detoxification pathways and their cellular and structural requirements in hepatocytes research...
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Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Research Program ”Systems of Life-Systems Biology”
A Systems Biology Approach to Detoxification Pathways and their
Cellular and Structural Requirementsin Hepatocytes
Coordination: Matthias ReussInstitute of Biochemical Engineering University of Stuttgart
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Direktor: Prof. Dr. M. Eichelbaum
Forschungsschwerpunkte:ArzneimittelmetabolismusPharmakogenetik
Mission:Individualisierte Pharmakotherapie
Dr. Margarete Fischer-Bosch-Institutfür Klinische Pharmakologie
Robert Bosch Stiftung, Stuttgart
PD Dr. Ulrich M. ZangerLeiter Molekular- und Zellbiologie
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
• Bader
Biomedizinisch-Biotechnologisches Zentrum (BBZ), University of Leipzig• Dauner
INSILICO biotechnology GmbH, Stuttgart• Eckerskorn
TECAN proteomics GmbH, München• Gasteiger
Computer Chemistry Center (CCC), University of Erlangen-Nürnberg• Reuss
Institute of Biochemical Engineering (IBVT), University of Stuttgart• Schmid
Institute of Technical Biochemistry (IBT), University of Stuttgart• Zanger & Eichelbaum
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology (IKP), Stuttgart
Project Partners
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Biotransformation of Foreign Substances
Phase I: FunctionalisationCytochrome P450 (~20 CYPs)Oxidases (z.B. MAO)Dehydrogenases (z.B. ADH)Esterases (z.B. Carboxylesterasen)Hydrolases (z.B. mEH)
Phase II: ConjugationUDP-Glucuronosyltransferases (UGT)N-Acetyltransferases (NAT)Glutathion-S-Transferases (GST)Methyltransferases (COMT, TPMT)Sulfotransferases (ST)
Phase III: TransportP-Glykoprotein (MDR1, MDR2)Multi-Drug Resistance Proteins (MRP)Organic Anion Transporters (OATP)Organic Cation Transporters (OCT)etc.
OH
Elimination
MetabolismUptake
OR
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
CYP2B6BupropionCyclophosphamidClopidogrelPropofol..........
CYP1A2ClozapineCaffeinePhenacetin..........
CYP3A4/5AmitriptylineCarbamazepineClarithromycinCyclosporinLignoscaineMidazolamNifedipineTerfenadine.......
CYP2E1ChlorzoxazonEthanolHalothan..........
CYP2D6ClomipramineCodeineFluoxetineMetoprololPropafenoneTamoxiphen........
Oxidative Drug Metabolism by the Cytochrome P450 System
CYP2C9DiclofenacIbuprofenLosartanPhenytoinTolbutamideWarfarin...........
CYP2C19DiazepamOmeprazolProguanilS-Mephenytoin........
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Complexity of Biotransformations
2C9
2C19
2C8
1A2
3A5
3A4
N
OCH3OCH3
OCH3CH3O
CN
H
N
OCH3OCH3
OCH3HO
CN
CH3
N
OCH3OH
OCH3CH3O
CN
CH3
NH
OCH3CH3O
CN
CH3
P450
N
OCH3O
OCH3CH3O
CN
CH3
CH3
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Complexity of Biotransformations
2C9
2C19
2C8
1A2
3A5
3A4
N
OCH3OCH3
OCH3CH3O
CN
H
N
OCH3OCH3
OCH3HO
CN
CH3
N
OCH3OH
OCH3CH3O
CN
CH3
NH
OCH3CH3O
CN
CH3
P450
N
OCH3O
OCH3CH3O
CN
CH3
CH3
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Complexity of Biotransformations
2C9
2C19
2C8
1A2
3A5
3A4
N
OCH3OCH3
OCH3CH3O
CN
H
N
OCH3OCH3
OCH3HO
CN
CH3
N
OCH3OH
OCH3CH3O
CN
CH3
NH
OCH3CH3O
CN
CH3
P450
N
OCH3O
OCH3CH3O
CN
CH3
CH3
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Variability in Biotransformations: Major Cause of Unexpected Drug Response
• Genetic Polymorphisms in Enzymes, Transporters, Receptors
• Reversible and Irreversible Inhibition (drug interactions)
• Regulation of Gene Expression by Xenobiotics (induction)
• Regulatory Networks (e.g. cholesterol homeostasis)
• Hormonal Regulation (e.g. sexual dimorphism)
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Tra
ns
po
rt
Drugs
Phase II
UDP - GPAPSGSHAminoA...Central
Metabolism
Tra
ns
po
rt
Regulatory andSignaling Network(Gene Expression)
Phase I
NADPHNADH
Intermediates
Metabolites
Endproducts
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
SYSTEMS BIOLOGY
Holistic Description of Cellular Functions
Connectionof Moduls
Modular Aggregationof Components
Analysis of Single Components
Holistic Functional Analysis:
Metabolic NetworksRegulatory NetworksSignalling Networks
Biological Information/Knowledge
Deductive
InductiveA
B
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Project Section A
Xenobiotic Metabolism and Transport:
Analysis, mathematical modeling andsimulation of the xenobiotic-metabolizing
system of the liver
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Quantitative experimental analysis of metabolism using model substrates to determine kinetic parameters in recombinant systems (ITB), human liver tissue (IKP) and in primary hepatocytes (BBZ)
