Receptor interaction profiles of novel psychoactive tryptamines compared with classic hallucinogens

Compounds and general assay approach: Test drugs included the novel tryptamines DiPT, 4-OH-DiPT, 4-OH-MET, 5-MeO-AMT and 5-MeO-MiPT, plus comparator classic hallucinogens (psilocin, LSD, DMT, mescaline) and MDMA. Purity was ≥98% and compounds were used as racemates. LSD and mescaline data were previously published but were re-used here because they were obtained concurrently using identical cell batches and assays. Radioligand binding assays: Human receptor and transporter binding was measured with membrane preparations from HEK293 cells overexpressing the targets (human genes except rodent TAAR1). Radioligands were used at concentrations equal to their Kd values; specific binding was defined as total minus nonspecific binding (determined with excess competitor). IC50s were obtained from three to five independent 10-point concentration–response curves and converted to Ki values using the Cheng–Prusoff equation. Functional 5-HT2A and 5-HT2B assays: 5-HT2A activity was measured in NIH-3T3 cells expressing human 5-HT2A using a calcium-fluorescence FLIPR assay; EC50 and maximal efficacy were estimated relative to 5-HT (set to 100%). 5-HT2B activity was assessed in HEK293 cells expressing human 5-HT2B using Fluo-4 calcium readout in a FLIPR setup, with EC50 and efficacy derived similarly. Transporter inhibition and release assays: NET, DAT and SERT inhibition were measured in HEK293 cells stably expressing the respective human transporters. Cells were preincubated with test compounds and then exposed to radiolabelled monoamines ([3H]NE, [3H]DA, [3H]5-HT); uptake was quantified and IC50 values obtained from variable-slope sigmoidal dose–response fits. Transporter-mediated release was tested at a single high concentration (100 µM) in preloaded cells; release was stopped after specified intervals and quantified by retained radioactivity. Nonspecific pseudo-efflux was controlled using transporter inhibitors and releasers were identified by ANOVA with Dunnett's test versus negative controls. Cytotoxicity: Cell integrity during assays was monitored with an adenylate-kinase release assay (ToxiLight) after 4 h exposure to 100 µM compound. Triton X-100 served as a positive control and 0.1% DMSO as vehicle control. Data analysis: Nonlinear regression was used to derive EC50, IC50 and Ki values. Correlations between receptor binding/activation metrics and reported psychoactive doses were assessed with Spearman rank correlations where reported.

Interactions with serotonin receptors: All tested tryptamines bound to and activated 5-HT receptors at mostly submicromolar concentrations. LSD showed the highest 5-HT2A receptor binding potency among the panel. Binding affinity at 5-HT2A was lower for all tryptamines, including the classics psilocin and DMT, compared with LSD; mescaline was the least potent. For the full set (n=10), 5-HT2A Ki values did not correlate with 5-HT2A EC50 values (Spearman R s =0.4, p>0.1). Analysing only the tryptamines (n=7), 5-HT2A binding affinity correlated with reported mean psychoactive doses (R s =0.9, p<0.05) whereas 5-HT2A activation potency did not (R s =0.6, p>0.1). Functional efficacy at 5-HT2A varied markedly. LSD and psilocin were partial agonists (28% and 16% efficacy, respectively). By contrast, several tryptamines displayed much higher activation efficacies, reaching up to 480% for DiPT and 5-MeO-MiPT. 5-MeO-AMT showed the highest 5-HT2A activation potency among the tryptamines. Selectivity of 5-HT2A over 5-HT2C binding was generally low (ratios <10) and several compounds also had comparable 5-HT1A potency; LSD uniquely exhibited single-digit nanomolar affinity at 5-HT1A. Binding to other monoamine receptors and transporters: Transporter binding was generally weak, except DiPT and 4-OH-MET, which showed submicromolar affinity for SERT. LSD was the only compound to bind α1-adrenergic receptors with submicromolar affinity; DMT had moderate α1 binding (Ki = 1.3 mM as reported in the extraction). LSD also bound α2-adrenergic receptors at submicromolar concentrations and was the sole compound with measurable affinity at dopamine D1–D3 receptors and rat TAAR1 in these assays. Monoamine uptake inhibition: Most substances (except mescaline and LSD) inhibited uptake at least at one transporter. Psilocin, DMT, DiPT and 4-OH-DiPT inhibited SERT with IC50 values reported in the low micromolar range, comparable to MDMA in these assays. Psilocin, DMT, DiPT and 4-OH-MET inhibited NET at lower potency than MDMA. Across the panel, DAT inhibition was absent or very weak for the tested compounds, in contrast to MDMA which inhibited DAT more strongly. Transporter-mediated monoamine release: Release assays were performed for tryptamines only. DMT induced 5-HT release, and 5-MeO-AMT induced release of both 5-HT and dopamine. None of the other tested tryptamines acted as substrate releasers under the conditions used. Releasers were identified as producing significantly greater efflux than non-releasing uptake inhibitors (statistical thresholds reported in the extraction). Cytotoxicity: No compound produced detectable cytotoxicity in the adenylate-kinase release assay after 4 h exposure to 100 µM, indicating preserved cell integrity during the pharmacology assays.

Rickli and colleagues interpret their findings as confirming that the tested novel tryptamines are 5-HT2A receptor agonists, a pharmacological property consistent with hallucinogenic activity. However, the tryptamines were generally less affine for 5-HT2A than LSD, which the authors relate to the lower in vivo potency of tryptamines compared with LSD. For the seven tryptamines examined, 5-HT2A binding affinity correlated significantly with estimated human psychoactive doses (R s =0.9, p<0.05), while 5-HT2A activation potency did not, and Ki and EC50 measures were not interchangeable across the series. Beyond 5-HT2A interaction, the authors emphasise that several tryptamines (notably DiPT and 4-OH-DiPT) inhibited SERT and in some cases NET, producing a pharmacological profile that resembles MDMA-like monoamine transporter effects in addition to 5-HT2A-mediated hallucinogenic activity. DMT and 5-MeO-AMT were shown to cause transporter-mediated 5-HT release (and for 5-MeO-AMT, DA release), which may further contribute to stimulant or empathogenic properties. By contrast, LSD differed in several respects: higher 5-HT2A binding affinity, partial 5-HT2A agonism with low efficacy, and substantial affinity at adrenergic and dopaminergic receptors (α1/α2 and D1–D3), features not shared by the tryptamines in this panel. The authors discuss structure–activity relationships consistent with prior literature: α-methylation and 5-methoxylation tended to increase 5-HT2A potency (illustrated by high potency of 5-MeO-AMT), 4-hydroxylation increased 5-HT2A binding (psilocin vs DMT; 4-OH-DiPT vs DiPT), and N-substitutions altered receptor interactions (N,N-isopropylation reduced 5-HT2 affinity compared with N,N-methylation, while asymmetrical N-methyl-N-isopropyl substitutions increased activity). TAAR1 binding was low-micromolar for some tryptamines; the authors note TAAR1 agonism has been linked to reduced stimulant properties in other compounds, but its relevance here remains uncertain. Limitations acknowledged by the investigators include the in vitro nature of the data: absorption, protein binding, brain penetration and metabolism will affect in vivo potency and toxicity and were not assessed. The authors cite the possibility that LSD may exhibit superior brain penetration compared with tryptamines as an example of such factors. Summarising their interpretation, the authors conclude that the in vitro pharmacology predicts that the studied tryptamines can produce both classical serotonergic hallucinogenic effects and MDMA-like psychoactive properties, whereas LSD’s broader adrenergic and dopaminergic interactions likely distinguish its in vivo profile.