Efficacy in CB 1 Cannabinoid Receptor Signal Transduction

1. Efficacy in CB1 Cannabinoid Receptor Signal Transduction Allyn Howlett, Ph.D. Neuroscience/Drug Abuse Research Program J. L. Chambers Biomedical/Biotechnology Research Institute North Carolina Central University Supported by Natl Institute on Drug Abuse NIDA November, 2003 Frontiers in Addiction Research .

2. Cannabinoid Receptor Subtypes • CB1 • Found in neuronal cells and brain; other non-innervated tissue? • Regulates neurotransmitter release • CB1(A) • Splice variant mRNA found in human brain, but not predicted in rodent gene (Sanofi Recherche) • Similar pharmacology and signal transduction as CB1 • CB2 • Found in immune tissue (B cells, macrophages, T cells) • Activity not fully characterized • CB?? or CB? • ? Antinociceptive effects of anandamide inCB1 (-/-) mice (Martin) • ? Vascular effects of anandamide not reproduced by other agonists (Kunos)

3. CB1 Cannabinoid Receptor Herkenham et al. (1991) J. Neurosci. 11: 563

4. CB1 Cannabinoid Receptor, A G-Protein Coupled Receptor EC2 EC3 EC1 extracellular intracellular IC1 IC2 IC3 3D structure recently determined (Biochemistry 2002, 41, 11344)

5. Homology Model of the CB1 Receptor extracellular E2 loop as a part of binding site intracellular Biopolymers(Peptide Sciences), 2003, 71, 169-189

6. Cannabinoid Receptor Agonists • Classical Cannabinoid (ABC-tricyclic) • Nonclassical Cannabinoid (AC-bicyclic; ACD-tricyclic) • CP55940; CP55244 (Pfizer) • Aminoalkylindole • WIN55212-2 (Sterling Research Inst.) • Eicosanoid • Arachidonylethanolamide (anandamide) • 2-Arachidonoylglycerol • Aryl Pyrazole analogs • Organon analogs (Razdan and Martin)

7. Some Other EndoCannabinoids >141 2.5 O OH OH O O OH OH 2-Arachidonylglyceryl ether (noladin ether) 2-Arachidonoylglycerol (2-AG) O CB1/CB2 Affiniy Ratio OH O ??? Devane et al. (1992) Science 258: 1946 Mechoulam et al. (1995) Biochem. Pharmacol. 50: 83 Hanus et al. (2001) PNAS 98: 3662 Porter et al. (2002) J. Pharmacol. Exp. Ther. 301: 1020 O-Arachidonoyl ethanol ester (Virodhamine)

8. CB1 Receptor Signal Via Gi/o Proteins Signal Transduction Effector G protein Subunit Ion Channels K+ currents Gi (1,2,3? Via cAMP?) Ca2+ currents Gi or Go beta-gamma? Mitogen-Activated Protein Kinase Gi (1,2,3?) beta-gamma? or Go(1,2)? Other? PLA2 ? Ca2+ mobility? Focal Adhesion Kinase? PI3Kinase? NO synthesis? Sphingomyelin hydrolysis and ceramide?

9. Adenylyl Cyclase (types 5,6) Gi (1,2,3?)alpha (types 1,3,8 to inhibit? Or types 2,4,7 to stimulate?) 250  N18TG2 Cells C6 glioma Cells 105  75   CB1 R 50  35  CP52444 CP55940 D9-THC CBN CBD (+)isomers

10. Cannabinoid receptor agonists inhibit N-typeCa2+ currents in differentiated N18 neuroblastoma cells Mackie et al., Mol.Phm.44:498’93

11. Cannabinoid Agonist-induced MAPK Phosphorylation Signal Transduction via Gi/o N18TG2 Neuroblastoma Cells - - - + + + Pertussis Toxin - + Serum MA WIN CP MA WIN CP C6 Glioma Cells + - + - + - Pertussis Toxin - + Serum WIN55212 Methanan- damide CP55940

12. CP55940 and Methanandamide induce Nitric Oxide (NO) production in N18TG2 neuroblastoma cells Control CP55940 Methanandamide L-NNA + Methanandamide

