Interaction of psychoactive tryptamines with biogenic amine transporters and serotonin receptor subtypes

This study (2014) analyzed the interaction of 21 different tryptamines with specific neurotransmitter transporters and receptor subtypes implicated in psychedelic effects in rodent brains. The authors found that all substances were 5-HT2A agonists, but that SERT activity may play an important role for some of the compounds.

Abstract

Rationale: Synthetic hallucinogenic tryptamines, especially those originally described by Alexander Shulgin, continue to be abused in the USA. The range of subjective experiences produced by different tryptamines suggests that multiple neurochemical mechanisms are involved in their actions, in addition to the established role of agonist activity at serotonin 2A (5-HT2A) receptors.

Objectives: This study evaluated the interaction of a series of synthetic tryptamines with biogenic amine neurotransmitter transporters and with serotonin (5-HT) receptor subtypes implicated in psychedelic effects.

Methods: Neurotransmitter transporter activity was determined in rat brain synaptosomes. Receptor activity was determined using calcium mobilization and DiscoveRx PathHunter® assays in HEK293, Gα16-CHO, and CHOk1 cells transfected with human receptors.

Results: Twenty-one tryptamines were analyzed in transporter uptake and release assays, and 5-HT2A, serotonin 1A (5-HT1A), and 5-HT2A β-arrestin functional assays. Eight of the compounds were found to have 5-HT-releasing activity. Thirteen compounds were found to be 5-HT uptake inhibitors or were inactive. All tryptamines were 5-HT2A agonists with a range of potencies and efficacies, but only a few compounds were 5-HT1A agonists. Most tryptamines recruited β-arrestin through 5-HT2A activation.

Conclusions: All psychoactive tryptamines are 5-HT2A agonists, but 5-HT transporter (SERT) activity may contribute significantly to the pharmacology of certain compounds. The in vitro transporter data confirm structure-activity trends for releasers and uptake inhibitors whereby releasers tend to be structurally smaller compounds. Interestingly, two tertiary amines were found to be selective substrates at SERT, which dispels the notion that 5-HT-releasing activity is limited only to primary or secondary amines.”

Authors: Bruce E. Blough, Antonio Landavazo, Ann M. Decker, John S. Partilla, Michael H. Baumann & Richard B. Rothman

Summary

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Synthetic hallucinogenic tryptamines are abused in the USA. Multiple neurochemical mechanisms are involved in their actions, in addition to agonist activity at serotonin 2A receptors.

Twenty-one tryptamines were analyzed in transporter uptake and release assays, 5-HT2A, serotonin 1A (5-HT.sub.1A) and 5-HT2A [beta]-arrestin functional assays, and HEK293, G[alpha]16-CHO, and CHOk1 cells transfected with human receptors.

All psychoactive tryptamines are 5-HT agonists, but 5-HT transporter (SERT) activity may contribute significantly to the pharmacology of certain compounds.

Synthetic hallucinogenic tryptamines, such as Foxy and Moxy, continue to be abused in the USA, despite the recent emergence of synthetic “bath salt” cathinone compounds. Psilocybin, a type of tryptamine, has shown efficacy in treating cluster headaches, psychotherapy, smoking cessation, and alcohol abuse.

Tryptamines are psychoactive compounds based on natural product psychedelics. The psychoactive properties of tryptamines are generally attributed to agonist activity at serotonin 2A (5-[HT.sub.2A]) receptors, but multiple neurochemical pathways may be involved.

Most psychedelics bind to 5-HT2A receptors, but not all 5-HT2A agonists are psychedelics. Hallucinogens activate different downstream effectors than non-hallucinogens, suggesting that functional selectivity at the 5-HT2A receptor is important in mediating the psychoactive behavioral effects of hallucinogenic compounds.

Although 5-HT2A receptor activity plays a major role in the pharmacology of psychedelic compounds, other signaling pathways, such as serotonin 2C receptor agonism, serotonin 1A receptor agonism, and 5-HT transporter (SERT) uptake inhibition, have also been suggested to be significant.

Synthetic psychoactive tryptamines are close analogs of the neurotransmitter 5-HT, and may act by blocking 5-HT uptake by the SERT or by acting as SERT substrates that induce 5-HT release via reversal of normal transporter flux. Psychoactive tryptamines are simple chemical structures that are relatively easy to synthesize. They have been shown to interact with biogenic amine transporters and 5-HT receptors.

