and their application in biotechnology and medicine In the human adrenal cortex glucocorticoids, mineralocorticoids, and androgens are being synthesized. The initial step is the side-chain cleavage of cholesterol to form pregnenolone, catalyzed by CYP11A1. The 11beta-hydroxylase (P45011B1) is responsible for the conversion of 11-deoxycortisol to cortisol. The aldosterone synthase (CYP11B2) catalyses the 11beta-hydroxylation, 18-hydroxylation and 18-oxidation of deoxycorticosterone to form aldosterone. Using site-directed mutagenesis we were able to change the regio- and stereoselectivity of steroid hydroxylation. The replacement of only one amino acid of CYP11B1 by the amino acid occurring in CYP11B2 (V320A, N335D) enabled the mutant to form aldosterone indicating on a potential role of point mutations of CYP11B1 as a cause of hyperaldosteronism. Moreover, patients with defects in mitochondrial steroid hydroxylases have been studied and new molecular genetic defects were found. Mitochondrial cytochromes P450 get the electrons for oxygen activation and following substrate conversion from an iron-sulfur protein (adrenodoxin) and a FAD-containing reductase. To study the role of distinct amino acids of adrenodoxin in protein protein interaction and in electron transfer, mutants of adrenodoxin have been prepared by site-directed mutagenesis, expressed in E. coli, and their structural and functional properties have been characterised in detail. Different, but partially overlapping binding sites for the partner proteins have been found and mutants with improved electron transfer ability were obtained. For biotechnological purposes these results are being used to construct strains of the fission yeast with high ability to produce cortisol. contact: Prof. Dr. Rita Bernhardt Universität des Saarlandes Biochemie [email protected] PF 15 11 50 66041 Saarbrücken (BRD) Andreas Bichet, Frank Hannemann, Rita Bernhardt Using the yeast two-hybrid system as in vivo screening-system for interactions between adrenodoxin and its redoxpartners Adrenodoxin (Adx) is a [2Fe - 2S] ferredoxin involved in steroid hormone biosynthesis in the adrenal gland mitochondrial matrix of mammals. It is a small soluble protein that transfers electrons from adrenodoxin reductase (AR) to different cytochrome P450 isoforms where they are consumed in hydroxylation reactions. The yeast two-hybrid system is a technique to detect interactions between two proteins. It was developed to provide a genetic means of identifying proteins that physically interact in vivo. We adapted this system to our purposes so that it can be used as a screening-system for adrenodoxin mutants and cytochrome P450 mutants to detect proteins with increased interaction to their respective partners. Different adrenodoxin mutants were generated by site-directed mutagenesis which lead to better interactions between adrenodoxin and its redox partners. On the one hand these results are important to complete the knowledge of the electron transfer from Adx to its redox partners by showing the importance of specified amino acid residues for this transfer. On the other hand there is a direct correlation between interaction and steroid production showing that the results obtained might be of relevance for biotechnological production of steroids. Literature Fields et al., Nature 1989; 340: 245-246 Frederickson, Current Opinion in Biotechnology 1998; 9:90-96 Schiffler et al., The Journal of Biological Chemistry 2001; 276: 36225-36232 contact: Diplom Biologe Andreas Bichet Universität des Saarlandes Biochemie Ak Prof.Bernhardt [email protected] im Stadtwald, Gebäude 9 Postfach 151150 66041 Saarbrücken (Deutschland) Calin-Aurel Dragan, Rita Bernhardt, Matthias Bureik Production of steroid hormones by fission yeast cells expressing human mitochondrial cytochromes P450 Genetically engineered microorganisms are being increasingly used for the industrial production of complicated chemical compounds such as steroids; however, there have been few reports on the use of the fission yeast Schizosaccharomyces pombe for this purpose. Mitochondrial cytochrome P450 enzymes play a crucial role in the steroid biosynthesis in human adrenals, catalyzing regio- and