Identification of synthetic ligands selective for muscarinic receptor subtypes has been challenging due to the high sequence identity and structural homology among the five muscarinic acetylcholine receptors. Here, we report the pharmacological characterization of PCS1055, a novel muscarinic M4 receptor antagonist. PCS1055 inhibited radioligand [(3)H]-NMS binding to the M4 receptor with a Ki=6.5nM. Though the potency of PCS1055 is lower than that of pan-muscarinic antagonist atropine, it has better subtype selectivity over previously reported M4-selective reagents such as the muscarinic-peptide toxins (Karlsson et al., 1994; Santiago and Potter, 2001a) at the M1 subtype, and benzoxazine ligand PD102807 at the M3-subtype (Bohme et al., 2002). A detailed head-to-head comparison study using [(3)H]-NMS competitive binding assays characterizes the selectivity profiles of PCS1055 to that of other potent muscarinic-antagonist compounds PD102807, tropicamide, AF-DX-384, pirenzapine, and atropine. In addition to binding studies, the subtype specificity of PCS1055 is also demonstrated by functional receptor activation as readout by GTP-γ-[(35)S] binding. These GTP-γ-[(35)S] binding studies showed that PCS1055 exhibited 255-, 69.1-, 342- and >1000-fold greater inhibition of Oxo-M activity at the M4 versus the M1-, M2(-), M3-or M5 receptor subtypes, respectively. Schild analyses indicates that PCS1055 acts as a competitive antagonist to muscarinic M4 receptor, and confirms the affinity of the ligand to be low nanomolar, Kb=5.72nM. Therefore, PCS1055 represents a new M4-preferring antagonist that may be useful in elucidating the roles of M4 receptor signaling.

中文翻译：

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新型毒蕈碱M4受体拮抗剂PCS1055的表征。

由于五个毒蕈碱乙酰胆碱受体之间的高度序列同一性和结构同源性，鉴定对毒蕈碱受体亚型具有选择性的合成配体一直具有挑战性。在这里，我们报告PCS1055，一种新型毒蕈碱M4受体拮抗剂的药理特性。PCS1055以Ki = 6.5nM抑制放射性配体[（3）H] -NMS与M4受体的结合。尽管PCS1055的效力低于泛毒蕈碱拮抗剂阿托品，但与先前报道的M4选择性试剂（例如毒蕈碱肽毒素）（Karlsson等，1994； Santiago and Potter，2001a）相比，它具有更好的亚型选择性。 M1亚型和M3亚型的苯并恶嗪配体PD102807（Bohme et al。，2002）。使用[（3）H] -NMS竞争结合试验进行的详细的头对头比较研究表明，PCS1055对其他有效毒蕈碱-拮抗剂化合物PD102807，托品酰胺，AF-DX-384，吡氮平和阿托品的选择性特征。除结合研究外，PCS1055的亚型特异性还通过功能性受体激活（通过GTP-γ-[（35）S]结合读出）得到证明。这些GTP-γ-[（35）S]结合研究表明，PCS1055对M4的Oxo-M活性的抑制作用是M1，M2（-）的255-，69.1-，342-和> 1000倍， M3或M5受体亚型。Schild分析表明，PCS1055充当毒蕈碱M4受体的竞争性拮抗剂，并证实该配体的亲和力很低，纳摩尔浓度Kb = 5.72nM。所以，