Rat brains were processed to yield synaptosomes, which were used to assess transport activity at dopamine transporter (DAT), norepinephrine transporter (NET), and serotonin transporter (SERT), respectively. To test the inhibitory effect of a drug on synaptic transmission, synaptosomes were preloaded with radiolabeled substrate in Krebs-phosphate buffer for 1 h (steady state), and then released into the solution containing the test drug.

HEK293 and CHO cells were plated into 96-well plates and incubated at 37°C, 5°C overnight. The growth medium was removed and the cells were gently washed with prewarmed assay buffer. Cells were incubated for 45 min at 37[degrees]C, 5 % C[O.sub.2] in 200 [micro]L of a calcium-sensitive fluorescent dye. The cells were then treated with 25 [micro]L of pretreatment, and the plate was read in a FlexStation II.

CHOk1 cells stably expressing the 5-[HT.sub.2A] receptor fused to the Prolink gene were incubated at 37 [degrees]C, 5 % C[O.sub.2] overnight, and the next day test compounds were added to the cells. Luminescence was measured at 1 h post detection reagent addition using a FlexStation III.

Compounds 1, 2, 6, 7, 19, 20, 21, 22, 23, 25, 26, and 28 were synthesized, and compounds 15 to 27 were purchased.

A set of 21 tryptamines were studied in biogenic amine uptake inhibition and release assays, as well as in 5-[HT.sub.2A] and 5-[HT.sub.1A] calcium mobilization and 5-[HT.sub.2A] [beta]-arrestin recruitment assays. These compounds differed by N-substitution and indole ring substituents. The activity of the compounds was assessed at biogenic amine transporters and 5-HT G protein-coupled receptors using an in vitro calcium mobilization assay and a stably transfected cell line expressing the human receptors.

The tryptamines binned into two groups, depending on their SERT activity. The smaller, less sterically encumbered compounds were found to be 5-HT releasers, while the larger N-alkyl groups were found to be 5-HT uptake inhibitors.

We found that all tryptamine compounds were active as 5-HT agonists and most were either SERT uptake inhibitors or transporter substrate releasers. The most potent and selective 5-HT releaser was N-ethyltryptamine (15, NETP), which was also a weak 5-HT uptake inhibitor. Psilocin, the active metabolite of psilocybin, was found to have reasonable uptake inhibitory properties at the SERT, and was converted from a SERT-mediated releaser to a SERT uptake inhibitor.

The uptake inhibition data for 5-MeO-DIPT, 5-MeO MIPT, and 5-MeO DMT in Table 2 correlate well with data reported by Nagai et al. All three 5-methoxy compounds had slightly lower IC.sub.50 values for 5-HT uptake inhibition than previously reported, though the rank order of potency was the same.

The 5-methoxy analogs of DET were much less potent at the SERT than their unsubstituted counterparts, but the diisopropyl compounds of DIPT and 5-MeO-DIPT inhibited 5-HT uptake much better than their monosubstituted analogs.

Our findings fit with the hypothesis that releasers are transporter substrates, similar to 5-HT, while uptake inhibitors are not. The addition of a second N-ethyl group to N-ethyltryptamine caused it to become a modestly potent 5HT uptake inhibitor.

The N, N-dimethyl analogs of DMT and 5-hydroxytryptamine were found to be SERT-selective releasers, while DPT, DIPT, and MIPT were found to be SERT uptake inhibitors. These results suggest that the N, N-dialkyl analogs are not SERT substrates.

All neurotransmitter-releasing tryptamines were potent and efficacious 5-[HT.sub.2A] agonists, except NMT (14) and DMT (6), which were inactive at 10 (M). The two methoxy derivatives (17 and 18) had similar potencies in the 5-[HT.sub.2A] calcium assay, but 17 was less efficacious than 18.

All of the compounds in Table 2 were potent and efficacious 5-HT2A agonists, with compound 25 being the most potent and efficacious (119 %). Interestingly, the compounds containing a methoxy group on the phenyl ring were weak 5-HT uptake-inhibitors, but were potent 5-HT2A agonists.

Functional selectivity is becoming an important aspect in GPCR ligand development, as the agonist may be able to activate one downstream signaling pathway over another leading to differences in pharmacological effects.

There was no correlation between in vitro activity and psychoactive effects for SERT-mediated activity, 5-[HT.sub.2A] receptor activity, or 5-[HT.sub.1A] receptor activity, and five compounds have not been reported to have psychoactive effects. Further pharmacological characterization would need to be conducted.

We have synthesized and studied a set of tryptamines, most of which have been reported to have psychoactivity in humans. We found no obvious relationships between the receptor/transporter activity and the reported in vivo effects of the compounds examined.

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