Selective Allosteric Modulation of the 5-HT3A Receptor by Halogenated Indole Derivates

G Grafton1, S Butterworth1, AD Powell1, NS Millar2, NM Barnes1. 1University of Birmingham, Birmingham, UK, 2University College London, London, UK

The human 5-HT3A receptor is a member of the cys-loop ligand-gated ion channel family. We have recently demonstrated positive allosteric modulation of this receptor by 5-chloroindole (Newman et al., Brit. J. Pharmacol. 169: 1228-1238, 2013) and 5-iodoindole.

In the present study we have examined functional responses to a variety of halogenated-indoles. Increases in intracellular calcium (assayed using a Flex Station) and current changes recorded by whole cell patch clamp techniques (see Newman et al., 2013 for methodologies) were used to explore the allosteric modulation of the human 5-HT3A receptor stably expressed in HEK293 cells. Selected experiments were also performed on HEK293 cells expressing the alpha-7 nicotinic receptor and HEK293 cells expressing a chimaeric nicotinic alpha-7/5-HT3A receptor to explore the potential binding site(s) for these modulators. Potency was calculated from the percentage change in the intracellular calcium response normalised to the response evoked by 300 nM 5HT. Responses evoked by the potential positive allosteric modulators ranged up to 700% of that evoked by 5HT alone.

We examined a range of halogenated indole derivatives and discovered eighteen novel potential positive allosteric modulators of the 5-HT3 receptor. Initial studies demonstrate an increase in potency associated with an increase of halogen size at the 5-position: 5-(trifluoromethyl)-indole > 5-Cl-indole > 5-F-indole:- EC50s respectively:3.1 ± 0.5 (n = 7) μμM, 13.5 ± 2.1 μM (n = 4), 177 ± 140 μM (n = 3). Halogenation of the 6-position reduced the activity of the allosteric modulator: 5-Cl-indole vs 6-Cl-indole: EC50 13.5 ± 0.5 μM (n = 4) vs 121 ± 59 μM (n = 3), respectively; p = 0.04 (t-test).

Recordings from voltage-clamped HEK293 cells expressing the h5-HT3A receptor showed that 5-Cl-indole (10 μM) potentiated the response to 10 μM 5HT. However in HEK293 cells expressing stably the human nicotinic alpha-7 receptor (as well as RIC-3), 5-Cl-indole (10 μM) failed to potentiate responses to the selective alpha-7 nicotinic receptor agonist PNU 282 977, suggesting specificity of 5-Cl-indole for the 5-HT3A receptor. Furthermore, in cells expressing a chimaeric (rat/mouse) alpha-7V201-5HT3A receptor, 5-Cl-indole failed to potentiate the response to PNU 282 977, suggesting that the binding site for 5-Cl-indole lies in the extracellular N-terminal portion of the 5-HT3A receptor.

Our preliminary data are allowing us to define the region of the receptor which binds the halogenated-indole allosteric modulators and to begin to define a structure-activity relationship for such allosteric modulators. These studies will aid rational drug design of potential therapeutic agents that modulate 5-HT3 receptor function.