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

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.

Approaching the 5-HT3 receptor heterogeneity by computational studies of the transmembrane and intracellular domains.

5-hydroxytryptamine type-3 receptor (5-HT3), an important target of many neuroactive drugs, is a cation selective transmembrane pentamer whose functional stoichiometries and subunit arrangements are still debated, due to the extreme complexity of the system. The three dimensional structure of the 5-HT3R subunits has not been solved so far. Moreover, most of the available structural and functional data is related to the extracellular ligand-binding domain, whereas the transmembrane and the intracellular receptor domains are far less characterised, although they are crucial for receptor function. Here, for the first time, 3D homology models of the transmembrane and the intracellular receptor domains of all the known human 5-HT3 subunits have been built and assembled into homopentameric (5-HT(3A)R, 5-HT(3B)R, 5-HT(3C)R, 5-HT(3D)R and 5-HT(3E)R) and heteropentameric receptors (5-HT(3AB), 5-HT(3AC), 5-HT(3AD) and 5-HT(3AE)), on the basis of the known three-dimensional structures of the nicotinic-acetylcholine receptor and of the ligand gated channel from Erwinia chrysanthemi. The comparative analyses of sequences, modelled structures, and computed electrostatic properties of the single subunits and of the assembled pentamers shed new light both on the stoichiometric composition and on the physicochemical requirements of the functional receptors. In particular, it emerges that a favourable environment for the crossing of the pore at the transmembrane and intracellular C terminus domain levels by Ca²âº ions is granted by the maximum presence of two B subunits in the 5-HT3 pentamer.

A first step towards the understanding of the 5-HT3 receptor subunit heterogeneity from a computational point of view.

The functional serotonin type-3 receptor (5-HT(3)-R), which is the target of many neuroactive drugs, is known to be a homopentamer made of five identical subunits A (5-HT(3A)-R) or a binary heteropentamer made of subunits A and B (5-HT(3A/B)-R) with a still debated arrangement and stoichiometry. This complex picture has been recently further complicated by the discovery of additional 5-HT(3)-R subunits, C, D, and E, which, similarly to the B subunit, are apparently able to form functional receptors only if co-expressed with subunit A. Being the binding site for both serotonin and antagonists (i.e. drugs) located at the extracellular interface between two adjacent subunits, the large variability of the 5-HT(3)-R composition becomes a crucial issue, since it can originate many different interfaces providing non-equivalent ligand binding sites and complicating the pharmacological modulation. Here, the different 5-HT(3)-R interfaces are analysed, on the bases of the structural conformations of previously built 3D homology models and of the known subunit sequences, by addressing their physicochemical characterization. The results confirm the presence of an aromatic cluster located in the core of the A-A interface as a key determinant for having an interface both stable and functional. This is used as a discriminant to make hypotheses about the capability of all the other possible interfaces constituted by the known 5-HT(3)-R sequences A, B, C, D, and E to build active receptors.

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.