2026 ECNP Congress

PS02-2206

Trace amine-associated receptor 1 as a target for monoaminergic activity enhancers: cyclic adenosine monophosphate profiling of 1-(indol-3-yl)-2-propylaminopentane versus beta-phenethylamine

Yashar Chalabiani — B.M. Kestecher, S. Ghosal, A.J. Ernyey, L.G. Hársing, X. Osteikoetxea, I. Miklya · Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest, Hungary

2026 ECNP Congress

Introduction

Trace amine–associated receptor 1 (TAAR1) is an intracellular, pleiotropic G protein–coupled receptor (GPCR) activated by endogenous trace amines such as β‑phenethylamine (β-PEA) and tryptamine (TRP), and it is also engaged by monoamine-releasing agents (MRAs) including amphetamine and methamphetamine [1, 2]. At low concentrations, β-PEA and TRP can act as monoaminergic activity enhancers (MAEs), preferentially facilitating vesicular neurotransmitter release [3], whereas MRAs trigger both vesicular release and non-vesicular, transporter-mediated monoamine efflux [4]. 1-(indol-3-yl)-2-propylaminopentane ((−)-IPAP) is a synthetic, tryptamine-derived MAE candidate designed to avoid MRA-like releasing properties [5]. Here, we investigate its mechanism of action (MOA) and assess its TAAR1-dependent signalling efficacy.

Aims

To investigate the MOA of the synthetic MAE compound (−)-IPAP at TAAR1 by measuring receptor-mediated cAMP accumulation in rat-TAAR1-expressing Expi293F suspension cells.

Methods

Dose–response curves (10⁻¹⁰–10⁻⁴M) were generated for β-phenethylamine (β-PEA; Sigma-Aldrich) and 1-(indol-3-yl)-2-propylaminopentane ((−)-IPAP; Fujimoto Pharmaceutical Corp, Japan) using seven-point serial dilutions prepared in assay buffer and applied to rTAAR1-expressing Expi293F cells to quantify TAAR1-mediated cAMP accumulation.

rTAAR1 cDNA (rTAAR1; GeneWiz) was cloned into the pcDNA3 mammalian expression vector with a CMV promoter. The plasmid was transformed into DH5α and grown using ampicillin selection. Plasmids were isolated and purified using a plasmid preparation kit, and sequenced prior to use. Expi293F suspension cells were transiently transfected using polyethyleneimine. After 48–72 h, receptor expression was confirmed by PCR, and cells were harvested immediately before the assay.

cAMP accumulation was measured using the Revvity cAMP-Gs Dynamic homogeneous time-resolved fluorescence (HTRF) assay, a competitive immunoassay based on HTRF. Fluorescence ratios (665 nm/620 nm) were plotted against a compound concentration, signals were inversely proportional to intracellular cAMP levels. EC₅₀ values were determined using 4-parameter logistic (4PL) non-linear regression.

Results

In rTAAR1-expressing Expi293F cells, cAMP responses were quantified across seven-point concentration–response curves and normalised to the maximal response evoked by β-phenethylamine (β-PEA; 100%). (−)-IPAP produced a robust, concentration-dependent increase in cAMP with slightly reduced maximal efficacy, reaching 89.99% of the β-PEA Emax. Concentration–response data were fitted using four-parameter logistic (4PL) non-linear regression with good fit (β-PEA: R² = 0.956; (−)-IPAP: R² = 0.964). The EC₅₀ of β-PEA was 8.96 × 10⁻⁷ M (logEC₅₀ = −6.048), whereas (−)-IPAP exhibited an EC₅₀ of 2.03 × 10⁻⁷ M (logEC₅₀ = −6.691), indicating an approximately 4.4-fold higher apparent potency for (−)-IPAP. Data are presented as mean ± SEM from n = 3 independent experiments.

Conclusions

In rTAAR1-expressing Expi293F cells, the synthetic MAE candidate (−)-IPAP elicited robust TAAR1-mediated cAMP signalling, displaying higher potency than β-PEA (~4.4-fold) with slightly reduced maximal efficacy(~90% of β-PEA). This profile supports (−)-IPAP as a potent TAAR1 agonist consistent with an MAE-like pharmacological signature rather than an MRA-like pattern. Further work should confirm TAAR1 specificity using pharmacological blockade with a selective TAAR1 antagonist and determine whether this signalling translates into MAE-relevant functional endpoints in native monoaminergic preparations, such as enhancement of electrically evoked monoamine release without inducing transporter-mediated efflux.

References

  1. .Pei, Y., A. Asif-Malik, and J.J. Canales, Trace Amines and the Trace Amine-Associated Receptor 1: Pharmacology, Neurochemistry, and Clinical Implications. Front Neurosci, 2016. 10: p. 148.
  2. .Miller, G.M., The emerging role of trace amine-associated receptor 1 in the functional regulation of monoamine transporters and dopaminergic activity. J Neurochem, 2011. 116(2): p. 164–76.
  3. .Shimazu, S. and I. Miklya, Pharmacological studies with endogenous enhancer substances: beta-phenylethylamine, tryptamine, and their synthetic derivatives. Prog Neuropsychopharmacol Biol Psychiatry, 2004. 28(3): p. 421–7.
  4. .Robertson, S.D., H.J. Matthies, and A. Galli, A closer look at amphetamine-induced reverse transport and trafficking of the dopamine and norepinephrine transporters. Mol Neurobiol, 2009. 39(2): p. 73–80.
  5. .Harsing, L.G., J. Knoll, and I. Miklya, Enhancer Regulation of Dopaminergic Neurochemical Transmission in the Striatum. International Journal of Molecular Sciences, 2022. 23(15): p. 8543.