1. Screening the ‘receptorome’ to discover the molecular targets responsible for psychoactive plant actions in humans Bryan Roth, MD, PhD Biochemistry Case Western Reserve University Medical School Director, NIMH Psychoactive Drug Screening Program

2. Outline Defining the receptorome Two case studies –Salvia divinorum –Ephedra sinensis and Catha edulis Proposal for future studies

3. Why should we discover how psychoactive plants mediate their actions in humans? Novel chemistries –New lead structures –Novel pharmacologies (de-orphanize molecular targets) Discovering molecular target(s) for psychoactive plants clarifies ‘chemistry of consciousness’ –Lewin, L. (1931) "Phantastica, Narcotic and Stimulating Drugs." (Translation of 1924, German edition.) Routledge and Kegan Paul, London Validates molecular targets for drug discovery –Psychoactive plants are, by definition, psychoactive –Wealth of anectodal data available: in essence uncontrolled clinical trials on a massive scale

4. The receptorome Receptorome: the entire complement of receptors in the genome (including non- GPCRs and transporters) Goal: to screen the receptorome to discover how psychoactive plants induce their actions in humans –Non-biased –Discovery based

5. Sequencing The Genome Has Uncovered A Large Amount Of (Mostly) Useful Information Venter et al. Science. 2001;291:1304. Chaperone (159, 0.5%) Cytoskeletal Structural Protein (876, 2.8%) Extracellular Matrix (437, 1.4%) Immunoglobulin (264, 0.9%) Ion Channel (406, 1.3%) Motor (376, 1.2%) Structural Protein Of Muscle (296,1.0%) Protooncogene (902, 2.9%) Select Calcium Binding Protein (34, 0.1%) Intracellular Transporter (350, 1.1%) Transporter (533, 1.7%) Molecular Function Unknown (12809, 41.7%) GO Categories Panther Categories Hydrolase (1227, 4.0%) Isomerase (163, 0.5%) Ligase (56, 0.2%) Lyase (117, 0.4%) Oxidoreductase (656, 2.1%) Synthase and Synthetase (313, 1.0%) Transferase (610, 2.0%) Select Regulatory Molecule (988, 3.2%) Kinase (868, 2.8%) Signaling Molecule (376, 1.2%) Receptor (1543, 5.0%) Nucleic Acid Enzyme (2308, 7.5%) Transcription Factor (1850, 6.0%) Transfer/Carrier Protein (203, 0.7%) Viral Protein (100, 0.3%) Miscellaneous (1318, 4.3%) Cell Adhesion (577, 1.9%) Enzyme Signal Transduction None Nucleic Acid Binding

6. Signal Transduction Signal-transducing molecules represent 1/8 of the genome Receptors represent 1/20 of the genome G-protein coupled receptors (GPCR) represent ~2% of the genome Transporters represent 1.7% of genome Signal Transduction

7. Kroeze, Sheffler and Roth, J Cell Sci, in press PDSP Psychoactive Drug Screening Program Group A GPCR’s comprises family of 274 receptors: biogenic amine receptors (5-HT, DA, NE) represent a small fraction of total

8. Receptors and transporters for biogenic amines (NE, 5HT, DA, Histamine) represent a small slice of the receptorome.

9. GPCR’s and transporters represent the proximal molecular targets (sites of action) for the majority of psychoactive plants

10. The in vitro approach Clones for human GPCRs, ion channels, transporters, and enzymes obtained Assays devised –Radioligand binding –Functional assays Compounds profiled

11. Ethnopharmacology Meets The Receptorome

12. Salvia Divinorum Grows naturally in Oaxaca, Mexico –all known plants propagated from the same parent –probably did not originate in Oaxaca Used by curanderos for many centuries for divination and shamanism Salvinorin A originally isolated in early 1960s by Leander Valdes One prior attempt at discovering molecular target using Novascreen ® was unsuccessful Sheffler and Roth. Trends Pharmacol Sci. 2003;24:107.

13. Salvinorin A is one of the components isolated Sheffler and Roth. Trends Pharmacol Sci. 2003;24:107. AB

14. 1st Account (Daniel Siebert) Of Effects Of Smoking ~2 mg Of Purified Salvinorin A “…Quite suddenly I found myself in a confused, fast-moving state of consciousness with absolutely no idea where my body or my universe had gone…. …I had the sudden realization that although I had managed to pull myself back into my body, I had somehow ended up back in the wrong spot in the timeline of my physical existence…. As the effects began to subside I managed to piece together what had happened. I remembered that I had tested a crystalline isolate, and realized that it must be responsible for what I had just been through. I realized that I had just made an important discovery. I felt ecstatic. I was literally jumping for joy. I wanted to say ‘EUREKA!!!’ I had stumbled upon the psychedelic essence of Salvia divinorum.”

