The binding characteristics and orientation of a novel radioligand with distinct properties at 5-HT(3)A and 5-HT(3)AB receptors

VUF10166 (2-chloro-3-(4-methyl piperazin-1-yl)quinoxaline) is a ligand that binds with high affinity to 5-HT(3) receptors. Here we synthesise [(3)H]VUF10166 and characterise its binding properties at 5-HT(3)A and 5-HT(3)AB receptors. At 5-HT(3)A receptors [(3)H]VUF10166 displayed saturable binding with a K(d) of 0.18 nM. Kinetic measurements gave monophasic association (6.25 × 10(7) M(−1) min(−1)) and dissociation (0.01 min(−1)) rates that yielded a similar K(d) value (0.16 nM). At 5-HT(3)AB receptors two association (6.15 × 10(−7), 7.23 M(−1) min(−1)) and dissociation (0.024, 0.162 min(−1)) rates were seen, yielding K(d) values (0.38 nM and 22 nM) that were consistent with values obtained in saturation (K(d) = 0.74 nM) and competition (K(i) = 37 nM) binding experiments respectively. At both receptor types, specific binding was inhibited by classical 5-HT(3) receptor-selective orthosteric ligands (5-HT, allosetron, d-tubocurarine, granisetron, mCPBG, MDL72222, quipazine), but not by non-competitive antagonists (bilobalide, ginkgolide B, picrotoxin) or competitive ligands of other Cys-loop receptors (ACh, bicuculline, glycine, gabazine). To explore VUF10166 ligand–receptor interactions we used in silico modelling and docking, and tested the predictions using site directed mutagenesis. The data suggest that VUF10166 adopts a similar orientation to 5-HT(3) receptor agonists bound in AChBP (varenicline) and 5HTBP (5-HT) crystal structures.