FUNCTIONAL CHARACTERIZATION OF SEROTONERGIC TRYPTAMINE ANALOGS VIA CALCIUM IMAGING: POTENCY AND EFFICACY PROFILING AT THE 5-HT₂A RECEPTOR

This study aimed to functionally characterize three serotonergic compounds, serotonin, psilocin, and a synthetic analog (X1), using a calcium imaging assay to evaluate their potency and efficacy at the 5-HT₂A receptor. This Gq-coupled GPCR plays a central role in perception, cognition, and mood and represents a key target for both psychedelic research and the development of novel neuropsychiatric therapies. U2OS HitSeeker cells stably expressing the 5-HT₂A receptor were lipofected with the genetic calcium indicator jGCaMP8m. Cells were stimulated with serotonin (10⁻⁶ µM - 100 µM), psilocin (10⁻⁴ µM - 200 µM), and X1 (10⁻⁴ µM - 200 µM). The intracellular calcium responses were recorded by wide-field fluorescence microscopy for 3 minutes. Fluorescence signals were normalized to the 60-second pre-stimulation baseline (ΔF/F₀), and the maximum post-stimulation response was extracted for each cell. Data were aggregated by replicate and concentration and fitted using a four-parameter logistic model (LL.4) implemented in the drc package in R software. Serotonin showed the highest potency (EC₅₀ = 3.2 nM), followed by psilocin (EC₅₀ = 15.4 nM) and X1 (EC₅₀ = 23.0 nM), with right-shifted dose–response curves relative to serotonin. Serotonin and psilocin achieved similar maximal efficacy (ΔF/F₀= 1.70), whereas X1 elicited a lower maximal response (ΔF/F₀ = 1.25), consistent with either partial agonism or reduced efficacy. Overall, this work demonstrates the utility of calcium imaging for characterizing the 5-HT₂A receptor, supports the presence of biased agonism, and underscores the need for multi-platform approaches in serotonergic drug development.