stereo-specific hydroxylations. Two of these enzymes (CYP11B1 and CYP11B2) catalyze the last steps of cortisol and aldosterone biosynthesis, respectively. We have expressed CYP11B2 in S.pombe using the strong nmt1 promotor and found, that the transformed yeasts show in vivo the inducible ability to convert deoxycortisol to cortisol as well as deoxycorticosterone to aldosterone, resp.. The bioconversion activity of these strains is sufficiently high to consider their use for industrial applications. Although in mammalian cells mitochondrial P450 steroid hydroxylases depend for their activity on an electron transport chain that consists of the two proteins adrenodoxin and adrenodoxin reductase, no coexpression of these proteins is needed for efficient substrate conversion by intact fission yeast cells, since S.pombe contains the adrenodoxin-homologue etp1 (Bureik et al., 2002). In order to improve this bioconversion process, we optimized the reaction conditions and created a fission yeast expression vector (pCAD1) that allows targeted integration and the fusion of a polyhistidine and a Pk-tag, resp. (Dragan et al., submitted). Transformation of fission yeast with the new vector containing the human CYP11B1 yielded a new strain that displays an increase in cortisol production rate by more than two orders of magnitude. Literature Bureik, M., et al., Functional expression of human mitochondrial CYP11B2 in fission yeast and identification of a new internal electron transfer protein, etp1. Biochemistry, 2002. 41(7): p. 2311-21. contact: Dr. Matthias Bureik Universität des Saarlandes FR 8.8 Biochemie [email protected] Im Stadtwald, Geb. 9.2 66041 Saarbrücken (Deutschland) additional information http://bernhardt.biochem.uni-sb.de/ F. Peter Guengerich Mechanisms of Oxidations of Drugs and Carcinogens Catalyzed by Human P450s Cytochrome P450 (P450) enzymes are the major catalysts involved in the metabolism of drugs, carcinogens, and other xenobiotic chemicals. A relatively small subset (6-8) of the total group of P450s account for the bulk of these transformations and have been studied extensively in regard to drug development and chemical carcinogenesis. The oxidations may be understood in the context of a general paradigm involving odd-electron abstraction by a high valent iron-oxygen complex, probably a perferryl oxygen in many cases. These oxidations include not only some common reactions such as C-hydroxylations and epoxidation but also more complex processes such as desaturation and ring formation and expansion. Understanding and predicting the course of P450 reactions is the focus of considerable interest. Some insight into ligand binding can be gained through crystallography and modeling. Another issue is the discernment of rate-limiting steps in P450 reactions. Recent studies indicate that several steps can contribute, including events leading to accumulation of iron-oxygen complexes and events following product formation. In particular, the existence of non-competitive intermolecular kinetic hydrogen isotope effects in several cases indicates the role of rate-limiting C-H bond breaking. Random mutagenesis approaches have been used to develop human P450 enzymes with catalytic activities greater than the wild type P450s, which may be of use in characterizing kinetic aspects of P450s. Literature Guengerich, F.P. (2001) Chem. Res. Toxicol. 14, 611-650. "Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity." Guengerich, F.P., and Macdonald, T.L. (1984) Acct. Chem. Res. 17, 9-16. "Chemical mechanisms of catalysis by cytochromes P-450: a unified view." Guengerich, F.P., and Macdonald, T.L. (1990) FASEB J. 4, 2453-2459. "Mechanisms of cytochrome P-450 catalysis." Guengerich, F.P., and Macdonald, T.L. (1993) Advances in Electron Transfer Chemistry 3, 191-241. "Sequential electron transfer reactions catalyzed by cytochrome P-450 enzymes." contact: Professor F. Peter Guengerich Vanderbilt University Dept. of Biochemistry [email protected] 23rd & Pierce Avenues, 638 RRB 37232-0146 Nashville, TN (U.S.A.) Annette Deul, Susanne Stevens, Uta Sander, Christian Kirchhoff, Joern Jungmann, Thomas Hesterkamp Use of cytochrome P450 isoenzyme assays in the hit-to-lead process of drug discovery