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
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
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
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
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
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Biotransformation of Foreign Substances
Uptake
Metabolism
OH
Phase I: Functionalisation
Cytochrome P450 (~20 CYPs)
Oxidases (z.B. MAO)
Dehydrogenases (z.B. ADH)
Esterases (z.B. Carboxylesterasen)
Hydrolases (z.B. mEH)
OR
Elimination
Phase II: Conjugation
UDP-Glucuronosyltransferases (UGT)
N-Acetyltransferases (NAT)
Glutathion-S-Transferases (GST)
Methyltransferases (COMT, TPMT)
Sulfotransferases (ST)
Phase III: Transport
P-Glykoprotein (MDR1, MDR2)
Multi-Drug Resistance Proteins (MRP)
Organic Anion Transporters (OATP)
Organic Cation Transporters (OCT)
etc.
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Oxidative Drug Metabolism by the Cytochrome P450 System
CYP1A2
CYP3A4/5
Amitriptyline
Carbamazepine
Clarithromycin
Cyclosporin
Lignoscaine
Midazolam
Nifedipine
Terfenadine
.......
CYP2E1
Chlorzoxazon
Ethanol
Halothan
..........
Clozapine
Caffeine
Phenacetin
..........
CYP2B6
Bupropion
Cyclophosphamid
Clopidogrel
Propofol
..........
CYP2C9
CYP2C19
Diazepam
CYP2D6 Omeprazol
Proguanil
Clomipramine
S-Mephenytoin
Codeine
........
Fluoxetine
Metoprolol
Propafenone
Tamoxiphen
........
Diclofenac
Ibuprofen
Losartan
Phenytoin
Tolbutamide
Warfarin...........
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Complexity of Biotransformations
OCH3
OCH3
OCH3
CH3O
CN
OCH3
N
CH3
OCH3
OH
CH3O
CN
N
CH3
2C8
2C9
2C19
OCH3
OCH3
OCH3
HO
CN
N
CH3
P450
1A2
OCH3
CH3O
CN
3A4
H
N
CH3
3A5
OCH3
OCH3
OCH3
CH3O
CN
N
H
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Complexity of Biotransformations
OCH3
OCH3
OCH3
CH3O
CN
OCH3
N
CH3
OCH3
OH
CH3O
CN
N
CH3
2C8
2C9
2C19
OCH3
OCH3
OCH3
HO
CN
N
CH3
P450
1A2
OCH3
CH3O
CN
3A4
H
N
CH3
3A5
OCH3
OCH3
OCH3
CH3O
CN
N
H
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Complexity of Biotransformations
OCH3
OCH3
OCH3
CH3O
CN
OCH3
N
CH3
OCH3
OH
CH3O
CN
N
CH3
2C8
2C9
2C19
OCH3
OCH3
OCH3
HO
CN
N
CH3
P450
1A2
OCH3
CH3O
CN
3A4
H
N
CH3
3A5
OCH3
OCH3
OCH3
CH3O
CN
N
H
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
Regulatory and
Signaling Network
(Gene Expression)
Drugs
Metabolites
Endproducts
Phase I
NADPH
NADH
Central
Metabolism
Transport
Transport
Intermediates
Phase II
UDP - G
PAPS
GSH
AminoA
...
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
SYSTEMS BIOLOGY
Holistic Description of Cellular Functions
A
Holistic Functional Analysis:
Inductive
Connection
of Moduls
Metabolic Networks
Regulatory Networks
Signalling Networks
Modular Aggregation
of Components
Deductive
Analysis of Single Components
Biological Information/Knowledge
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
B
Project Section A
Xenobiotic Metabolism and Transport:
Analysis, mathematical modeling and
simulation 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 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)
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
Human Liver Bank as a Tool
• Metabolic Pathways
• Identification of Metabolites and
Responsible Enzymes
• Variability of Expression
• Genotype-Phenotype Relationships
• Regulatory Networks
Clinical Documentation:
• Diagnosis
• Demogr. Data
• Drugs
• Nic & Alc
N > 300
• Life Style
Protein Fractions:
expression
function, kinetics
RNA:
DNA:
transkripts
splicing variants
polymorphisms
genotypes
µV
O C H
C H
3
O C H
3
O
3
O C H
3
C N
N
C H
3
t [min]
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
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
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
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)
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
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.
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Projektbereich A
Kinetik der
Detoxifikationsschritte
Projektbereich B
Untersuchungen zur Regulation
der Genexpression
Mathematische Modellierung und
dynamische Simulation des
Fremdstoffabbaus (Aggregation
der Einzelschritte)
Anbindung Reaktionsdatenbank
und Modell zur Vorhersage
des Metabolismus
Proteinmodellierung und Docking
Anbindung Datenbank
Genetische Polymorphismen
DNA-Arrays
Proteomics
Metabolomics
S YSTEMS BIOLOGY
Deductive
Inductive
Flussverteilungen
(Genomweite Zellmodelle)
(Reverse Engineering)
Biological Information/Knowledge
Geplante Aktivitäten für
die 2. und weitere Förderphasen
Projektbereich Z
Zellbiologie Primäre Zellkulturen
Leberbank
Modellierungswerkzeug und
Datenbanken
Projektkoordination
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Drug/Xenobiotics
lipophilic
polar
Transport
Phase I
Phase II
Transport
Products
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Drug/Xenobiotics
lipophilic
polar
Transport
Phase I
Phase II
Transport
Products
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Drug/Xenobiotics
lipophilic
polar
Transport
Phase I
Phase II
Transport
Products
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Drug/Xenobiotics
lipophilic
polar
Transport
Phase I
Phase II
Transport
Products
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes
Drug/Xenobiotics
lipophilic
polar
Transport
Phase I
Phase II
Transport
Products
Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes