TY - JOUR T1 - Development of a Radioligand, [<sup>3</sup>H]-LY2119620, to Probe the Human M<sub>2</sub> and M<sub>4</sub> Muscarinic Receptor Allosteric Binding Sites JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.114.091785 SP - mol.114.091785 AU - Douglas A. Schober AU - Carrie H. Croy AU - Hongling Xiao AU - Arthur Christopoulos AU - Christian C. Felder Y1 - 2014/01/01 UR - http://molpharm.aspetjournals.org/content/early/2014/05/07/mol.114.091785.abstract N2 - In this study, we described the characterization of a muscarinic acetylcholine receptor (mAChR) potentiator, LY2119620 (3-amino-5-chloro-N-cyclopropyl-4-methyl-6-[2-(4-methylpiperazin-1-yl)-2-oxoethoxy]thieno[2,3-b]pyridine-2-carboxamide) as a novel probe of the human M2 and M4 allosteric binding sites. Since the discovery of allosteric binding sites on G-protein coupled receptors (GPCRs), compounds targeting these novel sites are starting to emerge. For example, LY2033298 (Chan et al., 2008) and a derivative of this chemical scaffold, VU152100 (Brady et al., 2008), bind to the human M4 mAChR allosteric pocket. In the current study we characterized LY2119620, a compound similar in structure to LY2033298 that binds to the same allosteric site on the human M4 mAChRs. However, LY2119620 also binds to an allosteric site on the human M2 subtype. [3H]-NMS binding experiments confirm that LY2119620 does not compete for the orthosteric binding pocket at any of the five muscarinic receptor subtypes. Dissociation kinetic studies using [3H]-NMS further support that LY2119620 binds allosterically to the M2 and M4 mAChRs and was positively cooperative with muscarinic orthosteric agonists. In order to directly probe the allosteric sites on M2 and M4, we radiolabelled LY2119620. Cooperativity binding of [3H]-LY2119620 with mAChR orthosteric agonists detect significant changes in Bmax values with little change in Kd suggesting a G-protein dependent process. Furthermore, [3H]-LY2119620 was displaced by compounds of similar chemical structure but not by previously described mAChR allosteric compounds such as gallamine or WIN 62,577. Our results therefore demonstrate the development of a radioligand, [3H]-LY2119620, to probe specifically the human M2 and M4 muscarinic receptor allosteric binding sites. ER -