Development of a Radioligand, [3H]LY2119620, to Probe the Human M2 and M4 Muscarinic Receptor Allosteric Binding Sites

In this study, we characterized 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, compounds targeting these novel sites have been starting to emerge. For example, LY2033298 (3-amino-5-chloro-6-methoxy-4-methyl-thieno(2,3-b)pyridine-2-carboxylic acid cyclopropylamid) and a derivative of this chemical scaffold, VU152100 (3-amino-N-(4-methoxybenzyl)-4,6-dim​ethylthieno[2,3-b]pyridine carboxamide), bind to the human M₄ mAChR allosteric pocket. In the current study, we characterized LY2119620, a compound similar in structure to LY2033298 and 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 ([³H]N-methylscopolamine) 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. To probe directly the allosteric sites on M₂ and M₄, we radiolabeled LY2119620. Cooperativity binding of [³H]LY2119620 with mAChR orthosteric agonists detects significant changes in B_max values with little change in K_d, 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 (17-β-hydroxy-17-α-ethynyl-δ-4-androstano[3,2-b]pyrimido[1,2-a]benzimidazole). Our results therefore demonstrate the development of a radioligand, [³H]LY2119620 to probe specifically the human M₂ and M₄ muscarinic receptor allosteric binding sites.