Amit Kumar (CRS4, Università di Cagliari)

1. Computational Modelling of Receptor Ligand complex associated to Fibromyalgia Syndrome Amit Kumar [email protected] 27-05-2015

2. Outline Quick Introduction to problem of Fibromyalgia Motivation Key players Our methodological approach Building Model Docking Molecular Dynamics Results Conclusion 2

3. Quick Introduction 3

4. Motivation Fibromyalgia pathogenesis unclear and no biomarkers. Observed increase PC(14:0/0:0) and PC(16:0/0:0) in FMS patients noted using plasma lipidomic analysis approach metabolites. These metabolites had Platelet Activating Factor (PAF) like structure. PAF : a potent phospholipid activator mediator of many leukocyte (White Blood cell) functions, e.g. inflammation 4

5. Chemical structure of metabolites: lyso- phosphocholines ligands LMGP01050012: PC(14:O/O:O) 2 106 8 124 14 LMGP01050018: PC(16:O/O:O) 142 106 8 124 16 LMGP01020046: PAF (standard) 5

6. Search for Lipid ligands: Lipidomics Database 6

7. 3D-modelling-Ligands 7

8. CCCCCCCCCCCCCCCC(=O)OC[C@@H](O)COP([O-])(=O)OCC[N+](C)(C)C 3D modelling: Ligand 8

9. LMGP01050018: PC(16:O/O:O) Download 3D file 9 3D modelling: Ligand

10. Geometry optimisation: Attempt to find configuration of minimum energy of molecule 3D modelling: Ligand Free for academic use 10

11. 3D Modelling PAFr

12. 3D Models for PAFr and ligands Starting structure: Theoretical model PDBSum database: 2b0x (Gui et. al proteins 2005) 12

13. 3D Modelling for not known structures

14. DOCKING PAFr-Ligands GLIDE, GOLD, MOE: Commercial software

15. PAFR-Ligand Binding region Ligands G (Binding free energy) PAF -7.O kcal/mol PC(14:O/O:O) -7.1 kcal/mol PC(16:O/O:O) -5.1 kcal/mol Docking Energy 15

16. Molecular Dynamics: 1

17. Molecular Dynamics: 2 http://portfolio.scistyle.com/Publication-Quality-Science-Figures17 (1) (2) (3) (4)(5) Empirical Force Field equation Non-bonded terms Bonded terms Electrostatics Van der Waals

18. Molecular Dynamics: 3 18 MD Engine3D coordinates Topology file Parameter file New 3D coordinates at different time intervals NAMD, AMBER, CHARMM, GROMACS, DLPOLY

19. TOPOLOGY File Topology RESI ALA 0.00 GROUP ATOM N NH1 -0.47 ! | ATOM HN H 0.31 ! HN-N ATOM CA CT1 0.07 ! | HB1 ATOM HA HB 0.09 ! | / GROUP ! HA-CA--CB-HB2 ATOM CB CT3 -0.27 ! |ATOM HB1 HA 0.09 ! | HB3 ATOM HB2 HA 0.09 ! O=C ATOM HB3 HA 0.09 ! | GROUP ! ATOM C C 0.51 ATOM O O -0.51 BOND CB CA N HN N CA BOND C CA C +N CA HA CB HB1 CB HB2 CB HB3 DOUBLE O C IMPR N -C CA HN C CA +N O DONOR HN N ACCEPTOR O C c ct1 ct3 nh1

20. PARAMETER File BONDS CT3 CT1 222.00 1.538 CT3 HA 322 1.1 CE1 CE1 440.000 1.3400 ANGLE CT3 CT1 HB 30.000 110.00 CT3 CT1 C 48.00 123.50 HA CT3 CT1 32.00 112.20 NH1 CT1 HB 23.00 118.00 DIHEDRAL CT3 CT1 C O O.2 1 180.0 HA CT3 CT1 NH1 0.5 2 180.0 c ct1 ct3 ha hb nh1 o

21. Mimicking Biological membrane Insertion of PAFr-ligand complex into the membrane Start with a relaxed membrane-water system

22. Movies

23. RMSD Analysis: LIGAND Measure of the deviation between the atoms (usually the backbone atoms) with respect to starting structure

24. RMSD helix Gui. et. al found large deviation of Helix 3 and Helix 6: Related to PAFr activation

25. Hydrogen Bond interactions

26. Binding Free Energy Calculation MM-PBSA: Molecular Mechanics- Poisson Bolzmann Surface Area Gsolv = EPB/EGB + ECAVITY EPB/EGB = the electrostatic contribution to the free energy calculated by PB or GB ECAVITY = nonpolar contribution to the solvation free energy calculated by an empirical model. 26

27. Interaction Picture

28. Conclusions PC(14:0/0:0) and PC(16:0/0:0) ligands are slightly more flexible than PAF, but overall similar binding energy ~11 kcal/mol to PAFR. RMSD variations in key intra-membrane domains smaller than in another MD investigation, but show signals of a conformational change upon binding, which on turn could be linked to receptor activation. with PAF-like function, could role in the clinical manifestation of FMS. New disease biomarkers and therapeutic targets in Fibromyalgia; aid to design new diagnostic & therapeutic approaches & diets.