【摘要】 目的:建立内源荧光检测筛选烟酰胺磷酸核糖转移酶(NAMPT)抑制剂的方法。方法:以1,4-二马来酰亚胺基丁烷(BMB)交联G355C/D393C双突变NAMPT,封堵其催化活性中心,以野生型及交联型NAMPT蛋白的自发荧光变化(280 nm激发波长,333 nm发射波长)检测化合物与蛋白的结合,以核磁共振方法体外检测化合物对NAMPT催化作用的影响,以MTT方法检测化合物对人肺癌A549细胞活性的影响。结果:FK866在0.1～1.0μmol/L浓度范围内能浓度依赖地抑制野生型NAMPT自发荧光,但对交联型NAMPT的自发荧光没有影响。迷迭香酸、洋蓟素、1,3二咖啡奎宁酸对两种蛋白自发荧光的抑制率无显著差异。FK866能够显著抑制NAMPT的催化作用,但迷迭香酸、洋蓟素、1,3二咖啡奎宁酸对NAMPT的催化作用无抑制作用。FK866显著抑制A549细胞的活性,而迷迭香酸、洋蓟素、1,3二咖啡奎宁酸对A549细胞活性无抑制作用。结论:基于野生型和交联型NAMPT蛋白的内源性荧光检测法可以筛选与NAMPT蛋白结合的化合物,并分析化合物是否结合在NAMPT的催化活性部位,迷迭香酸、洋蓟素、1,3二咖啡奎宁酸可结合在NAMPT催化活性位点以外的部位。更多还原

【Abstract】 Objective:To establish a method for screening nicotinamide phosphoribosyl transferase(NAMPT) inhibitors based on endogenous fluorescence determination.Methods:The double mutants of NAMPT,G355 C/D393 C,was crosslinked by using 1,4-Bismaleimidobutane(BMB) to block the entrance of enzymatic active site of NAMPT.The binding of compounds to NAMPT was evaluated according to the change of spontaneous fluorescence of NAMPT and BMB-NAMPT with 280 nm excitation and 333 nm emmision.The in vitro enzamatic activity of NAMPT was determined by nuclear magnetic resonance.The cell viability was determined by MTT assay.Results:FK866 significantly decreased the spontaneous fluorescence of NAMPT but not of BMB-NAMPT.Rosmaric,cynarine and 1,3-dicaffeoylquinic acid also decreased the spontaneous fluorescence of both NAMPT and BMB-NAMPT.However,the inhibition on two proteins was equivalent.FK866 significantly inhibit the catalysis of NAMPT.Rosmarinic acid,cynarine and 1,3-dicaffeoylquinic acid failed to inhibit the catalysis of NAMPT.FK866 inhibited the viability of A549 cells,but rosmarinic acid,cynarine and 1,3-dicaffeoylquinic acid did not.Conclusion:Endogenous fluorescence spectrometry based on NAMPT and BMB-NAMPT protein can be used for screening compounds that bind with NAMPT,and distinguishing the binding site—either within the enzymatic active site or not.Rosmarinic acid,cynarine and 1,3-dicoffeoylquinic acid can bind to NAMPT out its enzymatic active site.更多还原