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Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes Research Program ”Systems of Life-Systems Biology” A Systems Biology.

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Presentation on theme: "Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes Research Program ”Systems of Life-Systems Biology” A Systems Biology."— Presentation transcript:

1 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 Requirements in Hepatocytes Coordination: Matthias Reuss Institute of Biochemical Engineering University of Stuttgart

2 Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes Direktor: Prof. Dr. M. Eichelbaum Forschungsschwerpunkte: Arzneimittelmetabolismus Pharmakogenetik Mission: Individualisierte Pharmakotherapie Dr. Margarete Fischer-Bosch-Institut für Klinische Pharmakologie Robert Bosch Stiftung, Stuttgart PD Dr. Ulrich M. Zanger Leiter Molekular- und Zellbiologie

3 Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes 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

4 Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes Biotransformation of Foreign Substances 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) Phase III: Transport P-Glykoprotein (MDR1, MDR2) Multi-Drug Resistance Proteins (MRP) Organic Anion Transporters (OATP) Organic Cation Transporters (OCT) etc. O H Elimination Metabolism Uptake O R

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

6 Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes Complexity of Biotransformations 2C9 2C19 2C8 1A2 3A5 3A4 P450 N OCH 3 O OCH 3 CH 3 O CN CH 3 CH 3

7 Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes Complexity of Biotransformations 2C9 2C19 2C8 1A2 3A5 3A4 P450 N OCH 3 O OCH 3 CH 3 O CN CH 3 CH 3

8 Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes Complexity of Biotransformations 2C9 2C19 2C8 1A2 3A5 3A4 P450 N OCH 3 O OCH 3 CH 3 O CN CH 3 CH 3

9 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)

10 Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes Transport Drugs Phase II UDP - G PAPS GSH AminoA... Central Metabolism Transport Regulatory and Signaling Network (Gene Expression) Phase I NADPH NADH Intermediates Metabolites Endproducts

11 Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes S YSTEMS B IOLOGY 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 B

12 Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes Project Section A Xenobiotic Metabolism and Transport: Analysis, mathematical modeling and simulation of the xenobiotic-metabolizing system of the liver

13 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)

14 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

15 Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes 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 Style N > 300 RNA: transkripts splicing variants DNA: polymorphisms genotypes Protein Fractions: expression function, kinetics Human Liver Bank as a Tool Clinical Documentation:

16 Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes 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

17 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 b 5 Analysis and modeling of protein-protein interactions by reconstitution of purified components (cooperation with Rebecca Wade, EML Heidelberg)

18 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

19 Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes 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

20 Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes S YSTEMS B IOLOGY 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 Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes Drug/Xenobiotics Phase I Phase II lipophilicpolar Transport Products

22 Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes Drug/Xenobiotics Phase I Phase II lipophilicpolar Transport Products

23 Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes Drug/Xenobiotics Phase I Phase II lipophilicpolar Transport Products

24 Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes Drug/Xenobiotics Phase I Phase II lipophilicpolar Transport Products

25 Detoxification Pathways and their Cellular and Structural Requirements in Hepatocytes Drug/Xenobiotics Phase I Phase II lipophilicpolar Transport Products


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