Drug-drug interactions at the level of cytochrome P450 (CYP) mediated drug metabolism are a frequent cause of failure of clinical candidates. Of outstanding medical relevance are the human CYP isoenzymes 1A2, 2C9, 2C19, 2D6, and 3A4. In order to routinely test inhibition of these isoenzymes during early stages of drug discovery, i.e. the hit-to-lead process, available fluorogenic assay principles were adapted to a medium throughput 384 well assay format. The novel assay format was validated using (i) known standard inhibitors for these isoenzymes and (ii) a library of 560 synthetic compounds made up of 20 different structural scaffolds with unknown interference with CYP activity. The good assay performance allowed for statistically significant correlations of inhibition results with coarse compound characteristics like clogP and molecular weight or volume. contact: Dr. Thomas Hesterkamp Evotec OAI AG [email protected] Schnackenburgallee 114 22525 Hamburg (Germany) Horst Honeck, Dominik N. Müller, Jürgen Theuer, Friedrich C. Luft, Wolf-H. Schunck, Eva Kärgel Cytochrome P450-Dependent Renal Metabolism of Arachidonic Acid in a Rat Model of Angiotensin II induced Hypertension and End-Organ Damage Metabolites produced by P450-dependent hydroxylation and epoxygenation of arachidonic acid (AA), such as 20-HETE and 11,12-EET, play an important role in the regulation of renal vascular tone and salt excretion. 11,12-EET may also have anti-inflammatory properties by inhibiting TNF±-induced NF-ºB activation. We compared AA-epoxygenase and hydroxylase activities in renal microsomes isolated from double transgenic rats (dTGR) overexpressing the human genes for renin and angiotensinogen and from normotensive Sprague-Dawley (SD) rats. Samples were prepared after the dTGR developed hypertension and renal damage (100 fold increased albuminuria), which is mainly caused by progressive inflammation in this model. A group of dTGR animals was treated with the peroxisome proliferator-activated receptor ± (PPAR±)- agonist fenofibrate. The present study shows that the P450-dependent AA-epoxygenation is downregulated in dTGR kidneys compared to SD controls. Western blotting revealed a strong decrease in the P450 2C11, 2C23 and 2J protein levels. Immunoinhibition studies identified P450 2C23 as the major AA- epoxygenase, both in dTGR and SD rats. Fenofibrate-treatment almost completely normalised blood pressure and abolished albuminuria. The AA-epoxygenase activity was strongly induced and, in parallel, the amount of immunodetectable P450 2C23 was increased. We propose, that the low 11,12-EET levels in dTGR may contribute to the uncontrolled inflammation featured in this model and that fenofibrate induced increases in P450 2C23-mediated 11,12-EET formation protects against angiotensin II-induced end-organ damage. contact: Dr. Eva Kärgel Max-Delbrück-Centrum [email protected] Robert-Rössle-Str. 10 13125 Berlin (Germany) André Förster, Jana Gerber, Thomas Juretzek, Gerold Barth, Stephan Mauersberger FUNCTIONAL EXPRESSION OF HETEROLOGOUS CYTOCHROME P450 IN YARROWIA LIPOLYTICA AND ITS USE IN BIOTRANSFORMATION OF STEROIDS High level expression of bovine P45017± (CYP17 cDNA) and of NADPH-P450 reductase (YlCPR gene) in haploid Y. lipolytica strains was obtained under control of the strong and regulated ICL1 promoter (inducible on ethanol and hydrocarbons) using multicopy integrative plasmids with ura3d4 as multicopy selection marker, LTR zeta of Ylt1 as integration target sequence, and expression cassettes [1]. Multicopy transformants (8-35 copies) were obtained with significantly increased P45017± content and CPR activity in correlation with the corresponding copy numbers. The P45017± expressed was found to be functionally highly active especially in alkane-growing cells, catalyzing the bioconversion of progesterone into 17±-hydroxy progesterone [2]. The overexpression of P45017± and CPR resulted in a strong proliferation of different ER types in Y. lipolytica (P450 - tubular network of ER membranes, karmellae-like stacks of up to 25 membranes, mostly in case of CPR overexpression). Selected haploid multicopy transformants and wild type strains were used to construct different lines of diploid strains