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Research Program ”Systems of Life-Systems Biology”

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. PD Dr. Ulrich M. Zanger

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Research Program ”Systems of Life-Systems Biology”

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  1. 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

  2. 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 • BaderBiomedizinisch-Biotechnologisches Zentrum (BBZ), University of Leipzig • DaunerINSILICO biotechnology GmbH, Stuttgart • EckerskornTECAN proteomics GmbH, München • GasteigerComputer Chemistry Center (CCC), University of Erlangen-Nürnberg • ReussInstitute of Biochemical Engineering (IBVT), University of Stuttgart • SchmidInstitute of Technical Biochemistry (IBT), University of Stuttgart • Zanger & EichelbaumDr. Margarete Fischer-Bosch Institute of Clinical Pharmacology (IKP), Stuttgart

  4. H O R O Biotransformation of Foreign Substances Uptake Metabolism 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) 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. O C H O C C H H 3 3 3 C H O O 3 C N N C H 3 Complexity of Biotransformations 2C8 2C9 2C19 P450 1A2 3A4 3A5

  7. O C H O C C H H 3 3 3 C H O O 3 C N N C H 3 Complexity of Biotransformations 2C8 2C9 2C19 P450 1A2 3A4 3A5

  8. O C H O C C H H 3 3 3 C H O O 3 C N N C H 3 Complexity of Biotransformations 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 Phase I Transport Transport Phase II NADPH NADH UDP - G PAPS GSH AminoA ... Central Metabolism

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

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

  13. Structure modeling at the molecular level including chemicals (CCC) as well as proteins (ITB) and their interactions 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) 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. Protein Fractions: expression function, kinetics RNA: transkripts splicing variants DNA: polymorphisms genotypes Human Liver Bank as a Tool • Diagnosis • Demogr. Data • Drugs • Nic & Alc • Life Style • Metabolic Pathways • Identification of Metabolites and Responsible Enzymes • Variability of Expression • Genotype-Phenotype Relationships • Regulatory Networks Clinical Documentation: N > 300

  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 Mathematische Modellierung und dynamische Simulation des Fremdstoffabbaus (Aggregation der Einzelschritte) Projektbereich B Anbindung Reaktionsdatenbank und Modell zur Vorhersage des Metabolismus DNA-Arrays Proteomics Metabolomics S B Y S T E M S I O L O G Y Untersuchungen zur Regulation der Genexpression Flussverteilungen Deductive Kinetik der Detoxifikationsschritte (Genomweite Zellmodelle) (Reverse Engineering) Inductive Proteinmodellierung und Docking Biological Information/Knowledge Geplante Aktivitäten für die 2. und weitere Förderphasen Anbindung Datenbank Genetische Polymorphismen Projektbereich Z Zellbiologie Primäre Zellkulturen Leberbank Modellierungswerkzeug und Datenbanken Projektkoordination

  21. Drug/Xenobiotics lipophilic polar Transport Phase I Phase II Transport Products

  22. Drug/Xenobiotics lipophilic polar Transport Phase I Phase II Transport Products

  23. Drug/Xenobiotics lipophilic polar Transport Phase I Phase II Transport Products

  24. Drug/Xenobiotics lipophilic polar Transport Phase I Phase II Transport Products

  25. Drug/Xenobiotics lipophilic polar Transport Phase I Phase II Transport Products

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