Novel allosteric modulation of the human 5-HT3A receptor by the clinically used drugs piperazine and hydroxyzine

Abstract

The 5-HT3 receptor is a serotonin-gated cation channel of the pentameric ligand-gated ion channel family andplays a key role in fast synaptic transmission within the central, peripheral, and enteric nervous systems. Dysregulationof this receptor has been linked to neurological, psychiatric, and gastrointestinal disorders. Whilecompetitive 5-HT3 antagonists, primarily setrons, are clinically used to alleviate gastrointestinal symptoms, thereis a growing interest in developing new negative allosteric modulators (NAMs) for this receptor. Here, we applieda drug-repositioning strategy targeting the human 5-HT3A receptor. Using macroscopic current recordings, weidentified a previously unrecognized NAM activity of two clinically used drugs, piperazine and its derivativehydroxyzine, on the 5-HT3A receptor. Computational analyses suggested two distinct binding sites at subunitinterfaces: one in the lower extracellular domain and another in the upper transmembrane domain. Functionalcharacterization of receptors carrying mutations at key residues within these sites (E75, E208, S248, D293)showed that some mutations abolished drug-induced inhibition, whereas others enhanced the inhibitory effect ofhydroxyzine. Altogether, these results validate the proposed allosteric sites and demonstrate that both contributeto the actions of these compounds. This work uncovers a new functional connection between 5-HT3A receptorsand piperazine-derived drugs, opening avenues for therapeutic repurposing.