containing multiple expression cassettes for P45017± only, or for both P45017± and CPR to perform co-expression. Selected strains were applied for P450 catalyzed steroid bioconversion using recombinant Y. lipolytica cells. Co-expression of P45017± and YlCPR in diploids resulted in an increased steroid bioconversion rate. Our results indicate significant advantages of the alkane-assimilating yeast Y. lipolytica as a host to perform P450 catalyzed biotransformation reactions with hydrophobic substrates in comparison with S. cerevisiae. Literature [1] Juretzek T, Le Dall M-T, Mauersberger S, Gaillardin C, Barth G, Nicaud J-M (2001) Vectors for gene expression and amplification in the yeast Yarrowia lipolytica. Yeast 18: 97-113 [2] Juretzek T, Prinz A, Schunck W-H, Barth G, Mauersberger S (1995, 1998) DE 19525282, DE 19932811A, and WO 000308. contact: Dr. Stephan Mauersberger Technische Universität Dresden Institut für Mikrobiologie [email protected] Mommsenstrasse 13 D-01062 Dresden (Germany) Kerstin Reichert, Matthias Rödel, Ralph Menzel Xenobiotically induced cytochrome P450 gene expression in Caenorhabditis elegans The nematode Caenorhabditis elegans possesses the remarkable number of 80 different cytochrome P450 genes. The functions of the encoding proteins are almost unknown. In order to study xenobiotically induced gene expression in C. elegans liquid cultures were exposed to different well-known xenobiotic inducers. The mRNA expression was detected by two different DNA arrays and semi-quantitative RT-PCR (1). In addition, using concentration-effect experiments, a classical reproduction test was compared with CYP gene expression screens. Beta-naphthoflavone, PCB52, Lansoprazol and Fluoranthene were the most active CYP inducers. They mainly induced almost all CYP35 isoforms. The reproduction test with nine different xenobiotics revealed Tributylzinn, Endosulfan and Fluoranthene as most toxic substances. The threshold of the semi-quantitative RT-PCR experiments showed a significant higher sensitivity than the reproduction tests. Obviously, the xenobiotic inducible gene expression of C. elegans is a very sensitive tool to reveal defense mechanisms against potential toxic substances and can be used to develop a biomonitoring system (2). Interestingly, the CYP35 isoforms promoter regions contain xenobiotic response elements (XRE) similar to mammalian CYP1 forms. A transgenic C. elegans line expressing GFP under control of the CYP35A2 promoter was constructed. With this tool it was possible to follow the GFP production in vivo depending on the added inducer. In the control worms a slight but distinct label was visible at the anterior and posterior part of the intestine. In beta-naphthoflavone treated worms the total gut emitted a very strong GFP fluorescence. Literature (1) Menzel, R., Bogaert T. and Achazi A. (2001) A systematical gene expression screen of the Caenorhabditis elegans cytochrome P450 genes revealed CYP35 as strong xenobiotic inducible. Arch. Biochem. Biophys. 395, 158-168 (2) Menzel, R., Reichert, K. and Achazi A. (2002) Nutzung der induzierbaren Genexpression des Nematoden Caenorhabditis elegans als Biomonitior. UWSF 14, 18-23. contact: Dr. Ralph Menzel Free University Berlin Institute of Biology - Ecotoxicology & Biochemistry [email protected] Ehrenbergstr. 26-28 10319 Berlin (Germany) Dietmar Pfeifer, Martina Kaufmann, Bernd Fiebich, Olivia Spleiss Pharmacogenetic profiling with Biochips: an innovative concept to improve the efficiency of clinical trials Due to their highly parallel nature, DNA chips are valuable tools for the simultaneous detection of known Single Nucleotide Polymorphisms (SNPs) in numerous genes. In the field of pharmacogenetics, SNPs with a well-described genotype-phenotype correlation in several drug metabolizing enzymes are known for quite a while, but their parallel detection was quite laborious in the past. GeneScan’s Pharm-O-Kin Chip was designed to meet the needs of Clinical research and Pharma customers as it presents a fast and reliable method to detect SNPs in several genes of pharmacogenetic relevance. In total, 39 polymorphisms from CYP2D6, CYP2C9 and CYP2C19 as well as NAT2 and MDR1 are represented on the chip, thus covering important enzymes from drug metabolism as well as a transporter protein known to play a role in the bioavailability of administered drugs. The high quality genotyping data of the Pharm-O-Kin Chip will be of great benefit for clinical trials in the future, as all the relevant polymorphisms known to result in variant phenotypes will be determined in one assay. This allows a better design of clinical trials in terms of minimizing the risks for probands and, in addition, will help to save costs in the drug development process. Literature Brockmoeller J., Kirchheiner J., Meisel C., Roots I. Pharmacogenetic diagnostics of cytochrome P450 polymorphisms in clinical drug development and in drug treatment. Pharmacogenomics 1(2): 125-151, 2000 Sachse C., Brockmoeller J., Bauer S., Roots I. Cytochrome P450 2D6 Variants in a Caucasian Population: Allele Frequencies and Phenotypic Consequences. Am.J.Hum.Genet. 60:284-295, 1997 Murphy M.P., Beaman M.E., Scott C., Cayouette M., Benson L., Morris D.M., Polli J.W. Prospective CYP 2D6 genotyping as an exclusion criterion for enrollment of a phase III clinical trial. Pharmacogenetics, 10:583-590, 2000 contact: Dr. Dietmar Pfeifer GeneScan Europe AG BU Diagnostics [email protected] Engesserstr.4 79108 Freiburg (Germany) additional information Adresse Dr. Bernd L. Fiebich Universitaetsklinik fuer Psychiatrie und Psychosomatik Abteilung fuer Psychiatrie und Psychotherapie mit Poliklinik Hauptstrasse 5, D-79104 Freiburg T. +49 761 270-6898/6501 Fax -6917 Tanja Richter, Kathrin Klein, Thomas E. Mürdter, Michel Eichelbaum, Matthias Schwab, Ulrich M. Zanger ThioTEPA and Clopidogrel are specific mechanism-based inhibitors of human CYP2B6 Human CYP2B6 catalyzes the metabolism of several drugs including cyclophosphamide and the antidepressant, bupropion. Investigation of this enzyme has long been hampered by the lack of suitable probes. The cytostatic ThioTEPA was recently shown to inhibit cyclophosphamide hydroxylation in vivo whereas the platelet aggregation inhibitor, clopidogrel, was suggested to be a substrate of CYP2B6. By measuring bupropion-hydroxylation in liver microsomes and in recombinant P450s, we found that these two substances strongly inhibit CYP2B6 in a time- and concentration-dependent manner by up to 90% and with IC50 values of about 5 and 0.5 µM, respectively. As shown by dialysis- and microfilter experiments, inhibition was irreversible and dependent on the presence of NADPH, strongly suggesting a mechanism-based mode of action for both thioTEPA and clopidogrel. High specificity of both inhibitors for CYP2B6 was demonstrated by analyzing enzyme activities selective for other human liver CYPs including ethoxycoumarin-O-dealkylation (CYP1A2 and CYP2E1), propafenon 5-hydroxylation (CYP2D6), coumarin 7-hydroxylation (CYP2A6), verapamil N-demethylation (CYP3A4/5), verapamil O-demethylation (CYP2C8), S-mephenytoin N-demethylation (CYP2C9) and S-mephenytoin 4'-hydroxylation (CYP2C19). These results suggest the possibility of drug interactions between these two drugs and other drug-substrates of CYP2B6. Clopidogrel in particular may prove to be a useful in-vitro probe for this enzyme. Supported by the DFG and by the Robert Bosch Foundation, Stuttgart. contact: Tanja Richter Dr. Margarete Fischer-Bosch Institut für Klinische Pharmakologie [email protected] Auerbachstr. 112 70376 Stuttgart (Deutschland) Ivar Roots, Julia Kirchheiner, Jürgen Brockmöller, Christian Meisel, Kersti Oselin, Thomas Gerloff Pharmacogenetic studies on drug-metabolizing cytochromes P450 and possibilities for individual drug therapy Drug metabolism and elimination is largely determined by functionally relevant cytochrome P450 genetic variants the most important of which are the polymorphic enzymes CYP2C9, CYP2C19, and CYP2D6. We studied the impact of the CYP2C9 polymorphisms on the pharmacokinetics of characteristic substrates in healthy volunteers. In carriers of the CYP2C9 *3/*3 genotype, clearances were one third (celecoxib, glibenclamide), one half (ibuprofen), and one sixth (tolbutamide) of those of the wild-type. Large inter-individual differences in pharmacokinetics are not necessarily reflected in pharmacodynamics, as tolbutamide-mediated insulin secretion did not differ between CYP2C9 genotypes, at least not in healthy volunteers. Many antipsychotics are metabolized by CYP2D6 and CYP2C19. From published studies with either patients or healthy volunteers, we derived dose-related parameters (clearance, AUC, trough concentrations) in order to calculate genotype-adapted dosages which are based on the principles of bioequivalence (1). Doses for carriers of the deficiency (PM) were often 50% or less of those in wild-type. PMs may have high plasma concentrations of drugs and run the risk of adverse effects and toxicity. Two percent of the German population carry a gene dublication of CYP2D6 and run the risk of therapeutic failure. Genotype-adapted dosage could reduce these risks, especially when the drug has a narrow therapeutic window. Literature 1) Kirchheiner J et al. Acta Psychiatr Scand 2001; 104: 173-92 contact: Prof. Dr. med. Ivar Roots Universitätsklinikum Charité, Humboldt-Universität zu Berlin Institut für Klinische Pharmakologie [email protected] Schumannstr. 20/21 10098 Berlin (D) additional information 3) Jürgen Brockmöller, Prof. Dr. med. Georg August Universität Göttingen Abteilung für Klinische Pharmakologie Robert-Koch-Str. 40 37075 Göttingen Ilme Schlichting Crystallographic studies on the reaction mechanism of P450 Cytochrome P450 enzymes form a family of ubiquitous heme proteins named after an absorption band at 450 nm when complexed to carbon monoxide (CO). P450 enzymes are mixed-function mono-oxygenases. They play a critical role in the synthesis and degradation of many physiologically important compounds and xenobiotics. This makes cytochrome P450s an attractive target for pharmaceutical or environmental industries. The biochemical relevance of the P450mono-oxygenases in general is their unique ability to catalyze the hydroxylation of non-activated aliphatic or aromatic hydrocarbons. We use conventional and kinetic crystallography to analyze the structural framework that allows this unusal chemistry. We are particularly interested in describing structures occuring along the reaction pathway to obtain insight in the enzymatic mechanism. Recent results will be described. contact: Dr. Ilme Schlichting MPI für med. Forschung Abteilung Biomolekulare Mechanismen [email protected] Jahnstr. 29 69120 Heidelberg (D) Birgit Lauterbach, Eduardo Barbosa, Eva Kaergel, Horst Honeck, Jürgen Theuer, Hermann Haller, Friedrich C. Luft, Maik Gollasch, Wolf-Hagen Schunck Cytochrome P450-dependent eicosapentaenoic acid epoxygenation produces novel vasoactive metabolites In the vasculature, CYP2C, 2J and 4A enzymes catalyze the formation of epoxyand hydroxy-derivatives of arachidonic acid (AA) which are important mediators in the regulation of vascular tone. Because eicosapentaenoic acid (EPA) has been reported to improve vascular function, we tested the hypothesis that P450-dependent EPA metabolism generates alternative vasoactive compounds. Using recombinant CYP2C and 2J enzymes, we found that EPA is metabolized to isoform-specific sets of regioisomeric epoxyeicosatetraenoic acids (EETeTrs) which involve 17,18-EETeTr as major product. CYP4A enzymes catalyzed 19- and 20-hydroxylation of EPA and produced, in addition, significant amounts of 17,18-EETeTr. With all P450 isoforms tested, the catalytic efficiencies were higher or at least equal comparing EPA and AA as substrates (see the contribution of E. Barbosa Sicard et al.). To test the physiological potential of the EPA epoxides, we examined them for effects on the calcium-activated potassium (BK) channel in rat cerebral artery vascular smooth muscle cells. The most potent AA metabolite, 11(R),12(S)-epoxyeicosatrienoic acid (50 nM), was used for comparison and stimulated the K+ current 6-fold at a membrane potential of 60 mV. This effect was exceeded by the EPA metabolite 17(R),18(S)-EETeTr which elicited a more than 14-fold increase under the same conditions. Indicating a high degree of stereo- and regiospecificity, the respective S,R enantiomer and the remaining four regioisomeric EETeTrs were almost inactive. We conclude that 17(R),18(S)-EETeTr represents a novel, potent activator of BK channels and that its P450-catalyzed formation may contribute to the beneficial