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

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

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

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.

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 ........

Complexity of Biotransformations H 3 2C8 2C9 2C19 P450 1A2 3A4 3A5

Complexity of Biotransformations H 3 2C8 2C9 2C19 P450 1A2 3A4 3A5

Complexity of Biotransformations H 3 2C8 2C9 2C19 P450 1A2 3A4 3A5

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)

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

A S B Holistic Description of Cellular Functions Y 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

Project Section A Xenobiotic Metabolism and Transport: Analysis, mathematical modeling and simulation 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 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)

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

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

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

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)

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

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.

Projektbereich A Projektbereich B Projektbereich Z S B Inductive Y 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

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

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

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

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

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