1. Research Program ”Systems of Life-Systems Biology”
A Systems Biology Approach to Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Coordination: Matthias Reuss Institute of Biochemical Engineering University of Stuttgart

2. Leiter Molekular- und Zellbiologie
PD Dr. Ulrich M. Zanger
Leiter Molekular- und Zellbiologie
Dr. Margarete Fischer-Bosch-Institut
für Klinische Pharmakologie
Robert Bosch Stiftung, Stuttgart
Direktor: Prof. Dr. M. Eichelbaum
Forschungsschwerpunkte:
Arzneimittelmetabolismus
Pharmakogenetik
Mission:
Individualisierte Pharmakotherapie

3. Project Partners
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

4. Biotransformation of Foreign Substances
Uptake
Metabolism O H
Phase I: Functionalisation
Cytochrome P450 (~20 CYPs)
Oxidases (z.B. MAO)
Dehydrogenases (z.B. ADH)
Esterases (z.B. Carboxylesterasen)
Hydrolases (z.B. mEH)
Phase II: Conjugation
UDP-Glucuronosyltransferases (UGT)
N-Acetyltransferases (NAT)
Glutathion-S-Transferases (GST)
Methyltransferases (COMT, TPMT)
Sulfotransferases (ST) O R
Elimination
Phase III: Transport
P-Glykoprotein (MDR1, MDR2)
Multi-Drug Resistance Proteins (MRP)
Organic Anion Transporters (OATP)
Organic Cation Transporters (OCT)
etc.

5. Oxidative Drug Metabolism by the Cytochrome P450 System
CYP1A2
Clozapine
Caffeine
Phenacetin
CYP2B6
Bupropion
Cyclophosphamid
Clopidogrel
Propofol
CYP3A4/5
Amitriptyline
Carbamazepine
Clarithromycin
Cyclosporin
Lignoscaine
Midazolam
Nifedipine
Terfenadine
CYP2C9
Diclofenac
Ibuprofen
Losartan
Phenytoin
Tolbutamide
Warfarin
CYP2C19
Diazepam
Omeprazol
Proguanil
S-Mephenytoin
CYP2E1
Chlorzoxazon
Ethanol
Halothan
CYP2D6
Clomipramine
Codeine
Fluoxetine
Metoprolol
Propafenone
Tamoxiphen

6. Complexity of BiotransformationsH 3
2C8
2C9
2C19
P450
1A2
3A4
3A5

7. Complexity of BiotransformationsH 3
2C8
2C9
2C19
P450
1A2
3A4
3A5

8. Complexity of BiotransformationsH 3
2C8
2C9
2C19
P450
1A2
3A4
3A5

9. 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)

10. Regulatory and
Signaling Network
(Gene Expression)
Drugs
Intermediates
Metabolites
Endproducts
Transport
Phase I
Transport
Phase II
NADPH
NADH
UDP - G
PAPS
GSH
AminoA
...
Central
Metabolism

11. A S B Holistic Description of Cellular FunctionsY T E M B I O L G
Holistic Description of Cellular Functions
Connection
of Moduls
Modular Aggregation
of Components
Analysis of Single Components
Holistic Functional Analysis:
Metabolic Networks
Regulatory Networks
Signalling Networks
Biological Information/Knowledge
Deductive
Inductive A

12. Project Section A Xenobiotic Metabolism and Transport:
Analysis, mathematical modeling and
simulation of the xenobiotic-metabolizing
system of the liver

13. 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 reactions
for functionalization and conjugation
Simulations of different individual situations regarding both
quantitative (enzyme expression levels) and qualitative differences
(polymorphism)
Simulation of regulation processes (induction)

14. 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

15. Human Liver Bank as a Tool
Diagnosis
Demogr. Data
Drugs
Nic & Alc
Life Style
Clinical Documentation:
Metabolic Pathways
Identification of Metabolites and Responsible Enzymes
Variability of Expression
Genotype-Phenotype Relationships
Regulatory Networks
N > 300
Protein Fractions:
expression function, kinetics
RNA:
transkripts
splicing variants
DNA:
polymorphisms
genotypes

16. Human Hepatocyte Models
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

17. Recombinant Expression Systems
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)

18. Project Section B Hepatic Differentiation and Dedifferentiation
Processes:
Holistic analysis of metabolic networks,
regulatory networks, signalling networks

19. INSILICO Biotechnology GmbH, Stuttgart
Global transcriptome analysis
Metabolite measurements (LC-MS)
Flux analysis based on labeling experiments (GC-MS)
Bioreactor cultivation
Modeling and simulation platform INSILICO discovery
TECAN Proteomics GmbH, München
Proteome analysis (automated global protein analysis)
Free-flow-electrophoresis for enrichment of rare proteins
Membrane proteins, phosphorylation patterns etc.

20. Projektbereich A Projektbereich B Projektbereich Z S B InductiveY T E M B I O L G
Biological Information/Knowledge
Inductive
Deductive
Projektbereich B
Projektbereich A
Flussverteilungen
(Genomweite Zellmodelle)
(Reverse Engineering)
Geplante Aktivitäten für
die 2. und weitere Förderphasen
Anbindung Datenbank
Genetische Polymorphismen
Proteinmodellierung und Docking
Kinetik der
Detoxifikationsschritte
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 und
Datenbanken
Projektkoordination

21. Drug/Xenobiotics Products
lipophilic
polar
Transport
Phase I
Phase II
Substrate is S-Mephenytoin. Protein is CYP2B6. Reaction is N-Demethylation.
Transport
Products

22. Drug/Xenobiotics Products
lipophilic
polar
Transport
Phase I
Phase II
Substrate is S-Mephenytoin. Protein is CYP2B6. Reaction is N-Demethylation.
Transport
Products

23. Drug/Xenobiotics Products
lipophilic
polar
Transport
Phase I
Phase II
Substrate is S-Mephenytoin. Protein is CYP2B6. Reaction is N-Demethylation.
Transport
Products

24. Drug/Xenobiotics Products
lipophilic
polar
Transport
Phase I
Phase II
Substrate is S-Mephenytoin. Protein is CYP2B6. Reaction is N-Demethylation.
Transport
Products

25. Drug/Xenobiotics Products
lipophilic
polar
Transport
Phase I
Phase II
Substrate is S-Mephenytoin. Protein is CYP2B6. Reaction is N-Demethylation.
Transport
Products