title: The binding characteristics and orientation of a novel radioligand with distinct properties at 5-HT3A and 5-HT3AB receptors.
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.
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge, UK.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Thompson
authors.affiliation: Amsterdam Institute for Molecules Medicines and Systems (AIMMS), Division of Medicinal Chemistry, Faculty of Sciences, VU University Amsterdam, Amsterdam, The Netherlands.
authors.forename: Mark H P
authors.initials: MH
authors.lastname: Verheij
authors.affiliation: VU University Medical Center, Dept Radiology & Nuclear Medicine, Amsterdam, The Netherlands.
authors.forename: Joost
authors.initials: J
authors.lastname: Verbeek
authors.affiliation: VU University Medical Center, Dept Radiology & Nuclear Medicine, Amsterdam, The Netherlands.
authors.forename: Albert D
authors.initials: AD
authors.lastname: Windhorst
authors.affiliation: Amsterdam Institute for Molecules Medicines and Systems (AIMMS), Division of Medicinal Chemistry, Faculty of Sciences, VU University Amsterdam, Amsterdam, The Netherlands.
authors.forename: Iwan J P
authors.initials: IJ
authors.lastname: de Esch
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge, UK. Electronic address: sl120@cam.ac.uk.
authors.forename: Sarah C R
authors.initials: SC
authors.lastname: Lummis
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D057809:HEK293 Cells; D006801:Humans; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010880:Piperidines / Q000138:chemical synthesis / Q000378:metabolism*; D011810:Quinoxalines / Q000138:chemical synthesis / Q000378:metabolism*; D011869:Radioligand Assay; D019275:Radiopharmaceuticals / Q000138:chemical synthesis / Q000378:metabolism*; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D014316:Tritium
chemical_list: C574388:2-chloro-(4-methylpiperazine-1-yl)quinoxaline; C509409:HTR3A protein, human; D010880:Piperidines; D011810:Quinoxalines; D019275:Radiopharmaceuticals; D044406:Receptors, Serotonin, 5-HT3; D014316:Tritium
keywords: 5-HT(3); Agonist; Antagonist; Cys-loop; Ion channel; Radioligand