15. Effects Of Salvinorin A Effects are nearly instantaneous (ie, before smoked material is exhaled) Memory of having ingested a “drug” is frequently lost (ie, anterograde amnesia) Users transported to “alternative/parallel universe” Effects wear off quickly (eg, rapid pharmacokinetics) Unlike effects of traditional LSD-like hallucinogens

16. Wooley and Shaw. Proc Natl Acad Sci USA. 1954;40:228. N N NH O H N HO NH 2

17. Roth et al. Proc Natl Acad Sci USA. 2002;99:11934. % Inhibition LSD Salvinorin A

18. Roth et al. Proc Natl Acad Su USA. 2002;99:11934. Sheffler and Roth. Trends Pharmacol Sci. 2003;24:107. Sheffler et al. Work in progress. A B

19. Jeremy Stuart and Jordan Zjawiony, U Miss

20. Jeremy Stuart and Jordan Zjawiony, U Miss

22. 0 25 50 75 100 % Max Sal-A Response * * *P<.01 Chavkin et al. In preparation. Salvinorin A Is An Extraordinarily Efficacious KOR Agonist DYN-AU69593U50488SAL-A HK SAL-A norBNI A HK U-69593 norBNI B

23. Implications Naloxone blocks salvinorin A effects in humans (anectodal reports) KORs are elevated in Alzheimer’s disease KOR antagonists to treat psychosis, which occurs in dementia Salvia divinorum reported to have antidepressant actions Salvia use reported to diminish abuse of other drugs

24. NATIONAL DESK | July 24, 2003, Thursday Complaints and Support for Diet Pill at Congressional Hearing By CHRISTOPHER DREW (NYT) 707 words Late Edition - Final, Section A, Page 12, Column 1 House subcommittee holds hearing to assess whether diet pills containing stimulant ephedra should be banned; hears testimony on complaints of dangerous side effects received by Metabolife International by users of the pills; company holds products are safe but executives do not testify, invoking Fifth Amendment; Nutraquest Inc, formerly Cytodyne Technologies, expresses similar confidence in its ephedra products, one of which was linked to death of Baltimore Orioles pitcher Steve Bechler earlier this year.

25. MA HUANG (Ephedra sinensis) Used in China for >5000 years (Ma Huang) to treat asthma and upper respiratory symptoms Used to increase metabolism and stamina –Frequently combined with caffeine and Guarana Potential anorectic actions Major public health issue at present

26. http://altnature.com/gallery/ephedra.htm

27. Catha edulis (Qat) Up to 80% of population of Yemen uses Khat on a daily basis –Growing abuse in US ‘Kat produces joyous excitation and gaiety.’ –Lewin, L. (1931) "Phantastica, Narcotic and Stimulating Drugs." (Translation of 1924, German edition.) Routledge and Kegan Paul, London Cathine and cathinone are major ingredients

28. http://www.uneue.org/Archive/DownloadableReports/khat_0501.pdf

29. Rothman et al, JPET, in press

30. Receptorome profile reveals affinity for NET, 5-HT7 and  2-adrenergic receptors Rothman et al, JPET, in press

31. Active compounds have nM affinity for NET and are substrates for NET Rothman et al, JPET, in press

32. Discriminant stimulus response for ephedrine-related compounds highly correlated with NE release activity Rothman et al, JPET, in press

33. Compounds inactive at adrenergic receptors Rothman et al, JPET, in press

34. Conclusions Effects of Qat and Ephedra likely related to actions as substrates at NET Compounds act as indirect adrenergic receptor agonists No significant activity at any other tested receptor Potential regulatory issues for NET substrates as a class?

35. Proposal Psychoactive plants exist for which mechanism of action is unclear (e.g. Tabernanthe iboga) It is likely that many more psychoactive plants exist –Expeditions aimed at obtaining extracts and documenting effects in humans likely to be highly productive F Novel chemical structures F Highly potent and selective compounds F Validated human targets for drug discovery –Technology exists to discover mechanism of action

36. * Rothlab V2003

37. Participating Labs Ernsberger lab (CWRU) –SeeAnna Steinberg –Ryan Strachan Ken Kellar lab (Georgetown University) Jarda Wroblewski lab (Georgetown University) Bryan Roth—Director Richard Rothman lab (NIDA) Richard Glennon lab (MCV) Charlie Chavkin lab (University of Washington) John Pintar lab (Rutgers University) Jeremy Stuart (University of Mississippi)