(-)-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

Abstract

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 (Ϫ)-7Ј-isothiocyanato-11-hydroxy-1Ј,1Ј-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 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.The CB1 and CB2 cannabinoid receptors are relatively new members in the G-protein-coupled receptor (GPCR) superfamily. They have been shown to play important physiological roles and represent targets for development of therapeutic medications. From a pharmacological standpoint, agonist activation of both receptors results in the release of G␣ iproteins, causing a concomitant reduction in intracellular This work has been supported by National Institute on Drug Abuse grants DA05955 (to R.P.P.) DA00355 (to A.D.K.), DA09158, DA03801, DA07215, DA07312 (to A.M.), DA03934, DA00489 (to P.H.R.), DA05274, and DA09978 (to M.E.A.).

Mutation Studies of Ser7.39 and Ser2.60 in the Human CB1Cannabinoid Receptor: Evidence for a Serine-Induced Bend in CB1Transmembrane Helix 7

“…This R* model construction was guided by the biophysical literature on the R-to-R* transition in rhodopsin (Rho), the ␤2-adrenergic and muscarinic M3 receptors, as discussed in the previous paragraph, and included a TMH6 conformer derived from our conformational memories study of CB 1 TMH6 . Further detail concerning this model is provided in our recent article (Picone et al, 2005).…”

“…Although current research efforts are directed toward obtaining a structure of the rhodopsin activated state, (Meta II) (Schertler, 2005;Salom et al, 2006;Szundi et al, 2006), our present primary structural information about the GPCR activation process comes from biophysical studies of Rho and the ␤2-adrenergic receptor. For this reason, we have created a model of the CB 1 activated state (R*) based upon the documented changes that occur during the R-to-R* transition (Picone et al, 2005). The importance of the generation of such models has been discussed by Gouldson et al (2004).…”

“…The conformational memories technique employs multiple Monte Carlo simulated annealing random walks using the CHARMM19 united atom force field. TMH7 from our published R* model of CB 1 (Picone et al, 2005) was used as a starting structure for the CM runs. All calculations were performed using a distance-dependent dielectric.…”

“…1B). Modeling studies have predicted that AM841, an isothiocyanate derivatized classic ligand for the CB 1 receptor, establishes a hydrogen bonding interaction through its phenolic hydroxyl group with Lys3.28 (192), its 11-hydroxyl group with Ser2.60(173), and its pyran oxygen with Ser7.39(383) (Picone et al, 2005). Our results demonstrate that mutation of Ser7.39 has a significant effect on ligand affinity and potency of the nonclassic agonist CP55,940, the classic agonist HU210, and AM4056.…”

Discriminative stimulus functions in rats of AM1346, a high-affinity CB1R selective anandamide analog

“…The currently known two cannabinoid receptors are G-coupled proteins. Data by Howlett and colleagues (e.g., Houston and Howlett 1998;Mukhopadhyay and Howlett 2005;Shim and Howlett 2006), as well as by others (e.g., Bonhaus et al 1998;Georgieva et al 2008;Picone et al 2005), suggest that different CB 1 R agonists can activate different signal transduction pathways down stream. Alternatively, CB 1 R agonists, especially those that are structurally related to AEA, may also interact with other Gcoupled-protein receptors such as the vanilloid type-1 receptor (TRPV1) or the recently deorphanized GPR55 (Brown 2007;Howlett 2004;Pacher et al 2006;Oz 2006 for reviews).…”