How 5-HT2A and 5-HT2B receptor reserves shape agonist pharmacology and implications for preclinical safety testing

This article and associated images are based on a poster originally authored by Sophie O’Neil, Kyle Thompson, Rita Pereira, Emily Russell and Stuart McElroy and presented at ELRIG Drug Discovery 2025 in affiliation with BioAscent Discovery Ltd and University of Glasgow.

This poster is being hosted on this website in its raw form, without modifications. It has not undergone peer review but has been reviewed to meet AZoNetwork's editorial quality standards. The information contained is for informational purposes only and should not be considered validated by independent peer assessment.

Introduction

• 5-HT_2A receptors are expressed abundantly in the CNS and are attractive clinical targets for anxiety, psychosis and OCD.
• 5-HT_2B receptors are expressed in the cardiovascular system, and their chronic activation is known to induce adverse cardiopulmonary effects.
• Many 5-HT_2A ligands lack specificity, leading to off-target 5-HT_2B effects.
• Differences in GPCR expression levels and the presence of receptor reserves can alter both the receptor coupling efficiency and the apparent pharmacological profile of ligands ^1,2.
• We use the 5-HT_2A/2B receptors as a case study to demonstrate how these factors may result in underestimation of compound activity and misrepresentation of clinical safety and efficacy, highlighting the need for careful mechanistic evaluation.

Methods

Multiple HEK-293T cell lines stably expressing human 5-HT_2A or 5-HT_2B receptors were generated at BioAscent via cationic lipid-mediated transfection and Hygromycin B selection. Receptor expression levels were quantified in all clones by RT-qPCR, and three clones per receptor were progressed, based on a range of expression levels (Figure 1), for full pharmacological characterisation using intracellular Ca²⁺ mobilisation FLIPR (Figure 2) and xCELLigence label-free impedance (Figure 3) assays.

Figure 1. Quantification of 5-HT_2A/5-HT_2B receptor expression by RT-qPCR. 10 ng of cDNA was used for qPCR with PowerUp^TM SYBR Green Master Mix. Transcript levels were normalised to GAPDH and expressed relative to the lowest expressing clone using the 2^-ΔΔCt method. Mean triplicate expression data ± SEM is shown. (n=3/N=3). Image Credit: Image courtesy of Sophie O’Neil et al., in partnership with ELRIG (UK) Ltd.

Figure 2. Schematic overview of the Ca²⁺ mobilization assay. Image Credit: Adapted from product insert R8190, Molecular Devices, 2019.

Figure 3. Principle of the cellular impedance xCELLigence assay. Image Credit: Adapted from Agilent, www.agilent.com/en/technology/cellular-impedance

FLIPR – Intracellular Ca²⁺ release assay

Figure 4. 5-HT₂ receptor agonists show expression-dependent potency and efficacy profiles at 5-HT_2A and 5-HT_2B receptors. Agonist DRCs were normalised to DMSO (min control) and 10 μM 5-HT (max control) and plotted for 5-HT_2A (A) and 5-HT_2B (B) stable cell clones. Mean duplicate data from three independent runs (n=2/N=3) is shown ± SEM. Mean agonist potencies (pEC50) and mean Emax ± SEM are tabulated for each clone. Image Credit: Image courtesy of Sophie O’Neil et al., in partnership with ELRIG (UK) Ltd.

xCELLigence – 5-HT_2A cellular impedance assay

Figure 5. 5-HT₂ agonists produce positive dose-dependent impedance responses. Normalised cell index traces (A, C) from 5-HT_2A Clone #1 following stimulation with 5-HT (A) and Psilocin (C). Normalised cell index values were converted to ‘% Impedance Response’ relative to the baseline and 10 μM 5-HT responses, and plotted as dose-response curves for 5-HT_2A Clones #1 and #2 (B; 5-HT, D; Psilocin). Image Credit: Image courtesy of Sophie O’Neil et al., in partnership with ELRIG (UK) Ltd.

Figure 6. The 5-HT_2A inverse agonist, Volinanserin, produces a negative dose-dependent impedance response in 5-HT_2A clones with high receptor reserves. Normalised cell index traces from 5-HT_2A Clone #1 (A), Clone #2 (B) and Clone #3 (C) following pretreatment with Volinanserin. Baseline normalised cell index values for all three clones were plotted against Volinanserin concentration, to generate inverse agonist dose-response curves (D) at the peak negative impedance response. Image Credit: Image courtesy of Sophie O’Neil et al., in partnership with ELRIG (UK) Ltd.

Conclusions

We show that differences in receptor levels significantly alters the pharmacological profiles of 5-HT_2A/2B ligands across different assay technologies. Failure to account for receptor density/reserves may lead to underestimation of partial agonism/inverse agonism and off-target toxicity in pre-clinical screens, especially for CNS-active compounds like psilocin/norpsilocin. These findings underscore the necessity to incorporate multiple physiologically-relevant models and assay approaches into pre-clinical drug safety/efficacy profiling, to ensure accurate predictions of clinical outcomes.

References

1. Jacobson, K.A. (2015). New paradigms in GPCR drug discovery. Biochemical Pharmacology, 98(4), pp.541–555. DOI: 10.1016/j.bcp.2015.08.085. https://www.sciencedirect.com/science/article/pii/S0006295215005146?via%3Dihub.
2. Li, A., et al. (2023). Loss of biased signaling at a G protein-coupled receptor in overexpressed systems. PLOS ONE, 18(3), e0283477. DOI: 10.1371/journal.pone.0283477. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0283477.

About BioAscent Discovery

Founded in 2013, BioAscent is a leading provider of integrated drug discovery services based at the former Organon / MSD R&D site in Newhouse, Scotland, UK. 

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Last Updated: Jan 6, 2026