1. Quantitative Modeling of Metabolic Networks Sai Jagan Mohan, Ph.D. Sonali Das, Ph.D. Anupama Bhat.

2. © Strand Life Sciences 2006 Nov-15 2 Overview  Problem definition and approach  Modules  The glutathione module  The bioenergetics module  Complementary modeling approaches  Constraint based modeling  Metabolic control analysis (MCA)  Summary

3. © Strand Life Sciences 2006 Nov-15 3 Genetic Physiology / Disease Drug / Dose Metabolism Hepatotoxicity intricate and dynamic 'system-level' interactions. Hepatotoxicity prediction is hard

4. © Strand Life Sciences 2006 Nov-15 4 Our Approach A comprehensive model of homeostasis metabolism in a liver cell Toxicity Drug-induced perturbations Hepatotoxicity: Mechanisms  Cell Death of Functional Liver Cells  Impaired Bile Flow  Faulty Fat Processing

5. © Strand Life Sciences 2006 Nov-15 5 Cytotoxicity Modules  The glutathione metabolism module  The bioenergetics module Characteristics  Non-linear ODE’s  Two compartments  Fluxes: Enzyme Kinetics/Mass Action

6. © Strand Life Sciences 2006 Nov-15 6 Enzymes => Non-linearity V GCS = V max {[ATP][Glu][Cys]/  Km ATP Km Glu (1+[GSH]/Ki GSH )Km Cys } {1+[Glu]/Km Glu (1+[GSH]/Ki GSH ) + [Glu] [Cys]/ Km Glu (1+[GSH]/Ki Glu )Km Cys +[Glu][ATP]/Km ATP Km Glu (1+[GSH]/Ki GSH ) + [Glu][Cys][ATP]/  Km ATP Km Glu (1+[GSH]/Ki GSH ) Km Cys }

7. © Strand Life Sciences 2006 Nov-15 7 Perturbations V enzyme = V max * [S] K M + [S] V max  [Enzyme] T

8. © Strand Life Sciences 2006 Nov-15 8 The Oxidative Stress Module

9. © Strand Life Sciences 2006 Nov-15 9 The Glutathione Module d [  -GC] /dt = V GCS – V GS d [GSH] /dt = V GS + V GR – V GPx – V gsh2ss – V gsh2ca – V GST – V gshC2M –V gshM2C

10. © Strand Life Sciences 2006 Nov-15 10 Validation Drug: Ethacrynic Acid (EA) Simulation 1 2 3 Experimental 2 1 3

11. © Strand Life Sciences 2006 Nov-15 11 Asymptotic Analysis Simulation: V gcs = 0Validation Toxin: Buthionine Sulfoximine (BSO) Target:  -GlutamylCysteine Synthetase (GCS) Depletes glutathione with a half –life of ~ 2 hours

12. © Strand Life Sciences 2006 Nov-15 12 Thought simulation

13. © Strand Life Sciences 2006 Nov-15 13 Metabolic Network for Cellular Energetics TCA cycle Glycolysis Malate-Aspartate shuttle ADP+Pi ATP NADHNAD NADH OXPHOS ADP+Pi ATP NADHNAD Energy Utilisation ATP ADP+Pi Cytosol Mitochondria PFK ANT MAL MAL- mito 21 state variables 17 differential equations 4 conservation laws

14. © Strand Life Sciences 2006 Nov-15 14 Total adenine pool in the cytosol = ATP e + ADP e +AMP e = constant Adenylate kinase (ADK) reaction is rapid (operates near equilibrium) 2ADP ATP + AMP ADK K eq = ATP*AMP ADP*ADP = constant (Ataullakhanov & Vitvitsky Bioscience Reports. 2002 22:501-511) Conservation Laws

15. © Strand Life Sciences 2006 Nov-15 15 Constraint Based Modeling

16. © Strand Life Sciences 2006 Nov-15 16 Metabolic Control Analysis (MCA)

17. © Strand Life Sciences 2006 Nov-15 17 Future Work  MCA for insights into control and regulation  Parameter estimation  Experimental validation  Scaling laws for metabolic networks

18. © Strand Life Sciences 2006 Nov-15 18 The Linear Approximation

19. © Strand Life Sciences 2006 Nov-15 19 The Linear Approximation Homeostasis Asymptotic Simulation V GCS = 0 Asymptotic Simulation V GS = 0 SS continuation analysis Parameter : V maxGS