【摘要】 目的初步研究福氏志贺菌组氨酸蛋白激酶EnvZ抑制剂对福氏志贺菌毒力的影响。方法通过构建志贺菌:EnvZ胞内段(SF-EnvZc)蛋白的原核表达质粒,表达并纯化SF-EnvZc蛋白,在SPECS公司的小分子化合物库中模拟筛选出的10个候选EnvZ抑制剂的基础上,通过体外磷酸化的方法筛选出能够有效地抑制SF- EnvZc蛋白磷酸化的小分子化合物(EnvZ抑制剂),然后采用表面等离子共振(SPR)技术检测EnvZ抑制剂与SF-EnvZc蛋白的结合力,并采用小鼠角结膜志贺菌感染模型检测EnvZ抑制剂对福氏志贺菌株Sf9380毒力的影响。结果在10个候选EnvZ抑制剂中,化合物1、2、3能够有效地抑制SF-EnvZc蛋白的磷酸化,其IC50值分别为(70．17±5．83)、(10．18±0．86)和(37．66±9．64)μmol／L,且上述3个化合物表现出对SF-EnvZc蛋白的高亲和力,其平衡解离常数(Kd)分别为4．08、3．57和2．94μmol／L,其中化合物1和2能够明显抑制福氏志贺菌株Sf9380导致的小鼠角结膜炎症反应(由■变为-),化合物3能够明显降低其炎症反应(由■降为+),化合物1、2、3的最小有效浓度分别为12．5、12．5和100μmol／L结论筛选出3个EnvZ抑制剂,化合物1、2、3能够不同程度地抑制或降低福氏志贺菌导致的小鼠角结膜炎症反应,提示EnvZ抑制剂能够降低福氏志贺菌的毒力。更多还原

【Abstract】 Purpose To explore the effects of the EnvZ histidine kinase inhibitors on the virulence of Shigella flexneri. Methods Recombinant S. flexneri SF-EnvZc was cloned into the expression vector pQE30 and was expressed and purified in Escherichia coli system. By using Kinase-Glo^TMLuminescent Kinase assay to screen against the chemical library, compounds which inhibit the autophospho-rylation activity of SF-EnvZc were selected as EnvZ inhibitors. The binding affinities of the inhibitors to SF-EnvZc protein were determined by using surface plasmon resonance （SPR） in vitro. The mouse sereny test was used to evaluate the effect of EnvZ inhibitors on the virulence of the Shigella flexneri sf9380. Results Compounds 1,2, and 3 inhibited the activity of SF-EnvZc autophosphorylation with IC50, values of （70. 17±5. 83）μmol/L, （10. 18±0. 86）μmol/L, and （37. 66±9. 64）μmol/L, respectively. Their high binding affinities to SF-EnvZc were evaluated with Kd values of 4. 08μmol/L, 3. 57μmol/L, and 2. 94μmol/L, respectively. In mouse sereny test, the mice inoculated with sf9380 which was treated with compounds 1 and 2 displayed no disease, the group inoculated with sf9380 which was treated with compound 3 displayed a slight conjunctival inflammation （ + ） , while the control group inoculated with sf9380 which was treated with dimthyl sulfoxide displayed a severe keratoconjunctival inflammation （■）. The minimum effective concentrations of compounds 1,2, and 3 to release mouse sereny reaction were 12.5μmol/L, 12.5μmol/L, and 100μmol/L, respectively. Conclusions The three EnvZ inhibitors, compounds 1,2, and 3, can release the the mouse sereny reaction of Shigella flexneri, suggesting that these three EnvZ inhibitors can reduce the virulence of Shigella flexneri.更多还原