Allosteric Modulation of the Human 5-HT3A Receptor by 5-(trifluoromethyl)-indole

JC Palandri, S Butterworth, G Grafton, NM Barnes. University of Birmingham, Birmingham, UK

The 5-HT3 receptor belongs to the cys-loop ligand gated ion channel superfamily of receptors. The allosteric binding site of this receptor is a potential target for the treatment of irritable bowel syndrome. In a companion study we have identified a series of halogenated indole derivatives as potential allosteric modulators of the 5-HT3A receptor (Grafton et al., this meeting). One of these molecules, 5-(trifluoromethyl)-indole (5-TFMI) has been examined in more detail. The aim of this study was to investigate the effects of this molecule on the binding of full and partial orthosteric agonists and antagonists.

[3H]-Granisetron binding experiments were conducted using HEK293 cells expressing stably the human 5-HT3A receptor, with 5-HT (10-10-10-4 M), ondansetron (10-12-10-6 M), quipazine (10-12-10-6 M) and (S)-zacopride (10-13-3x10-7 M) competing for the radioligand in the absence and presence of 5-TFMI (3 and 10 µM). See Newman et al., Brit. J. Pharmacol. 169: 1228-1238, 2013 for detailed methodology.

Table 1. Ability of 5-TFMI to alter the interaction of agonists and partial agonists with the [3H]-granisetron-labelled 5-HT3A receptor. Data represent mean ± SEM, n = 4-6 independent experiments. * p < 0.05. ** p<0.01, Mann-Whitney U test

5-TFMI (concentrations up to 10 µM) did not enhance 5-HT-induced calcium release in fluorescence-based assays (data not shown), suggesting a lack of binding of 5-TFMI at the orthosteric site at these concentrations. 5-TFMI failed to impact the ability of ondansetron, a selective 5-HT3 receptor antagonist to compete for [3H]-granisetron (p=0.1). In contrast, the presence of 5-TFMI (3 µM and 10 µM) significantly increased the apparent affinities of 5-HT and quipazine (at 10 µM only) – the latter a 5-HT3 receptor partial agonist – for the [3H]-granisetron-labelled 5-HT3A receptor (Table 1). A slight leftward shift in (S)-zacopride competition curve was noticeable but not significant. 10µM 5-TFMI also significantly increased the Hill coefficients for the 5-HT and quipazine competition curves, suggesting modified cooperativity.

The data show 5-TFMI is able to impact agonist affinity for the radiolabelled 5-HT3 receptor at concentrations that do not interact with the orthosteric site of the receptor. 5-TFMI could prove a useful tool to help locate the molecular site of the allosteric binding site within the 5-HT3 receptor complex, which will aid rational drug design of suitable allosteric modulators of the 5-HT3 receptor with therapeutic potential.