title: Functional characterization and high-throughput screening of positive allosteric modulators of α7 nicotinic acetylcholine receptors in IMR-32 neuroblastoma cells.
abstract: α7 nicotinic acetylcholine receptors (nAChRs) are characterized by relatively low ACh sensitivity, rapid activation, and fast desensitization kinetics. ACh/agonist evoked currents at the α7 nAChR are transient, and, typically, calcium flux responses are difficult to detect using conventional fluorometric assay techniques. One approach to study interactions of agonists with the α7 nAChR is by utilizing positive allosteric modulators (PAMs). In this study, we demonstrate that inclusion of type II PAMs such as PNU-120596, but not type I, can enable detection of endogenous α7 nAChR-mediated calcium responses in human neuroblastoma (IMR-32) cells. Using this approach, we characterized the pharmacological profile of nicotine, epibatidine, choline, and other nAChR agonists such as PNU-282987, SSR-180711, GTS-21, OH-GTS21, tropisetron, NS6784, and A-582941. The rank order potency of agonists well correlated with α7 nAChR binding affinities measured in brain membranes. Inhibition of calcium response by methyllycaconitine in the presence of increasing concentrations of PNU-282987 or PNU-120596 revealed that the IC(50) value of methyllycaconitine was sensitive to varying concentrations of the agonist, but not that of the PAM. This format demonstrated the feasibility of this approach for high-throughput screening to identify small molecule, PAMs, which were further confirmed in electrophysiological assays of human α7 nAChR expressed in oocytes.
authors.affiliation: Advanced Technology, Global Pharmaceutical Research & Development, Abbott Laboratories, Abbott Park, Illinois 60064, USA. sujatha.m.gopalakrishnan@abbott.com
authors.forename: Sujatha M
authors.initials: SM
authors.lastname: Gopalakrishnan
authors.forename: Betsy M
authors.initials: BM
authors.lastname: Philip
authors.forename: Jens Halvard
authors.initials: JH
authors.lastname: Gronlien
authors.forename: John
authors.initials: J
authors.lastname: Malysz
authors.forename: David J
authors.initials: DJ
authors.lastname: Anderson
authors.forename: Murali
authors.initials: M
authors.lastname: Gopalakrishnan
authors.forename: Usha
authors.initials: U
authors.lastname: Warrior
authors.forename: David J
authors.initials: DJ
authors.lastname: Burns
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects / Q000502:physiology; D000818:Animals; D000831:Animals, Newborn; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005260:Female; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D009447:Neuroblastoma; D018722:Nicotinic Agonists / Q000737:chemistry* / Q000494:pharmacology*; D018733:Nicotinic Antagonists / Q000737:chemistry* / Q000494:pharmacology*; D051381:Rats; D011978:Receptors, Nicotinic / Q000502:physiology*; D014982:Xenopus laevis; D064569:alpha7 Nicotinic Acetylcholine Receptor
chemical_list: C579167:Chrna7 protein, human; C579165:Chrna7 protein, rat; D018722:Nicotinic Agonists; D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor