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

A.J. Thompson, M.H.P. Verheij, J. Verbeek, A.D. Windhorst, I.J.P. de Esch, S.C.R. Lummis

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7 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)378-388
Number of pages11
Publication statusPublished - Nov 2014


  • Amino Acid Sequence
  • Binding, Competitive
  • HEK293 Cells
  • Humans
  • Journal Article
  • Molecular Docking Simulation
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Piperidines
  • Quinoxalines
  • Radioligand Assay
  • Radiopharmaceuticals
  • Receptors, Serotonin, 5-HT3
  • Research Support, Non-U.S. Gov't
  • Sequence Alignment
  • Tritium

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