以nNOS和PSD-95相互作用为靶标的解耦联剂筛选模型的建立  

Establishment of a screening model for compounds uncoupling the interaction of nNOS with PSD-95

作　　者：张蕾 周鹤鸣 李晓芸 张楚 张雅卓 周苗 陈立娜[1] 张宇[1] ZHANG Lei;ZHOU He-ming;LI Xiao-yun;ZHANG Chu;ZHANG Ya-zhuo;ZHOU Miao;CHEN Li-na;ZHANG Yu(School of Pharmacy, Nanjing Medical University, Nanjing 211166, China)

机构地区：[1]南京医科大学药学院,江苏南京211166

出　　处：《药学学报》2019年第4期638-644,共7页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(81373895);江苏省高等学校大学生创新创业训练计划项目(201610312048Y)

摘　　要：复合物神经元型一氧化氮合酶(neuronal nitric oxide synthase, nNOS)-突触后密度蛋白-95 (postsynaptic density protein-95, PSD-95)形成的增加在缺血性脑卒中引发的神经损伤中起着重要作用,本研究以nNOS和PSD-95相互作用为靶点,建立nNOS-PSD-95解耦联剂筛选模型。模型采用转染的方式将质粒pCDH-Flag-nNOS和pcDNA3.1-PSD-95分别转染入人胚胎肾-293T (human embryonic kidney-293T, HEK-293T)细胞,通过真核表达的方式富集Flag-nNOS和PSD-95。随后,通过nNOS和PSD-95体外共孵育获得nNOS-PSD-95复合物,以ZL006为阳性药干预nNOS-PSD-95的结合,考察ZL006在该系统的解耦联效率,并对解耦联模型进行优化。结果表明,在该模型中, ZL006显示出较明显的解耦联效果,并通过戊二醛法获得抗体耦联磁珠,从而优化筛选模型,提高了筛选效率,降低了成本。最后运用该模型对另一个已知的nNOS-PSD-95解耦联化合物IC87201及候选ZL006结构类似物进行了解耦联活性评价,发现IC87201在该模型中同样具有解耦联活性,并且在候选化合物中也筛选出了潜在的解耦联化合物。In ischemic stroke, increased level of neuronal complex of nitric oxide synthase (nNOS)-postsynaptic density protein-95 (PSD-95) plays an important role in neuronal damage. We aimed to establish a screening model to identify compounds capable of uncoupling nNOS interaction with PSD-95. In this model, human embryonic kidney-293T (HEK-293T) cells were transfected with either pCDH-Flag-nNOS or pcDNA3.1-PSD-95 plasmid to obtain the protein of Flag-nNOS or PSD-95. Incubating Flag-nNOS with PSD-95 causes formation of the nNOS-PSD-95 complex. ZL006, a known uncoupler of nNOS-PSD-95 interaction, can disturb the interaction between Flag-nNOS and PSD-95, serving as a positive control. The method coupling antibodies to magnetic beads with glutaraldehyde was used to decrease the cost and increase the efficiency. To establish that our model is suitable for selecting nNOS-PSD-95 uncouplers, we evaluated the ability of IC87201, another reported uncoupler of nNOS-PSD-95 interaction, and structural analogs of ZL006. IC87201 and one structure analog of ZL006 showed uncoupling effect, supporting that our model can be used to select different types uncoupler blocking nNOS-PSD-95 interaction.

关 键 词：nNOS-PSD-95 解耦联剂 ZL006 筛选模型 

分 类 号：R965.1[医药卫生—药理学]