Croy Carrie H, Chan Wai Y, Castetter Andrea M, Watt Marla L, Quets Anne T, Felder Christian C
Lilly Research Laboratories, Neuroscience Division, Indianapolis, IN, U.S.A.
Neuroscience Discovery Research, Lilly Research Centre, Surrey, United Kingdom.
Eur J Pharmacol. 2016 Jul 5;782:70-6. doi: 10.1016/j.ejphar.2016.04.022. Epub 2016 Apr 13.
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.
由于五种毒蕈碱型乙酰胆碱受体之间存在高度的序列同一性和结构同源性，因此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。在此，我们报告了新型毒蕈碱M4受体拮抗剂PCS1055的药理学特性。PCS1055抑制放射性配体[³H]-NMS与M4受体的结合，其Ki = 6.5 nM。尽管PCS1055的效力低于泛毒蕈碱拮抗剂阿托品，但在M1亚型上，它比先前报道的M4选择性试剂如毒蕈碱肽毒素（卡尔松等人，1994年；圣地亚哥和波特，2001a）具有更好的亚型选择性，在M3亚型上比苯并恶嗪配体PD102807（博梅等人，2002年）具有更好的亚型选择性。使用[³H]-NMS竞争性结合试验进行的详细的直接比较研究，表征了PCS1055与其他强效毒蕈碱拮抗剂化合物PD102807、托吡卡胺、AF-DX-384、哌仑西平和阿托品的选择性概况。除了结合研究外，PCS1055的亚型特异性还通过以GTP-γ-[³⁵S]结合作为读出的功能性受体激活来证明。这些GTP-γ-[³⁵S]结合研究表明，PCS1055对M4受体的抑制作用分别比对M1、M2（-）、M3或M5受体亚型的Oxo-M活性高255倍、69.1倍、342倍和>1000倍。Schild分析表明，PCS1055作为毒蕈碱M4受体的竞争性拮抗剂，并证实该配体的亲和力为低纳摩尔，Kb = 5.72 nM。因此，PCS1055代表了一种新的偏向M4的拮抗剂，可能有助于阐明M4受体信号传导的作用。
