Salvinorin A: Allosteric Interactions at the -Opioid Receptor

Salvinorin A [(2S,4aR,6aR,7R,9S,10aS,10bR)-9-(acetyloxy)-2(3-furanyl)-dodecahydro-6a,10b-dimethyl-4,10-dioxo-2h-naphtho[2,1-c]pyran-7-carboxylic acid methyl ester] is a hallucinogenic -opioid receptor agonist that lacks the usual basic nitrogen atom present in other known opioid ligands. Our first published studies indicated that Salvinorin A weakly inhibited -receptor binding, and subsequent experiments revealed that Salvinorin A partially inhibited -receptor binding. Therefore, we hypothesized that Salvinorin A allosterically modulates -receptor binding. To test this hypothesis, we used Chinese hamster ovary cells expressing the cloned human opioid receptor. Salvinorin A partially inhibited [H]Tyr-D-Ala-Gly-N-MePhe-Gly-ol (DAMGO) (0.5, 2.0, and 8.0 nM) binding with EMAX values of 78.6, 72.1, and 45.7%, respectively, and EC50 values of 955, 1124, and 4527 nM, respectively. Salvinorin A also partially inhibited [H]diprenorphine (0.02, 0.1, and 0.5 nM) binding with EMAX values of 86.2, 64, and 33.6%, respectively, and EC50 values of 1231, 866, and 3078 nM, respectively. Saturation binding studies with [H]DAMGO showed that Salvinorin A (10 and 30 M) decreased the -receptor Bmax and increased the Kd in a dose-dependent nonlinear manner. Saturation binding studies with [H]diprenorphine showed that Salvinorin A (10 and 40 M) decreased the -receptor Bmax and increased the Kd in a dose-dependent nonlinear manner. Similar findings were observed in rat brain with [H]DAMGO. Kinetic experiments demonstrated that Salvinorin A altered the dissociation kinetics of both [H]DAMGO and [H]diprenorphine binding to receptors. Furthermore, Salvinorin A acted as an uncompetitive inhibitor of DAMGO-stimulated guanosine 5 -O(3-[S]thio)-triphosphate binding. Viewed collectively, these data support the hypothesis that Salvinorin A allosterically modulates the -opioid receptor. Salvia divinorum is a plant from the sage family that has been used in the traditional spiritual practices by the Mazatec Indians of Oaxaca, Mexico to produce “mystical” or hallucinogenic experiences. The active ingredient isolated from the leaves of S. divinorum is Salvinorin A, a neoclerodane diterpene. Current evidence suggests that Salvinorin A-induced hallucinogenic effects are mediated by activation of -opioid receptors (for review, see Sheffler and Roth, 2003). Salvinorin A, a -opioid receptor agonist (Roth et al., 2002), is a unique opioid receptor ligand. It bears little structural similarity to other structural classes of nonpeptidic opioid receptor ligands, including agonists, such as U50,488H and U69,593 (Harding et al., 2005). The common structural motif among all of these compounds is the presence of a basic amino group. Until recently, it had been assumed that the presence of a positively charged nitrogen atom in opioid compounds represented an absolute requirement for their interaction with opioid receptors (Rees and Hunter, 1990). The This work was supported by the Intramural Research Program of the National Institutes of Health and National Institute on Drug Abuse Grant R01 DA018151-01A2 (to T.E.P.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.113167. ABBREVIATIONS: CHO, Chinese hamster ovary cells; hMOR-CHO, CHO cells expressing the cloned human -opioid receptor; DAMGO, Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol; herkinorin, (2S,4aR,6aR,7R,9S,10aS,10bR)-9-(benzoyloxy)-2-(3-furanyl)dodecahydro-6a,10b-dimethyl-4, 10-dioxo-2H-naphtho-[2,1-c]pyran-7-carboxylic acid methyl ester; [S]GTP S, guanosine 5 -O-(3-[S]thio)triphosphate; U50,488H, ( )trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide; U69,593, 5 ,7 ,8 -( )-N-methyl-N-[7-(1-pyrrolidinyl)-1oxaspiro(4,5)dec-8-yl]-phenyl-benzeneacetamide; Salvinorin A, (2S,4aR,6aR,7R,9S,10aS,10bR)-9-(acetyloxy)-2-(3-furanyl)-dodecahydro6a,10b-dimethyl-4,10-dioxo-2H-naphtho[2,1-c]pyran-7-carboxylic acid methyl ester; SA, specific activity; MAPK, mitogen-activated kinase protein; KRBG, Krebs-Ringer bicarbonate buffer with glucose; [I]IOXY, 6 -iodo-3,14-dihydroxy-17-cyclopropylmethyl-4,5 -epoxymorphinon. 0022-3565/07/3202-801–810 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 320, No. 2 U.S. Government work not protected by U.S. copyright 113167/3166090 JPET 320:801–810, 2007 Printed in U.S.A.