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![Page 1: Screening the ‘receptorome’ to discover the molecular targets responsible for psychoactive plant actions in humans Bryan Roth, MD, PhD Biochemistry Case.](https://reader036.fdocuments.us/reader036/viewer/2022062314/56649d015503460f949d36da/html5/thumbnails/1.jpg)
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
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Outline• Defining the receptorome
• Two case studies
– Salvia divinorum
– Ephedra sinensis and Catha edulis
• Proposal for future studies
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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
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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
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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
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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
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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
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Receptors and transporters for biogenic amines (NE, 5HT, DA, Histamine) represent
a small slice of the receptorome.
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GPCR’s and transporters represent the proximal molecular targets (sites of
action) for the majority of psychoactive plants
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The in vitro approach
• Clones for human GPCRs, ion channels, transporters, and enzymes obtained
• Assays devised
– Radioligand binding
– Functional assays
• Compounds profiled
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Ethnopharmacology MeetsThe Receptorome
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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.
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Salvinorin A is one of the components isolatedSheffler and Roth. Trends Pharmacol Sci. 2003;24:107.
A B
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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.”
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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
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Wooley and Shaw. Proc Natl Acad Sci USA. 1954;40:228.
N N
NH
O
H
N
HO NH2
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Roth et al. Proc Natl Acad Sci USA. 2002;99:11934.
% Inhibition
LSDSalvinorin A
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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
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Jeremy Stuart and JordanZjawiony, U Miss
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Jeremy Stuart and JordanZjawiony, U Miss
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0.1 1 10 100 1000 100000
25
50
75
100
125
Concentration (nM)
U69593
U50488
Salvinorin A
Propionyl Sal-A
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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
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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
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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
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http://altnature.com/gallery/ephedra.htm
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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
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http://www.uneue.org/Archive/DownloadableReports/khat_0501.pdf
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Rothman et al, JPET, in press
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Receptorome profile reveals affinity for NET, 5-HT7 and 2-adrenergic receptors
Rothman et al, JPET, in press
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Active compounds have nM affinity for NET and are substrates for NET
Rothman et al, JPET, in press
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Discriminant stimulus response for ephedrine-related compounds highly correlated
with NE release activity
Rothman et al, JPET, in press
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Compounds inactive at adrenergic receptors
Rothman et al, JPET, in press
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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?
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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
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*
Rothlab V2003
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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)