QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: mol.105.014407v1 68/6/1623    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Picone, R. P. Articles by Makriyannis, A. Search for Related Content PubMed PubMed Citation Articles by Picone, R. P. Articles by Makriyannis, A.

(-)-7'-Isothiocyanato-11-hydroxy-1',1'-dimethylheptylhexahydrocannabinol (AM841), a High-Affinity Electrophilic Ligand, Interacts Covalently with a Cysteine in Helix Six and Activates the CB1 Cannabinoid Receptor

Center for Drug Discovery (R.P.P., A.D.K., W.X., L.A.A., G.A.T., D.J.F., A.M.), Departments of Pharmaceutical Sciences (R.P.P., A.D.K., W.X., L.A.A., G.A.T., D.J.F., A.M.) and Molecular & Cell Biology (A.M.), University of Connecticut, Storrs, Connecticut; Department of Chemistry & Biochemistry, University of North Carolina at Greensboro, Greensboro, North Carolina (D.P.H., P.H.R.); and Forbes Norris Amyotrophic Lateral Sclerosis/Muscular Dystrophy Association Research Center, California Pacific Medical Center, San Francisco, California (M.E.A.)

The CB1 cannabinoid receptor has been shown to play important physiological roles in the central nervous system, as well as peripherally, and is a target for development of therapeutic medications. To gain insight on the ligand binding site(s) and structural features of activation, we designed and synthesized (-)-7'-isothiocyanato-11-hydroxy-1',1'-dimethylheptylhexahydrocannabinol (AM841), a classical cannabinoid affinity label that incorporates an isothiocyanate substituent as an electrophilic reactive group capable of interacting irreversibly with a suitably located and properly oriented nucleophilic amino acid residue at or near the binding site. To obtain evidence for the site of covalent attachment of AM841, C6.47, identified in part by interactive ligand docking, was mutated to serine, alanine, and leucine to reduce or eliminate the nucleophilic character. Wild-type (WT) and mutant CB1 receptors were evaluated for their abilities to recognize a series of cannabinergic ligands. Each bound comparably to WT, excluding C6.47L, which displayed a reduced affinity for ³H-labeled (1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol (CP55940), AM841, 11-hydroxy-1',1'-dimethylheptylhexahydrocannabinol (AM4056), and (-)-7'-bromo-11-hydroxy-1',1'-dimethylheptylhexahydrocannabinol (AM4043) and an improvement in affinity for (-)-trans-⁹-tetrahydrocannabinol (⁹-THC). The affinity of ³H-labeled [2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](naphthyl)methanone (WIN55212-2) was unchanged across all mutants. It is noteworthy that AM841 was shown to bind irreversibly to WT CB1 but exhibited no covalent attachment with the mutants and behaved as an agonist suggesting irreversible attachment to C6.47 maintains CB1 in its active state. The evidence presented identifies C6.47 as the site of covalent bond formation with AM841 and combined with the binding data fully supports the molecular modeling. These studies present the first report of tandem applications of affinity labeling, site-directed mutagenesis, and interactive ligand docking for CB1.

Address correspondence to: Alexandros Makriyannis, Center for Drug Discovery, Northeastern University, Boston, MA 02115. E-mail: a.makriyannis{at}neu.edu

This article has been cited by other articles:

				A. Kapur, D. P. Hurst, D. Fleischer, R. Whitnell, G. A. Thakur, A. Makriyannis, P. H. Reggio, and M. E. Abood Mutation Studies of Ser7.39 and Ser2.60 in the Human CB1 Cannabinoid Receptor: Evidence for a Serine-Induced Bend in CB1 Transmembrane Helix 7 Mol. Pharmacol., June 1, 2007; 71(6): 1512 - 1524. [Abstract] [Full Text] [PDF]