Abstract

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.