Screening the ‘receptorome’ to discover

the molecular targets responsible for

psychoactive plant actions in humans

Bryan Roth, MD, PhDBiochemistry

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 CategoriesHydrolase (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

Sig

nal

Tran

sdu

ction

None

Nucleic AcidBinding

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

Sig

nal

Tran

sdu

ction

Kroeze, Sheffler and Roth, J Cell Sci, in pressPDSP 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

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 MeetsThe 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 isolatedSheffler and Roth. Trends Pharmacol Sci. 2003;24:107.

A B

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

Wooley and Shaw. Proc Natl Acad Sci USA. 1954;40:228.

N N

NH

O

H

N

HO NH2

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

% Inhibition

LSDSalvinorin 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 JordanZjawiony, U Miss

Jeremy Stuart and JordanZjawiony, U Miss

0.1 1 10 100 1000 100000

25

50

75

100

125

Concentration (nM)

U69593

U50488

Salvinorin A

Propionyl Sal-A

HK

SAL-A

norBNI

HK

U-69593

norBNI

A B

200

nA

2min

0

25

50

75

100

%Max

Sal-AResponse

**

*P<.01Chavkin et al. In preparation.

Salvinorin A Is An Extraordinarily Efficacious KOR Agonist

DYN-AU69593U50488 SAL-A

HK

SAL-A

norBNI

AHK

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.

                                                                                                

                       

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

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

Rothman et al, JPET, in press

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)