新型M5胆碱受体选择性拮抗剂的发现及药理活性研究

Discovery of Novel Subtype-selective M5 Acetylcholine Receptor Antagonists and Evaluation of Pharmacological Activities

目的:选择性拮抗M5胆碱受体可成为治疗药物成瘾的新途径,本文旨在利用虚拟筛选、结构优化、分子与细胞水平的药理活性评价,以期获得新型M5胆碱受体选择性拮抗剂。方法:通过虚拟筛选获得具有新型骨架的候选化合物,以该结构为基础进行结构优化;利用放射配基实验对系列化合物进行活性测定;进一步评价其亲和力对各胆碱受体亚型的选择性,并利用细胞内钙离子释放实验评价其拮抗活性。结果:通过虚拟筛选得到具有新型骨架的M5胆碱受体拮抗剂WXY-1-1,对其进行三轮结构优化共合成20个化合物;放射配基实验表明,其中化合物WXY-3-5对M5胆碱受体亚型具有亚微摩尔级亲和力(Ki=0.7μM)且具有亚型选择性(高出其他4个亚型9-35倍);钙流实验表明,化合物WXY-3-5对M5胆碱受体亚型具有拮抗活性(IC50=6.1μM)。结论:通过虚拟筛选、结构优化以及药理活性评价,获得具有新骨架的M5胆碱受体选择性拮抗剂,为后续药物开发提供了新的先导结构。

Objective: M5 muscarinic acetylcholine receptor(M5 R)selective antagonists provide a new approach for the treatment of drug addiction. By using virtual screening, structural optimization, and molecular/cell-based pharmacological activity evaluation, we aimed to obtain novel M5 R-selective antagonists. Methods: Virtual screening was used to obtain hits with new scaffolds and structural modification was carried out to optimize the hit structure. By using radioligand binding assay, the binding affinities were measured and the subtype-selectivities were evaluated on all five muscarinic receptor subtypes. Then, cell-based calcium mobilization assay was performed to characterize the antagonistic activity. Results: Through virtual screening, WXY-1-1 was identified to be an M5 R antagonist with a new scaffold. Twenty new structures were synthesized during three rounds optimization. According to radioligand binding assay,WXY-3-5 displayed submicromolar affinity(Ki=0.7 μM) to M5 R and relative subtype-selectivity, which was 9-35 folds higher than other 4 subtypes. By using calcium mobilization assay, WXY-3-5 was identified as an antagonist(IC50=6.1 μM). Conclusions: Through virtual screening, structural optimization, and pharmacological activity evaluation, an M5 R selective antagonist was discovered with a new scaffold and well characterized, which provided a promising novel lead structure for further drug development.