新型Ⅰ型大麻素受体拮抗剂LMJ07的体外生物活性

In vitro activity of a novel cannabinoid receptor Ⅰ selective antagonist LMJ07

目的 LMJ07为靶向大麻素Ⅰ型受体(CB1R)设计合成的选择性拮抗剂,本文从分子水平受体结合、细胞水平CB1R受体内在化、CB1R诱导的细胞骨架和信号分子的变化等方面对其CB1R拮抗活性进行了评价。方法以已知的CB1R选择性拮抗剂利莫那班(SR141716A)为对照,采用放射性配体竞争结合和增强型绿色荧光蛋白(EGFP)示踪大麻素受体(CBR)内在化的高内涵分析实验分析了LMJ07对2种CBR亚型(CB1R和CB2R)的结合及G蛋白不依赖的受体激活的拮抗活性及选择性;分别采用CELLKEY无标记检测系统和均相时间分辨荧光(HTRF)测定了LMJ07对CBR激活诱导的CHO-CB1细胞骨架和胞内环腺苷酸(c AMP)含量的影响;最后采用连续荧光检测技术使用表达CB1R的原代海马细胞检测LMJ07对胞内Ca2+的影响。结果 LMJ07高亲和、高选择地与CB1R结合,选择性地拮抗CB1R的激活导致的受体内吞,且选择性和拮抗活性与利莫那班相当;在CHO-CB1R细胞上,LMJ07(0.01~10μmol/L)剂量依赖地逆转CBR激动剂Win55212-2诱导的细胞骨架变化相关的细胞电阻改变、剂量依赖地逆转Win55212-2降低胞内c AMP含量效应;在原代培养的海马细胞上,LMJ07(10 nmol/L^1μmol/L)可拮抗Win55212-2诱导的胞内Ca2+增加效应。在上述实验中,LMJ07拮抗CB1R通路的信号分子和功能的活性与利莫那班相当。结论 LMJ07是一个与利莫那班活性相当的新结构类型CB1R选择性拮抗剂。

Objective LMJ07 is a novel cannabinoid receptorⅠ（CB1R）selective antagonist discovered by our lab. In the present study,its affinity and antagonistic activity against CB1 R were evaluated at the molecular and cellular levels by receptor binding experiment,CB1 R internalization experiment and by monitoring the change in cytoskeletal and intracellular signal induced by CB1 R activation. Methods With the CB1 R selective antagonist rimonabant（SR141716A） as control,the affinity and selectivity of LMJ07 to CB1 R and CB2 R were assayed by radioligand binding assays,and the G protein-independent antagonistic activity against cannabinoid receptor（CBR） was assayed by enhanced green fluorescein protein（EGFP）-CBR internalization with hierarchiae cluscer anclysis（HCA） analysis. At the same time,we evaluated the changes in cytoskeletal and the intracellular c AMP levels in response to LMJ07 treatment in CHO-CB1 cells by Cellkey label-free assays and homogeneous time-resolved fluorescence（HTRF）.Additionally,we also confirmed the CB1 R antagonistic efficacy of LMJ07 by detecting the content of Ca2 ＋in primary cultured hippocampal neuronal cells which could express CB1 R with continuous fluorescence detection technology. Results LMJ07 is a selective CB1 R antagonist with high affinity,which can selectively antagonize receptor endocytosis induced by CB1 R activation。It＇s affinity and antagonistic efficacy to CB1 R were equal to those of rimonabant. In CHO-CB1 cells,LMJ07（0.01-10μmol/L）could dosedependently inverse the change in cytoskeletal as well as the increase in intracellular c AMP induced by CBR agonist Win55212-2. In the primary cultured hippocampal neuronal cells,LMJ07（10 nmol/L-1 μmol/L） could block the increase in [Ca^2＋]induced by CB1 R agonist Win55212-2. Conclusion LMJ07 is a new selective CB1 R antagonist,which shows equal affinity and antagonistic activity against CB1 R as the widely accepted CB1 R antagonist rimonabant. In addition,the combination of high content analysis（HCS） and Cellkey label-free assay provides a better research tool for rapid and high throughput screening of novel CB1 R antagonists.