1. Efficacy in CB 1 Cannabinoid Receptor Signal Transduction. NIDA November, 2003 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 Frontiers in Addiction Research

2. Cannabinoid Receptor Subtypes CB 1 – Found in neuronal cells and brain; other non-innervated tissue? –Regulates neurotransmitter release CB 1(A) –Splice variant mRNA found in human brain, but not predicted in rodent gene(Sanofi Recherche) –Similar pharmacology and signal transduction as CB 1 CB 2 –Found in immune tissue (B cells, macrophages, T cells) –Activity not fully characterized CB ?? or CB? –? Antinociceptive effects of anandamide in CB 1 (-/-) mice (Martin) –? Vascular effects of anandamide not reproduced by other agonists (Kunos)

3. Herkenham et al. (1991) J. Neurosci. 11: 563 CB 1 Cannabinoid Receptor

4. CB 1 Cannabinoid Receptor, A G-Protein Coupled Receptor EC1 EC2 EC3 IC1 IC2 IC3 3D structure recently determined (Biochemistry 2002, 41, 11344) extracellular intracellular

5. Homology Model of the CB 1 Receptor Biopolymers (Peptide Sciences), 2003, 71, 169-189 E2 loop as a part of binding site extracellular intracellular

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. 2-Arachidonylglyceryl ether (noladin ether) Some Other EndoCannabinoids O-Arachidonoyl ethanol ester (Virodhamine) 2-Arachidonoylglycerol (2-AG) O OH O 2.5>141 O OH 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 CB 1 /CB 2 Affiniy Ratio O OH O ???

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

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

10. Cannabinoid receptor agonists inhibit N-type Ca 2+ currents in differentiated N18 neuroblastoma cells Mackie et al., Mol.Phm.44:498’93

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

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

13. CB 1 Receptor Signaling via G-proteins The domains of the CB 1 receptor selective for interaction with G-proteins Agonists can affect CB 1 receptor – G-protein association differentially Speculation on conformational induction & G-protein activation

14. Peptides Synthesized from the IL3 and C-terminal Domains CB 1 301 begins IL3;3 peptides span the loop CB 1 401 begins at membrane interface, extend beyond cys- palmitoyl anchor Peptides Derived from the Intracellular CB1 Receptor

15. Peptide CB 1 401 Disrupts the Association Between CB 1 Receptor and Gi3 but not Gi1 or Gi2 in Rat Brain Membrane Extracts Peptide 401: - + - + - + Gi1 Gi2 Gi3

16. Peptide CB 1 401 Disrupts the CB1 Receptor Association with Go but not Gi1/2 in Rat Brain Membrane Extracts

17. Peptide CB 1 401 Disrupts the Association Between CB 1 Receptor and Gi3 but not Gi1 or Gi2 in Rat Brain Membrane Extracts Peptide 401: - + - + - + Gi1 Gi2 Gi3

18. Peptides from IL3 Disrupt the CB 1 R Association with Gi1 & 2 but not Gi3 in N18TG2 membrane extracts IL3 peptides: - + - + - + CB1R Gi alpha Conclusions CB 1 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. CB 1 IL3 domain is involved in the association with Gi1 & Gi2 but not Gi3.

19. CB 1 Receptor Signaling via G-proteins The domains of the CB 1 receptor selective for interaction with G-proteins Agonists can affect CB 1 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/CB 1 R Association

22. CB 1 Receptor Signaling via G-proteins The domains of the CB 1 receptor selective for interaction with G-proteins Agonists can affect CB 1 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-down1 aroyl-up1 TM2 TM3 TM5 TM7

25. WIN55212-2 and CP55244 Binding to CB 1 Receptor F5.42(278) V3.32(196) E(258) K3.28(192) CP55244WIN55212-2 H-bonding: Y5.39(275) T5.38(274)

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

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

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

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 CB 1 receptor can direct selective signal transduction pathways. CB 1 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