title: Salvinorin A: allosteric interactions at the mu-opioid receptor.
abstract: 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 kappa-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 mu-receptor binding, and subsequent experiments revealed that Salvinorin A partially inhibited mu-receptor binding. Therefore, we hypothesized that Salvinorin A allosterically modulates mu-receptor binding. To test this hypothesis, we used Chinese hamster ovary cells expressing the cloned human opioid receptor. Salvinorin A partially inhibited [(3)H]Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (DAMGO) (0.5, 2.0, and 8.0 nM) binding with E(MAX) values of 78.6, 72.1, and 45.7%, respectively, and EC(50) values of 955, 1124, and 4527 nM, respectively. Salvinorin A also partially inhibited [(3)H]diprenorphine (0.02, 0.1, and 0.5 nM) binding with E(MAX) values of 86.2, 64, and 33.6%, respectively, and EC(50) values of 1231, 866, and 3078 nM, respectively. Saturation binding studies with [(3)H]DAMGO showed that Salvinorin A (10 and 30 microM) decreased the mu-receptor B(max) and increased the K(d) in a dose-dependent nonlinear manner. Saturation binding studies with [(3)H]diprenorphine showed that Salvinorin A (10 and 40 microM) decreased the mu-receptor B(max) and increased the K(d) in a dose-dependent nonlinear manner. Similar findings were observed in rat brain with [(3)H]DAMGO. Kinetic experiments demonstrated that Salvinorin A altered the dissociation kinetics of both [(3)H]DAMGO and [(3)H]diprenorphine binding to mu receptors. Furthermore, Salvinorin A acted as an uncompetitive inhibitor of DAMGO-stimulated guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding. Viewed collectively, these data support the hypothesis that Salvinorin A allosterically modulates the mu-opioid receptor.
authors.affiliation: Clinical Psychopharmacology Section, Intramural Research Program, National Institute of Drug Abuse, National Institutes of Health, Baltimore, MD 21224, USA. rrothman@mail.nih.gov
authors.forename: Richard B
authors.initials: RB
authors.lastname: Rothman
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Murphy
authors.forename: Heng
authors.initials: H
authors.lastname: Xu
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Godin
authors.forename: Christina M
authors.initials: CM
authors.lastname: Dersch
authors.forename: John S
authors.initials: JS
authors.lastname: Partilla
authors.forename: Kevin
authors.initials: K
authors.lastname: Tidgewell
authors.forename: Matthew
authors.initials: M
authors.lastname: Schmidt
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Prisinzano
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000096:biosynthesis; D004174:Diprenorphine / Q000378:metabolism; D004224:Diterpenes / Q000494:pharmacology*; D045785:Diterpenes, Clerodane; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009019:Morphinans / Q000378:metabolism; D017473:Receptors, Opioid, kappa / Q000819:agonists*; D017450:Receptors, Opioid, mu / Q000187:drug effects* / Q000378:metabolism
chemical_list: D004224:Diterpenes; D045785:Diterpenes, Clerodane; D009019:Morphinans; D017473:Receptors, Opioid, kappa; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; C075927:6-iodo-3,14-dihydroxy-17-(cyclopropylmethyl)-4,5-epoxymorphinan; D004174:Diprenorphine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP; C090499:salvinorin A