Salvinorin A: A potent naturally occurring nonnitrogenous ␬ opioid selective agonist Bryan L. Roth*†‡§¶, Karen Baner*, Richard Westkaemperሻ, Daniel Siebert**, Kenner C. Rice††, SeAnna Steinberg*, Paul Ernsberger*‡‡, and Richard B. Rothman§§ *National Institute of Mental Health Psychoactive Drug Screening Program, and Departments of †Biochemistry, ‡Psychiatry, §Neurosciences, and ‡‡Pharmacology and Nutrition, Case Western Reserve University Medical School, Cleveland, OH 44106; §§Clinical Psychopharmacology Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD 21224; ࿣Department of Medicinal Chemistry, Medical College of Virginia, Richmond, VA 23298; **The Salvia divinorum Research and Information Center, Malibu, CA 90263; and ††Laboratory of Medicinal Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 Edited by Erminio Costa, University of Illinois, Chicago, IL, and approved July 9, 2002 (received for review April 18, 2002) Salvia divinorum, whose main active ingredient is the neoclero- pharmacological properties of rodent and human molecular dane diterpene Salvinorin A, is a hallucinogenic plant in the mint targets are frequently distinct (7), and that tissue-based radio- family that has been used in traditional spiritual practices for its ligand binding assays frequently yield inaccurate estimates of psychoactive properties by the Mazatecs of Oaxaca, Mexico. More drug potency and selectivity. Accordingly, we reexamined the recently, S. divinorum extracts and Salvinorin A have become more molecular pharmacological profile of the novel diterpene Salvi- widely used in the U.S. as legal hallucinogens. We discovered that norin A at a large number of cloned human G protein-coupled Salvinorin A potently and selectively inhibited 3H-bremazocine receptors (GPCRs), channels, and transporters. We report here binding to cloned ␬ opioid receptors. Salvinorin A had no signifi- that Salvinorin A is a potent and selective ␬ opioid receptor cant activity against a battery of 50 receptors, transporters, and ion (KOR) agonist and represents, to our knowledge, the first channels and showed a distinctive profile compared with the nonalkaloid opioid subtype-selective drug. We suggest that prototypic hallucinogen lysergic acid diethylamide. Functional because the KOR has long been recognized as a target for studies demonstrated that Salvinorin A is a potent ␬ opioid agonist psychotomimetic agents, KOR antagonists may represent a at cloned ␬ opioid receptors expressed in human embryonic novel class of psychotherapeutic compounds. Our results also kidney-293 cells and at native ␬ opioid receptors expressed in suggest that the KOR͞dynorphin peptide system functions to guinea pig brain. Importantly, Salvinorin A had no actions at the modulate human perception. 5-HT2A serotonin receptor, the principal molecular target respon- sible for the actions of classical hallucinogens. Salvinorin A thus Materials and Methods represents, to our knowledge, the first naturally occurring nonni- Materials. Two sources of Salvinorin A were used for the studies trogenous opioid-receptor subtype-selective agonist. Because described here: Biosearch and the Salvia divinorum Research Salvinorin A is a psychotomimetic selective for ␬ opioid receptors, and Information Center, Malibu, CA; both samples were iden- ␬ opioid-selective antagonists may represent novel psychothera- tical by thin-layer chromatography and mass spectroscopy and peutic compounds for diseases manifested by perceptual distor- showed the expected molecular ion in the mass spectrum. In tions (e.g., schizophrenia, dementia, and bipolar disorders). Addi- addition, the Biosearch sample showed the reported melting tionally, these results suggest that ␬ opioid receptors play a point (6), and the Varian 300 MHz NMR spectrum was identical prominent role in the modulation of human perception. with that reported. The coding region of the KOR was cloned via PCR-amplification of ‘‘Quick-Clone’’ cDNA (CLONTECH) alvia divinorum, a member of the mint family, is a psycho- and subcloned into the eukaryotic expression vector pIRESNEO Sactive plant that has been used in traditional spiritual via NotI adaptors to yield pIRESNEO-KOR. The entire insert practices by the Mazatec people of Oaxaca, Mexico for many was verified by automated double-stranded DNA sequencing centuries (1). S. divinorum also grows in California and has been (Cleveland Genomics, Cleveland). A stable human embryonic used as a legal hallucinogen for several years (2). Traditionally, kidney-293 cell line expressing the KOR was also constructed S. divinorum is ingested as a quid or smoked for its psychoactive (KOR-293) and was used for radioligand-binding and functional properties (1) and has been reported to have potent hallucina- assays. GF-62 cells, a stable cell line expressing the 5-HT2A receptor (8), was used for functional studies of 5-HT receptors. tory actions (1, 3). The main active ingredient of S. divinorum is 2A All other receptors were obtained as previously described (9, 10) Salvinorin A (Fig. 1), a novel neoclerodane diterpene of known as part of the National Institute of Mental Health Psychoactive absolute configuration (4) whose structure was determined by Drug Screening Program (NIMH-PDSP) resource. single-crystal x-ray analysis in two independent studies (5, 6). Frozen guinea pig brains and rat brains were purchased from Salvinorin A is structurally distinct from the naturally occurring Harlan Bioproducts for Science (Indianapolis). [D-Ala-2- hallucinogens N,N-dimethyltryptamine, psilocybin, and mesca- MePhe4,Gly-ol5]enkephalin (DAMGO), D-Phe-Cys-Tyr-D-Trp- line and synthetic hallucinogens such as lysergic acid diethylam- Arg-Thr-Pen-Thr-NH (CTAP), and H-Tyr-Tic-Phe-Phe-OH ide (LSD), 4-bromo-2,5-dimethoxyphenylisopropylamine 2 (TIPP) were obtained from Multiple Peptide Systems (San (DOB), and ketamine. Salvinorin A has been reported to be the Diego) through arrangement with Paul Hillery of the Research most potent naturally occurring hallucinogen, with an effective Technology Branch, National Institute on Drug Abuse. SNC-80 dose in humans in the 200- to 1,000-␮g range when smoked (1, was obtained from K.C.R. (Ϫ)-Nor-binaltorphine 2HCl (Nor- 3). Salvinorin A thus rivals the synthetic hallucinogens LSD and BNI) and (ϩ)-U69593 were obtained from Research Biochemi- DOB in potency. Salvinorin A has been reported to induce an intense hallucinatory experience in humans, with a typical duration of action being several minutes to an hour or so (1). This paper was submitted directly (Track II) to the PNAS office. Several prior investigations attempted unsuccessfully to iden- Abbreviations: KOR, ␬ opioid receptor; MOR, ␮ opioid receptor; DOR, ␦ opioid receptor; tify the molecular and cellular targets responsible for the actions LSD, lysergic acid diethylamide; NIMH-PDSP, National Institute of Mental Health Psycho- of Salvinorin A (1, 3) by using mainly nonhuman molecular active Drug Screening Program; GPCR, G protein-coupled receptor. targets. Since then, it has become widely recognized that the ¶To whom reprint requests should be addressed. E-mail: [email protected]. 11934–11939 ͉ PNAS ͉ September 3, 2002 ͉ vol. 99 ͉ no. 18 www.pnas.org͞cgi͞doi͞10.1073͞pnas.182234399 Fig. 1. Molecular modeling predicts Salvinorin A is a structurally novel ␬ opioid ligand. A shows the structure of Salvinorin A, enadoline, U69593 and LSD whereas B shows a superimposition of the structures of Salvinorin A and U69593. C shows potential residues on the KOR identified by molecular modeling, which might interact with Salvinorin A, and D shows a model of Salvinorin A’s interactions with the KOR (see supporting information for further details). NEUROBIOLOGY 35 Ј ␥ ␮ Ͼ cals (Natick, MA). [ S]Guanosine 5 -( -thio)-triphosphate 10 M test compound inhibited 50% of specific binding, Ki ([35S]-GTP[␥S], 45 TBq͞mmol) was purchased from DuPont͞ determinations were performed by using six concentrations of ␥ NEN. BSA, naloxone, GDP, and GTP[ S] were purchased from unlabeled ligand spanning a 10,000-fold dose range. Kis were Sigma. calculated by using GRAPHPAD PRIZM and represent the mean Ϯ SEM of quadruplicate determinations. Radioligand-Binding Assays. Radioligand-binding assays at human cloned GPCRs, ion channels, and transporters were performed Functional Assays. Phosphoinositide hydrolysis assays at 5-HT2A as previously detailed (9, 10) by using the resources of the receptors were performed as previously described (8, 12). ␬ NIMH-PDSP. Detailed on-line protocols are available for all opioid agonist-dependent inhibition of adenylate cyclase was assays at the NIMH-PDSP web site (http:͞͞pdsp.cwru.edu). ␬ performed by using the KOR-cell line. Briefly, cells were split opioid radioligand-binding assays in situ in guinea pig brain and into polylysine-coated 24-well plates and then incubated over- ␮ opioid receptor (MOR)- and ␦ opioid receptor (DOR)-binding night in serum-free medium. The next day, medium was replaced assays in rat brain were performed as previously detailed (11). with Hanks’ F12 medium containing 100 ␮M isobutylmethyl- Initial screening assays were performed by using 10 ␮M Salvi- xanthine and 100 ␮M forskolin together with various concen- norin A or 10 ␮M LSD by using quadruplicate determinations trations of test agent. After incubation at 37°C for 15 min, the and the percent inhibition of specific binding determined. Where reaction