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

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

Author information

1
Department of Biochemistry, University of Cambridge, Cambridge, UK.
2
Amsterdam Institute for Molecules Medicines and Systems (AIMMS), Division of Medicinal Chemistry, Faculty of Sciences, VU University Amsterdam, Amsterdam, The Netherlands.
3
VU University Medical Center, Dept Radiology & Nuclear Medicine, Amsterdam, The Netherlands.
4
Department of Biochemistry, University of Cambridge, Cambridge, UK. Electronic address: sl120@cam.ac.uk.

Abstract

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

PMID:
25174552
PMCID:
PMC4220016
DOI:
10.1016/j.neuropharm.2014.08.008
[Indexed for MEDLINE]
Free PMC Article

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