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

Andrew J Thompson, Mark H P Verheij, Joost Verbeek, Albert D Windhorst, Iwan J P de Esch, Sarah C R Lummis

Research output: Contribution to JournalArticleAcademicpeer-review

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.

Original languageEnglish
Pages (from-to)378-388
Number of pages11
JournalNeuropharmacology
Volume86
DOIs
Publication statusPublished - Nov 2014

Fingerprint

Receptors, Serotonin, 5-HT3
Ligands
ginkgolide B
Cysteine Loop Ligand-Gated Ion Channel Receptors
Serotonin 5-HT3 Receptor Agonists
Quipazine
Granisetron
Tubocurarine
Quinoxalines
Picrotoxin
Bicuculline
Site-Directed Mutagenesis
Computer Simulation
Glycine
2-chloro-(4-methylpiperazine-1-yl)quinoxaline

Keywords

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

Cite this

Thompson, Andrew J ; Verheij, Mark H P ; Verbeek, Joost ; Windhorst, Albert D ; de Esch, Iwan J P ; Lummis, Sarah C R. / The binding characteristics and orientation of a novel radioligand with distinct properties at 5-HT3A and 5-HT3AB receptors. In: Neuropharmacology. 2014 ; Vol. 86. pp. 378-388.
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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.",
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author = "Thompson, {Andrew J} and Verheij, {Mark H P} and Joost Verbeek and Windhorst, {Albert D} and {de Esch}, {Iwan J P} and Lummis, {Sarah C R}",
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language = "English",
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The binding characteristics and orientation of a novel radioligand with distinct properties at 5-HT3A and 5-HT3AB receptors. / Thompson, Andrew J; Verheij, Mark H P; Verbeek, Joost; Windhorst, Albert D; de Esch, Iwan J P; Lummis, Sarah C R.

In: Neuropharmacology, Vol. 86, 11.2014, p. 378-388.

Research output: Contribution to JournalArticleAcademicpeer-review

TY - JOUR

T1 - The binding characteristics and orientation of a novel radioligand with distinct properties at 5-HT3A and 5-HT3AB receptors

AU - Thompson, Andrew J

AU - Verheij, Mark H P

AU - Verbeek, Joost

AU - Windhorst, Albert D

AU - de Esch, Iwan J P

AU - Lummis, Sarah C R

N1 - Copyright © 2014. Published by Elsevier Ltd.

PY - 2014/11

Y1 - 2014/11

N2 - 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.

AB - 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.

KW - Amino Acid Sequence

KW - Binding, Competitive

KW - HEK293 Cells

KW - Humans

KW - Molecular Docking Simulation

KW - Molecular Sequence Data

KW - Mutagenesis, Site-Directed

KW - Piperidines

KW - Quinoxalines

KW - Radioligand Assay

KW - Radiopharmaceuticals

KW - Receptors, Serotonin, 5-HT3

KW - Sequence Alignment

KW - Tritium

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1016/j.neuropharm.2014.08.008

DO - 10.1016/j.neuropharm.2014.08.008

M3 - Article

VL - 86

SP - 378

EP - 388

JO - Neuropharmacology

JF - Neuropharmacology

SN - 0028-3908

ER -