Ligand docking studies of Metabotropic Glutamate Receptor Type 6 (mGluR6)

1. Ligand docking studies of Metabotropic Glutamate Receptor Type 6 (mGluR6) Kalyan C. Tirupula Graduate Student MBSB Program University of Pittsburgh Dipeptide docking update – 5/21/10

2. G-Protein Coupled Receptors • Three major classes • Class A • Rhodopsin • Class B • Secretin like • Class C • Metabotropic Glutamate receptors Figure from Bockaert and Pin, 1999 GPCRs are an extremely important class of receptors

3. Metabotropic Glutamate Receptors (mGluRs) • Bind to glutamate • most important excitatory neurotransmitter • Modulatory role in many neurological processes • memory, learning, sensory processing, synaptogenesis, pain transmission mGluR6 specifically expresses in retina

4. Function of mGluR6 Na+, K+, Ca2+,Mg2+ mGluR6 mGluR6* • Rod cells synapse on to ON bipolar cells • ON bipolar cells specifically express mGluR6 • Activation of mGluR6 results in negative regulation of TRPM1 Glu TRPM1 Rod cell β αo* αo γ β γ ON Bipolar cell L-Glutamate (Glu)

5. Role of mGluR6 in vision • Defects in mGluR6 result in night blindness • Characterized by absence of b-wave in electroretinograms • Knock out mice • human subjects with mutations in mGluR6 Non-functional mGluR6 results in congenital stationary night blindness (CSNB)

6. Potential role of mGluRs in addiction • mGluR6 and mGluR8 are associated with increased risk of heroin addiction • Concluded from genome-wide association studies • Glutamatergic signal transduction is implicated in modulation of drug addiction by dipeptideGly-Gln • But actual mGluR targets are unknown We will test Gly-Gln binding to mGluR6 Nielsen et al, 2008 Gly-Gln studies : Dr. Millington’s lab

7. Structural organization of mGluRs Extracellular Ligand binding Domain (EC) • mGluRs are disulfide linked dimers • Two ligand binding sites • Orthosteric: Glutamate, competitive agonists and antagonists (EC domain) • Allosteric : Non-Competitive ligands (TM domain) Glutamate Binding site (Open / Closed) Cysteine-rich Domain (CR) Allosteric Binding site (Active / inactive) Transmembrane Domain (TM)

8. Available EC domain structures There is no structural data for mGluR6

9. Crystal structures of mGluRs (EC)

10. Functional assay

11. Functional assay for mGluR6: cAMP assay mGluR6* Adenylyl Cyclase (AC) β γ Forskolin (FK) stimulates cAMP production αo/i* cAMP • mGluR6 is a Gαocoupled receptor • Active (Gαi/o*) negatively regulates AC • inhibits AC activity • decreases cAMP PDE inhibitor (IBMX) blocks cAMP hydrolysis AMP mGluR6 activation decreases cAMP

12. Effect of agonists on mGluR6 L-glutamate L-ap4 EC50 = 1.2 - 7.5 μM EC50 = 0.1 – 0.3 μM • Determined EC50 matches with the values in literature Stably expressed mGluR6 in HEK293 cells is active

13. Effect of antagonist on mGluR6 LY341495 • Addition of LY341495 right shifts the dose response curves for • L-AP4 and L-Glu mGluR6 activity is diminished in presence of antagonist

14. Effect of UBP1112 on mGluR6 • Uninduced • Induced • UBP1112 is reported to be selective antagonist for group III mGluRs UBP1112 appears to be an inverse agonist

15. Summary of ligand effects on mGluR6 activity: cAMP assay Calcualted EC50 values for agonists is similar to reported values

16. Modeling and docking studies of orthostericligand binding pocket

17. Approach Specific Aim 1

18. Generation of Homology models • Modeller software package • Multiple templates are used to generate average models • Closed mGluR6 • 1EWV_B, 1EWK_B, 1EWT_A, 1EWT_B, 1ISS_A, 1ISS_B • Open mGluR6 and mGluR3 • 1EWV_A, 1EWK_A, 1ISR_A, 2E4U_A, 2E4U_B, 2E4V_A, 2E4V_B, 2E4W_A, 2E4W_B, 2E4X_A, 2E4X_B, 2E4Y_A, 2E4Y_B • Model evaluation • Modeling scores and PROCHECK • Ramachandran plots Models generated: open & closed mGluR6, open mGluR3

