Exploring a potential palonosetron allosteric binding site in the 5-HT 3 receptor

Palonosetron (Aloxi) is a potent second generation 5-HT3 receptor antagonist whose mechanism of action is not yet fully understood. Palonosetron acts at the 5-HT3 receptor binding site but recent computational studies indicated other possible sites of action in the extracellular domain. To test this hypothesis we mutated a series of residues in the 5-HT3A receptor subunit (Tyr73, Phe130, Ser 163, and Asp165) and in the 5-HT3B receptor subunit (His73, Phe130, Glu170, and Tyr143) that were previously predicted by in silico docking studies to interact with palonosetron. Homomeric (5-HT3A) and heteromeric (5-HT3AB) receptors were then expressed in HEK293 cells to determine the potency of palonosetron using both fluorimetric and radioligand methods to test function and ligand binding, respectively. The data show that the substitutions have little or no effect on palonosetron inhibition of 5-HT-evoked responses or binding. In contrast, substitutions in the orthosteric binding site abolish palonosetron binding. Overall, the data support a binding site for palonosetron at the classic orthosteric binding pocket between two 5-HT3A receptor subunits but not at allosteric sites previously identified by in silico modelling and docking

Bivalent Ligands for the Serotonin 5-HT3 Receptor

The serotonin 5-HT3 receptor is a ligand-gated ion channel, which by virtue of its pentameric architecture, can be considered to be an intriguing example of intrinsically multivalent biological receptors. This paper describes a general design approach to the study of multivalency in this multimeric ion channel. Bivalent ligands for 5-HT3 receptor have been designed by linking an arylpiperazine moiety to probes showing different functional features. Both homobivalent and heterobivalent ligands have shown 5-HT3 receptor affinity in the nanomolar range, providing evidence for the viability of our design approach. Moreover, the high affinity shown by homobivalent ligands suggests that bivalency is a promising approach in 5-HT3 receptor modulation and provides the rational basis for applying the concepts of multivalency to the study of 5-HT3 receptor function