13. CB1 Receptor Signaling via G-proteins • The domains of the CB1 receptor selective for interaction with G-proteins • Agonists can affect CB1 receptor – G-protein association differentially • Speculation on conformational induction & G-protein activation

14. Peptides Derived from the Intracellular CB1 Receptor Peptides Synthesized from the IL3 and C-terminal Domains CB1301 begins IL3;3 peptides span the loop CB1401 begins at membrane interface, extend beyond cys-palmitoyl anchor

15. Peptide CB1401 Disrupts the Association Between CB1 Receptor and Gi3 but not Gi1 or Gi2 in Rat Brain Membrane Extracts Gi1 Gi2 Gi3 Peptide 401: - + - + - +

16. Peptide CB1401 Disrupts the CB1 Receptor Association with Go but not Gi1/2 in Rat Brain Membrane Extracts

17. Peptide CB1401 Disrupts the Association Between CB1 Receptor and Gi3 but not Gi1 or Gi2 in Rat Brain Membrane Extracts Gi1 Gi2 Gi3 Peptide 401: - + - + - +

18. Peptides from IL3 Disrupt the CB1R Association with Gi1 & 2 but not Gi3 in N18TG2 membrane extracts CB1R Gi alpha IL3 peptides: - + - + - + • Conclusions • CB1 Receptor-G alpha complexes exist in the absence of agonists, but can be disrupted by pertussis toxin or GTP analogs. • The juxtamembrane C-terminal domain is involved in the association with Go & Gi3, but not Gi1 & G2. • CB1 IL3 domain is involved in the association • with Gi1 & Gi2 but not Gi3.

19. CB1 Receptor Signaling via G-proteins • The domains of the CB1 receptor selective for interaction with G-proteins • Agonists can affect CB1 receptor – G-protein association differentially • Speculation on conformational induction & G-protein activation

20. Conformational changes in the intracellular surface may direct interaction with selective G proteins

21. Agonist Regulation of Gi/CB1R Association

22. CB1 Receptor Signaling via G-proteins • The domains of the CB1 receptor selective for interaction with G-proteins • Agonists can affect CB1 receptor – G-protein association differentially • Speculation on conformational induction & G-protein activation

23. CP55244 Binding Model Biopolymers(Peptide Sciences), 2003, 71, 169 Assumption: H-bonding between K3.28(192) and phenolic OH blue/green: less lipophilic brown: more lipophilic

24. WIN55212-2 Binding Model aroyl-up1 TM5 TM3 TM2 TM7 aroyl-down1

25. WIN55212-2 and CP55244 Binding to CB1 Receptor E(258) K3.28(192) Y5.39(275) T5.38(274) F5.42(278) V3.32(196) CP55244WIN55212-2 H-bonding:

26. G-protein Activation Mechanism by Receptor Conformational Change By breaking H-bonding network By breaking H-bonding network WIN55212-2 CP55244 By breaking hydrophobic interaction

27. Conformational Induction of R-G Complex by A Response Phosphorylation by GRK Arrestin association Internalization

28. Agonist Directed “Trafficking” of Signal Transduction Response 1 Response 2 A1 A2 Response 3 Inverse Agonist Response A3 InARiG InA

29. ` • Signal transduction pathways will depend upon the G-proteins and effector pathways present in the cell. • Domain specificity for G-proteins suggests that induction or selection of different conformations of the CB1 receptor can direct selective signal transduction pathways. • CB1 receptor signaling through a given pathway may be directed by agonist-specific conformational changes in the receptor.

30. Prospectus • Few CB receptor subtypes limits use of pharmacophoric distinctions in ligand affinities to separate therapeutic from untoward effects. • Can we develop agonists that induce receptor conformations that activate specific G proteins ? • Manipulation of G protein coupling may promote signal transduction pathways limited to cell types that regulate therapeutic responses.

31. Collaborators & Acknowledgements • JLC-BBRI at NCCU Derek Norford, Skyla Carney, Abdel-Azim Assi John Joong-Youn Shim Somnath Mukhopadhyay CMDNJ-RWJMS William Welsh J Nehru Univ, Delhi Sudha Cowsik • $$ National Institute on Drug Abuse