19. Receptor structures for docking Crystal structures used as docking controls

20. Docking studies of Closed mGluR structures / models

21. Testing docking approach and receptor models • Primary validation • Verify if ligands from crystal structures when docked recapitulate their binding • Secondary validation • Prepared a database of all the ligands from literature • Docked ligands from database and compare results to experimental binding data

22. Docking studies • AutoDockVina is used • Rigid docking of receptor, but flexible ligand • Grid box: 30 x 30 x 30 • Exhaustiveness = 64 • Total of 10 docking poses generated • Best hit is the top ranked structures • Successful docking • Top ranked structure docks in binding pocket

23. Positive controls for docking mGluR1 mGluR3 LYS-409 LYS-389 ARG-68 SER-186 ALA-172 TYR-74 ALA-187 SER-173 Crystal Structure THR-188 THR-174 SER-151 SER-165 LYS-409 LYS-389 ARG-68 SER-186 Docked Structure ALA-172 TYR-74 ALA-187 SER-173 ARG-64 ARG-323 THR-188 THR-174 SER-165 SER-151 -6.2 kcal/mol -6.2 kcal/mol Docking results with glutamate are close to crystal structures

24. Optimization of Glutamate binding pocket in mGluR6 MODEL OPTIMIZED MODEL ARG-68 SER-155 GLN-64 LYS-400 ASN-281 LYS-312 L-Glu docking -5.7 kcal/mol (Docking pose 3) L-Glu docking -6.1 kcal/mol (Docking pose 2) Optimized model is used for mGluR6 docking studies

25. Glutamate binding pocket in mGluR6 mGluR6 LYS-400 ARG-68 LYS-409 LYS-389 ALA-175 ARG-68 SER-186 ALA-172 SER-176 LYS-312 TYR-74 ALA-187 Docked Structure SER-173 THR-177 THR-188 THR-174 SER-154 -6.1 kcal/mol SER-151 SER-165 ASN-281 mGluR1 mGluR3 Crystal Structure Glutamate docking pocket in mGluR6 is analogous to that of mGluR1 and3

26. Docking summary for database ligands

27. Docking of dipeptides to closed mGluR6 model • Gly-Glu and Gly-Gln • did not dock • cyclo-Gly-Gln • docked (-6.7 kcal/mol) Gly-L-Glu Gly-L-Gln cyclo-Gly-Gln Cyclo Gly-Gln possible agonist?

28. Effects of Dipeptides on mGluR6 function Gly-Gln Gly-Glu Cyclo Gly-Gln • cyclo Gly-Gln • No effect • Gly-Glu and Gly-Gln • Antagonist effect Gly-Glu and Gly-Gln have effects comparable to UBP1112 which is an inverse agonist

29. Docking studies of Open mGluR structures / models

30. Positive controls for docking (L-Glu) ALA-187 ALA-187 SER-186 TYR-74 SER-186 TYR-74 THR-188 THR-188 SER-165 SER-165 ASP-208 ASP-208 TYR-74 ASP-218 TYR-74 ASP-218 L-Glu docking has poor binding energy (-4.3 kcal/mol )

31. Positive controls for docking (S-MCPG) ALA-187 ALA-187 SER-186 TYR-74 SER-186 TYR-74 LYS-409 LYS-409 THR-188 THR-188 SER-165 SER-165 ASP-208 ASP-208 TYR-74 ASP-218 TYR-74 ASP-218 S-MCPG docking does not recapitulate the docking pose seen in the crystal structure

32. mGluR1: Open (LY341495 docking) LYS-409 TYR-74 SER-186 ALA-187 ALA-187 SER-186 TYR-74 LYS-409 THR-188 THR-188 SER-165 SER-165 ASP-208 ASP-208 TYR-74 ASP-218 TYR-74 ASP-218

33. Plan for next 3 months • Develop homology model for mGluR6 based on one template alone • Include glutamate in docking • Generate models for all mGluRs which lack crystal strucures • Optimize docking to open structures • Arguslab, Autodock, MOE • Dock ligands from literature and perform comparative analysis • How well do the computational studies correlate with experimental? • What are the key differences between binding pockets of different mGluRs? • Strategy for developing selective ligands

34. EXTRA SLIDES

35. Orhtostericligand binding pocket Group I Group II Group III Green: Identical across all groups; Yellow: Partially identical across groups Red: Different residues within group; None: Different across groups; Grey: Bulky residue in group I and group II not present in group III