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

Abstract

Palonosetron (Aloxi) is a potent second generation 5-HT(3) receptor antagonist whose mechanism of action is not yet fully understood. Palonosetron acts at the 5-HT(3) 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 (Tyr(73), Phe(130), Ser(163), and Asp(165)) and in the 5-HT3B receptor subunit (His(73), Phe(130), Glu(170), and Tyr(143)) that were previously predicted by in silico docking studies to interact with palonosetron. Homomeric (5-HT(3)A) and heteromeric (5-HT(3)AB) 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.

Keywords: 5-HT; 5-hydroxytryptamine (serotonin); Allosteric binding site; Computational studies; EC(50); FlexStation assays; HEK; IC(50); K(d); Palonosetron; Radioligand binding; Serotonin receptor; Site-directed mutagenesis; affinity constant; concentration of agonist required for half-maximal response; concentration of antagonist required for half-maximal inhibition; human embryonic kidney.

Graphical abstract

Figure 1

(A) A homology model of the human 5-HT₃A receptor showing the 5-HT binding site (orange square), a potential allosteric binding site (blue square) and the location of the 5-HT₃A subunit residues (stick representation) mutated in this study; inset: the potential allosteric binding site predicted by homology modelling and in silico docking, showing the 5-HT₃A subunit residues mutated in this study; (B) homology model of the human 5-HT₃B subunit showing the residues mutated in this study.

Figure 2

Previous predicted binding mode of palonosetron into the A+A− (A), and B+A− (B) interfaces, showing the interactions identified these in silico studies.