title: On-line screening of conformationally constrained nicotines and anabasines for agonist activity at the alpha3beta4- and alpha4beta2-nicotinic acetylcholine receptors using immobilized receptor-based liquid chromatographic stationary phases.
abstract: Liquid chromatography columns containing stationary phases based upon immobilized nicotinic acetylcholine receptors (nAChRs) were used to screen a series of conformationally constrained nicotine and anabasine derivatives for agonist activity. The alpha3beta4 nAChR and alpha4beta2 nAChR subtypes were used to prepare the chromatographic columns and [(3)H] epibatidine dihydrochloride ([(3)H] EB) was used as the marker ligand. Single displacement experiments were conducted with the test ligands and with nicotine and carbachol. Nicotine was used as an internal control for compounds with agonist activity and carbachol was used as an internal control for compounds with very weak agonistic activity (K(d) > 4700 nM for alpha3beta4). The displacement of [(3)H] EB by each of the test compounds and internal controls was calculated and expressed as Deltaml. Functional studies were then conducted using a stably transfected cell line that expresses the alpha3beta4 nAChR and EC(50) values were determined for the test compounds and the internal controls. A comparison of the Deltaml and EC(50) values indicated that 9/11 compounds had been correctly identified as agonists or non-agonists of the alpha3beta4 nAChR. A similar comparison could not be made for the alpha4beta2 nAChR, since the intact cell line was not available for testing. The results of the study suggest that the immobilized nAChR columns can be used for the rapid on-line screening of compounds for their relative affinities for the immobilized receptor and as an initial determination of qualitative functional activities.
authors.affiliation: Bioanalytical and Drug Discovery Unit, National Institute on Aging, National Institutes of Health, Gerontology Research Center, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825, USA.
authors.forename: Ruin
authors.initials: R
authors.lastname: Moaddel
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Jozwiak
authors.forename: Rika
authors.initials: R
authors.lastname: Yamaguchi
authors.forename: Christopher
authors.initials: C
authors.lastname: Cobello
authors.forename: Kevin
authors.initials: K
authors.lastname: Whittington
authors.forename: Tarun K
authors.initials: TK
authors.lastname: Sarkar
authors.forename: Sankar
authors.initials: S
authors.lastname: Basak
authors.forename: Irving W
authors.initials: IW
authors.lastname: Wainer
mesh_terms: D000691:Anabasine / Q000737:chemistry / Q000494:pharmacology*; D002853:Chromatography, Liquid / Q000295:instrumentation* / Q000379:methods; D009538:Nicotine / Q000737:chemistry / Q000494:pharmacology*; D018722:Nicotinic Agonists / Q000737:chemistry / Q000494:pharmacology*; D011978:Receptors, Nicotinic / Q000187:drug effects*
chemical_list: D018722:Nicotinic Agonists; D011978:Receptors, Nicotinic; C419362:nicotinic receptor alpha3beta2; C403530:nicotinic receptor alpha3beta4; D009538:Nicotine; D000691:Anabasine