effects on vascular function attributed to EPA-rich diets. contact: Dr. Wolf-Hagen Schunck Max Delbrueck Centre for Molecular Medicine [email protected] Robert-Roessle-Str. 10 13092 Berlin (Germany) Hannemann Frank, Bernhardt Rita, Zearo Silvia Activities of CYP11B isoforms from different species. Cytochromes P450 of the CYP11B family are involved in the final steps of glucocorticoid and mineralocorticoid synthesis. In humans CYP11B1 catalyses the production of cortisol and CYP11B2 the synthesis of aldosterone. In many other mammals similar enzymes were found, which catalyze the same reactions. The alignment of the DNA sequences shows high similarity (between 70% and 95%) among the members of the CYP11B family. But the enzyme activities of the various species are found to be different. For a better understanding of the structural basis of the selectivity of CYP11B- dependent reactions we tried to clone and expressed CYP11B genes from various mammalian species (human, monkey, cow, rat and guinea pig) under the same conditions. For the expression we always used the same pNMT1-TOPO vector. With this vector we transformed S. pombe and then expressed the various CYP11Bs proteins. Because of a recently discovered electron transfer protein in S. pombe this cellular system can also be used as a new model for the comparison of the enzyme activity of CYP11B family members. Together with data from computer modelling of the 3D-structure this system might help to get new information about the relationship between sequence, structure and function of CYP11B proteins. Literature Bernhardt R., Rev Physiol Biochem Pharmacol. 1996;127:137-221. Review. Bureik M., et al., Biochemistry. 2002 Feb 19;41(7):2311-21. Hampf M, et al., Endocr Res. 1996 Nov;22(4):495-9 Nomura M, et al., J Biochem (Tokyo). 1993 Feb;113(2):144-52. Nonaka Y, et al., J Steroid Biochem Mol Biol. 1992 Mar;41(3-8):779-80. Bulow HE, et al., Biochem Biophys Res Commun. 1996 Apr 16;221(2):304-12. Denner K, et al., Endocr Res. 1995 Feb-May;21(1-2):443-8. Bechtel S, et al., Eur J Biochem. 2002 Feb;269(4):1118-27. Belkina NV, et al., J Inorg Biochem. 2001 Dec 15;87(4):197-207. Maundrell K., J Biol Chem. 1990 Jul 5;265(19):10857-64. contact: Diplom Biologin Zearo Silvia Universität des Saarlandes Biochemie [email protected] Im Stadtwald, Gebäude 9, Postfach 151150 66041 Saarbrücken (Deutschland) Ulrich M. Zanger, Kathrin Klein, Tanja Richter, Thomas E. Mürdter, Ute Hofmann, Thomas Lang, Michel Eichelbaum, Matthias Schwab FUNCTIONAL SIGNIFICANCE OF GENETIC POLYMORPHISMS OF HUMAN CYP2B6 CYP2B6 is the human ortholog to the rat phenobarbital-inducible CYP2B1. In contrast to other human P450s, CYP2B6 has not been well investigated, due to initially underestimated expression and lack of specific probes. However, identification of several clinically important drug-substrates including cyclophosphamide, bupropion, and propofol increased the interest in this enzyme. We recently found that the CYP2B6 gene is highly polymorphic in the population with at least 6 mutant alleles which occur at frequencies between 0.5% and over 30% and which differ with respect to protein expression in human liver samples (Lang et al., Pharmacogenetics 11:399-415, 2001; http://www.imm.ki.se/CYPalleles/). We now extended our studies by using newly established LC-MS assays based on deuterated metabolite standards synthesized for the two CYP2B6-selective drug-substrates, S-mephenytoin and bupropion. Measurement of enzyme activities in human liver microsomes demonstrated functional differences between genotypes and indicated surprising features of some alleles. In particular, the results indicate a functional impairment of allele CYP2B6*2 (R22C), which shows quite normal expression in human livers, whereas allele CYP2B6*5 (R487C) appears to partially compensate low expression by higher specific activity. Extensive further characterization of recombinant enzyme variants in insect cells is currently in progress. Supported by BMBF (grant 01GG9846) and by the Robert Bosch Foundation, Stuttgart. contact: Priv.-Doz. Dr. Ulrich M. Zanger Dr. Margarete Fischer-Bosch-Institut für Klinische Pharmakologie [email protected] Auerbachstr. 112 70376 Stuttgart (Baden-Württemberg)