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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
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Bulow HE, et al., Biochem Biophys Res Commun. 1996 Apr 16;221(2):304-12.
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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)