An investigation of allosteric modulation of the human 5-HT3A receptor using 5-halo-indoles

Gillian Grafton1, Alessandra Chiarenza1,2, Silvia Gambassi1,2, Andrew Powell1, Nicholas Barnes1. 1University of Birmingham, Birmingham, UK, 2University of Siena, Siena, Italy

The human 5-HT3A receptor is a member of the cys-loop ligand-gated ion channel family. Various molecules had been identified as positive allosteric modulators of this receptor, including alcohols, volatile anaesthetics and most recently 5-chloroindole. In the present study we have used binding assays, fluorescence indicators and patch clamp techniques to explore the allosteric modulation of human 5-HT3A receptor stably expressed in HEK293 cells. Affinity (Ki) and Hill slope values arising from various 5-HT3 receptor agonists (DDP 733, (s)-zacopride and quipazine) and an antagonist (ondansetron) were consistent with the pharmacology of the human 5-HT3 receptor. Neither 5-chloroindole nor 5-iodoindole competed with [3H]-granisetron for the 5-HT3A receptor orthosteric site (concentrations up to 100 µM). Furthermore neither of these halogenated indoles (10-30 µM) altered the affinity of ondansetron for the receptor. In contrast, 5-chloroindole provoked a concentration dependent increase in the affinity of the partial agonist quipazine for the 5-HT3A receptor from 19 ± 3 nM to 11 ± 1 nM and 6 ± 1 nM at 10 µM and 30 µM, respectively. DDP 733 displayed an affinity for the 5-HT3A receptor of 2.3 ± 0.5 nM and 5-chloroindole increased the affinity to 2.2 ± 0.04 (10 μM 5-chloroindole), 1.6 ± 0.05 (30 μM 5-chloroindole), and 1.6 ± 0.05 (100 μM 5-chloroindole). Similarly 5-iodoindole provoked an increase in the affinity of quipazine from 23 ± 1 nM to 8 ± 1 nM and 3 ± 0.2 nM at 10 µM and 30 µM, respectively. 5-Iodoindole also increased the affinity of DDP773 for the receptor from 3.9 ± 0.1 nM to 1.9 ± 0.4 nM (10 μM 5-iodoindole) and 1.5 ± 0.2 nM (30 μM 5-iodoindole). Finally, 10 and 30 μM 5-iodoindole provoked an increase in affinity of (S)-zacopride from 0.98 ± 0.11 nM to 0.61 ± 0.07 nM and 0.64 ± 0.07 nM, respectively. Determination of functional responses (increase in intracellular calcium assayed by fluorescence dye on Flex Station and current recorded by whole cell patch clamp) demonstrated the ability of 5-chloroindole and 5-iodoindole (10-30 µM) to potentiate responses to the partial agonists.

In this study we have extended our previous observations of the usefulness of 5-chloroindole (and now 5-iodoindole) as a pharmacological tool for exploring the allosteric modulation of the 5-HT3A receptor. Further investigations with chimeric receptors to map the binding site(s) for these halogenated indoles will allow rational design of drugs that may become useful therapeutic agents via modulation of 5-HT3 receptor function.