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目的 通过计算机虚拟筛选和体外活性测定,筛选自噬相关蛋白ATG4B的特异性小分子抑制剂.方法 基于ATG4B蛋白的晶体结构(PDB ID:2CY7)找出合适的对接口袋;用分子对接方法对SPECS数据库20万化合物进行虚拟筛选并确定候选化合物;用荧光共振能量转移方法(FRET)检测候选化合物对ATG4B的体外抑制活性并对其特异性进行验证.结果 确定受体蛋白2CY7的site 5为最适的对接口袋;分子对接后经综合分析选择并购买30个代表性化合物做进一步活性测定.活性测定结果显示,AG-690/10400046能有效抑制ATG4B蛋白的活性,而对半胱氨酸蛋白酶家族的另一成员caspase-3无酶切抑制作用.结论 建立了一种ATG4B蛋白抑制剂的虚拟筛选方法.筛选得到的化合物AG-690/10400046具有明显的体外抑制活性,为后续ATG4B抑制剂的生理功能研究奠定基础.“,”Aim ToscreenoutnovelATG4Binhibitors based on the computer-aided drug screening and inves-tigate the in vitro activities of these inhibitors.Methods Byperforminginsilicodockingbasedonthecrystal structure of ATG4B(PDB ID:2CY7),the SPECS da-tabase with 200000 compounds were screened.The inhibitory effect on ATG4B of those candidate com-pounds was verified by fluorescence resonance energy transferassay(FRET).Results Site5ofthe2CY7 was the most suitable pocket for molecular docking. After screening,30 compounds were purchased fromSPECS database for further bioassay.Among them, AG-690/10400046 effectively inhibited ATG4B activity invitrowithhighspecificity.Conclusion Apromising method is established for screening ATG4B inhibitors. Compound AG-690/10400046 is screened out with high activity and specificity.This work lays a founda-tion for virtual screening and later physiological func-tion studies of ATG4B inhibitors.