RhoA蛋白小分子抑制剂的活性筛选及其舒张血管作用的研究

发布时间：2018-07-25 07:07

【摘要】：目的：拟在前期RhoA蛋白小分子抑制剂HL07号化合物的基础上，通过结构优化，在细胞、组织、整体水平筛选出在蛛网膜下腔出血后脑血管痉挛模型上有舒张作用的化合物，为发现新的有利于心血管疾病治疗的先导化合物提供基础，并为推动RhoA相关通路在疾病病理生理中作用机制的研究提供理论依据。 方法：1）人脑血管平滑肌细胞（thehuman cerebrovascular smooth muscle cells,HBVSMCs）的培养，采用G-LISA方法；定量评价结构优化的33个化合物对溶血磷脂酸（LPA）引起RhoA激活作用的抑制，观察化合物对RhoA蛋白GTP结合域竞争性抑制活性；2）采用SD大鼠离体血管条实验方法观察化合物HL47/47K、HL79/79K、HL82/82K对预收缩血管条的张力的影响；3）大鼠SAH-CVS动物模型采用视交叉池二次注血法，实验随机分为7组：正常对照组、SAH模型组、SAH模型组+DMSO、SAH模型组+Fasudil、SAH模型组+HL47（L）、SAH模型组+HL47（M），，SAH模型组+HL47（H），每组9只，比较各组大鼠基底动脉(basilar artery, BA)细胞形态学及内径周长的差异。 结果：1）细胞筛选实验中，33个结构改造后的化合物中，筛选出3个小分子抑制剂活性强于HL07（IC50=1.79±0.17μM），分别是HL47（IC50=1.66±0.28μM）、HL79（IC50=1.26±0.13μM）和HL82（IC50=1.17±0.19μM）；2）血管条组织水平：HL47（IC50=70.90±1.33μM）、HL82（IC50=73.43±2.61μM）对100nM苯肾上腺素（phenylephrine, PE）预收缩的离体去内皮胸主动脉（thoracic aorta, TA）血管条呈现浓度依赖的明显舒张作用（P0.05），且活性均强于HL0（7IC50=123.22±8.93μM）;HL79（0~180μM）舒张作用微弱；HL47K（IC50=59.92±5.40μM）、HL82K（IC50=66.64±2.32μM）、HL79K（IC50=111.88±15.71μM）较未成盐时，活性均有不同程度的增强，HL79K活性增加最为显著（P0.05）；3）大鼠SAH-CVS动物模型实验中，各组血管内径周长比较：SAH模型组（386.25±11.25μm）较正常对照组（472.50±53.47μm）、SAH模型组+Fasudil（468.83±43.53μm）减小，其差异有统计学意义（P0.05）；SAH模型组与SAH模型组+DMSO（390.93±20.60μm）之间差异无统计学意义（P0.05）；、SAH模型组+HL47(L、M、H)（464.00±50.46μm、475.60±63.79μm、513.42±40.15μm）均较SAH模型组增大，其差异有统计学意义(P0.05)，与SAH模型组+Fasudil（468.83±43.53μm）差异无统计学意义（P0.05）。SAH模型组+HL47(L)、SAH模型组+HL47(M)、SAH模型组+HL47(H)三组之间相互差异均无统计学意义（P0.05）。 结论：1）部分经过HL07结构改造后的化合物（HL47、HL79、HL82），其抑制LPA对RhoA激活作用的活性有所增强；2）HL47/47K、HL82/82K对PE预收缩的血管条有显著的舒张作用，且活性均强于HL07，结果与细胞实验一致；小分子抑制剂的水溶性对其活性有较大的影响；3）HL47对大鼠脑血管痉挛有缓解作用。 本实验结果提示：小分子化合物HL47可更好地通过抑制RhoA蛋白活性，舒张大鼠蛛网膜下腔出血后脑血管痉挛。
[Abstract]:Objective: on the basis of HL07, a small molecular inhibitor of RhoA protein, we selected the vasodilator on the model of cerebral vasospasm after subarachnoid hemorrhage by structural optimization, cell, tissue and whole level. It provides the basis for the discovery of new leading compounds which are beneficial to the treatment of cardiovascular disease and provides the theoretical basis for the study of the mechanism of RhoA related pathway in pathophysiology of the disease. Methods (thehuman cerebrovascular smooth muscle cells of human vascular smooth muscle cells (thehuman cerebrovascular smooth muscle cells) were cultured by G-LISA method. The inhibitory effects of 33 compounds on RhoA activation induced by lysophosphatidic acid (LPA) were evaluated quantitatively. To observe the effect of compounds on the competitive inhibitory activity of RhoA protein GTP binding domain in SD rat vascular strips in vitro, we observed the effect of HL47 / 47K / 79KN HL82 / 82k on the tension of preconstricted vascular strips. The rat model of SAH-CVS was injected with the second injection of blood into the optic chiasma cistern. The experiment was randomly divided into 7 groups: normal control group: SAH model group: DMSO SAH model group, Fasudiline SAH model group, HL47 (L) group, SAH model group, HL47 (M) group, SAH model group, HL47 (H), group, 9 rats in each group. The differences of (basilar artery, BA) cell morphology and inner diameter perimeter of basilar artery in each group were compared. Results in the cell screening experiment, three small molecular inhibitors (IC50=1.79 卤0.17 渭 M),) were found to be HL47 (IC50=1.66 卤0.28 渭 M), HL79 (IC50=1.26 卤0.13 渭 M) and HL82 (IC50=1.17 卤0.19 渭 M);) in 33 structurally modified compounds. 2)琛

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