# Presentation: 570462

## 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

## Outline	

- Defining the receptorome
- Two case studies
*Salvia divinorum*
*Ephedra sinensis *and *Catha edulis*
- Proposal for future studies

## 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***

## 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

## 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**

## 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**

## Kroeze, Sheffler and Roth, J Cell Sci, in press

- Group A GPCR’s comprises family of 274 receptors: biogenic amine receptors
- (5-HT, DA, NE) represent a small fraction of total

## Receptors and transporters for biogenic amines (NE, 5HT, DA, Histamine) represent

- a small slice of the receptorome.

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

## The in vitro approach

- Clones for human GPCRs, ion channels, transporters, and enzymes obtained
- Assays devised
- Radioligand binding
- Functional assays
- Compounds profiled

## Ethnopharmacology MeetsThe Receptorome

## 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.

## Salvinorin A is one of the components isolated

- Sheffler and Roth.  *Trends Pharmacol Sci*.  2003;24:107.

## 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*.”

## 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

## N

**N**

**N**

**NH**

**O**

**H**

**N**

**HO**

**NH****2**

## Roth et al.  Proc Natl Acad Sci USA.  2002;99:11934.

- Roth et al.  *Proc Natl Acad Sci USA*.  2002;99:11934.

**% ****Inhibition**

- LSD

- Salvinorin A

## A

- 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**

## Jeremy Stuart and Jordan

- Zjawiony, U Miss

## Jeremy Stuart and Jordan

- Zjawiony, U Miss

## Slide 21

## Salvinorin A Is An Extraordinarily Efficacious KOR Agonist

**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-A**

**U69593**

**U50488**

**SAL-A**

**HK**

**SAL-A**

**norBNI**

**A**

**HK**

**U-69593**

**norBNI**

**B**

## 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

## 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.

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## 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

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

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

## 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

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

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

## Rothman et al, JPET, in press

**Rothman et al, JPET, in press**

## Receptorome profile reveals affinity for NET, 5-HT7 and 2-adrenergic receptors

- Receptorome profile reveals affinity for NET, 5-HT7 and 2-adrenergic receptors

**Rothman et al, JPET, in press**

## Active compounds have nM affinity for NET and are substrates for NET

**Rothman et al, JPET, in press**

## Discriminant stimulus response for ephedrine-related compounds highly correlated

- with NE release activity

**Rothman et al, JPET, in press**

## Compounds inactive at adrenergic receptors

**Rothman et al, JPET, in press**

## 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?

## 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
- Novel chemical structures
- Highly potent and selective compounds
- Validated human targets for drug discovery
- Technology exists to discover mechanism of action

## *

*****

- Rothlab V2003

## 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)