research program ”systems of life-systems biology”

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


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


• 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


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


CYP2B6BupropionCyclophosphamidClopidogrelPropofol..........

CYP1A2ClozapineCaffeinePhenacetin..........

CYP3A4/5AmitriptylineCarbamazepineClarithromycinCyclosporinLignoscaineMidazolamNifedipineTerfenadine.......

CYP2E1ChlorzoxazonEthanolHalothan..........

CYP2D6ClomipramineCodeineFluoxetineMetoprololPropafenoneTamoxiphen........

Oxidative Drug Metabolism by the Cytochrome P450 System

CYP2C9DiclofenacIbuprofenLosartanPhenytoinTolbutamideWarfarin...........

CYP2C19DiazepamOmeprazolProguanilS-Mephenytoin........


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


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)


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


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


Project Section A

Xenobiotic Metabolism and Transport:

Analysis, mathematical modeling andsimulation of the xenobiotic-metabolizing

system of the liver


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)


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


• 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:


• 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


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)


Project Section B

Hepatic Differentiation and Dedifferentiation Processes:

Holistic analysis of metabolic networks, regulatory networks, signalling networks


• 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


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


Drug/Xenobiotics

Phase I

Phase II

lipophilic polar

Transport

Transport

Products

research program ”systems of life-systems biology”

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