The binding characteristics and orientation of a novel radioligand with distinct properties at 5-HT3A and 5-HT3AB receptors

Neuropharmacology. 2014 Nov;86:378-88. doi: 10.1016/j.neuropharm.2014.08.008. Epub 2014 Aug 28.


VUF10166 (2-chloro-3-(4-methyl piperazin-1-yl)quinoxaline) is a ligand that binds with high affinity to 5-HT3 receptors. Here we synthesise [(3)H]VUF10166 and characterise its binding properties at 5-HT3A and 5-HT3AB receptors. At 5-HT3A receptors [(3)H]VUF10166 displayed saturable binding with a Kd 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 Kd value (0.16 nM). At 5-HT3AB 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 Kd values (0.38 nM and 22 nM) that were consistent with values obtained in saturation (Kd = 0.74 nM) and competition (Ki = 37 nM) binding experiments respectively. At both receptor types, specific binding was inhibited by classical 5-HT3 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-HT3 receptor agonists bound in AChBP (varenicline) and 5HTBP (5-HT) crystal structures.

Keywords: 5-HT(3); Agonist; Antagonist; Cys-loop; Ion channel; Radioligand.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Binding, Competitive
  • HEK293 Cells
  • Humans
  • Molecular Docking Simulation
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Piperidines / chemical synthesis
  • Piperidines / metabolism*
  • Quinoxalines / chemical synthesis
  • Quinoxalines / metabolism*
  • Radioligand Assay
  • Radiopharmaceuticals / chemical synthesis
  • Radiopharmaceuticals / metabolism*
  • Receptors, Serotonin, 5-HT3 / chemistry
  • Receptors, Serotonin, 5-HT3 / genetics
  • Receptors, Serotonin, 5-HT3 / metabolism*
  • Sequence Alignment
  • Tritium


  • 2-chloro-(4-methylpiperazine-1-yl)quinoxaline
  • HTR3A protein, human
  • Piperidines
  • Quinoxalines
  • Radiopharmaceuticals
  • Receptors, Serotonin, 5-HT3
  • Tritium