Structure modeling at the molecular level including chemicals (CCC)as well as proteins (ITB) and their interactions
Mathematical modeling of the biotransformation system by integrating experimental data and structure models (IBVT)
Dynamic simulation of the most important metabolic reactionsfor functionalization and conjugation
Simulations of different individual situations regarding bothquantitative (enzyme expression levels) and qualitative differences(polymorphism)
Simulation of regulation processes (induction)
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Biological Model Systems
• Human Liver Tissue Bank with Clinical Documentation (N>300)
quantitative data on variability of function, protein, mRNA,
polymorphisms
• Human Hepatocytes in Primary Culture
dynamics of metabolism and transport, all aspects of
regulation
• Recombinant Proteins
substrate selectivities and kinetic parameters of individual
components
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
• Metabolic Pathways• Identification of Metabolites and
Responsible Enzymes• Variability of Expression• Genotype-Phenotype Relationships• Regulatory Networks
• Metabolic Pathways• Identification of Metabolites and
Responsible Enzymes• Variability of Expression• Genotype-Phenotype Relationships• Regulatory Networks
• Diagnosis• Demogr. Data • Drugs• Nic & Alc• Life Style
• Diagnosis• Demogr. Data • Drugs• Nic & Alc• Life StyleN > 300
RNA:transkripts
splicing variants
DNA:polymorphisms
genotypes
Protein Fractions:expression function, kinetics
t [min]
µV
N
O C H3
O
O C H3
C H3
O
C N
C H3
C H3
Human Liver Bank as a Tool
Clinical Documentation:
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
• Organotypical culture model • Membrane / sandwich reactor (Bader, BBZ)
• Microcarriers (INSILICO)
Objectives:
• Kinetics of overall biotransformation (model substrates)
• Dynamics of regulation processes (e.g. induction)
• Global analysis of cellular changes associated with regulation processes
Human Hepatocyte Models
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Recombinant Expression Systems
NADPH + H+ NADPH-P450-
reductase
FADFMN Fe+++
NADP+
2x1e-
O2 H2O OH
CytochromeP450
endoplasmic reticulum membrane lumen
cytoplasm
The Cytochrome P450 Monooxygenase System
2e-
• Various yeast strains (Schmid, ITB)
• Baculovirus / insect cells (Zanger, IKP)
Objectives:
• Kinetic parameters of individual proteins by coexpressing P450-reductase and cytochrome b5
• Analysis and modeling of protein-protein interactions by reconstitution of purified components (cooperation with Rebecca Wade, EML Heidelberg)
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Project Section B
Hepatic Differentiation and Dedifferentiation Processes:
Holistic analysis of metabolic networks, regulatory networks, signalling networks
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
• Global transcriptome analysis • Metabolite measurements (LC-MS)• Flux analysis based on labeling experiments (GC-MS)• Bioreactor cultivation• Modeling and simulation platform INSILICO discovery
INSILICO Biotechnology GmbH, Stuttgart
• Proteome analysis (automated global protein analysis)• Free-flow-electrophoresis for enrichment of rare proteins• Membrane proteins, phosphorylation patterns etc.
TECAN Proteomics GmbH, München
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
SYSTEMS BIOLOGY
Biological Information/Knowledge
Inductive
Deductive
Projektbereich B
Projektbereich A
Flussverteilungen
(Genomweite Zellmodelle)
(Reverse Engineering)
Geplante Aktivitäten für
die 2. und weitere Förderphasen Anbindung DatenbankGenetische Polymorphismen
Proteinmodellierung und Docking
Kinetik derDetoxifikationsschritte
Untersuchungen zur Regulation
der Genexpression
Anbindung Reaktionsdatenbank
und Modell zur Vorhersage
des Metabolismus
Mathematische Modellierung und
dynamische Simulation des
Fremdstoffabbaus (Aggregation
der Einzelschritte)
DNA-Arrays
Proteomics
Metabolomics
Projektbereich Z
Primäre Zellkulturen
Leberbank
Zellbiologie
Modellierungswerkzeug undDatenbanken
Projektkoordination
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Drug/Xenobiotics
Phase I
Phase II
lipophilic polar
Transport
Transport
Products
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Drug/Xenobiotics
Phase I
Phase II
lipophilic polar
Transport
Transport
Products
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Drug/Xenobiotics
Phase I
Phase II
lipophilic polar
Transport
Transport
Products
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Drug/Xenobiotics
Phase I
Phase II
lipophilic polar
Transport
Transport
Products