目的 基于荧光共振能量转移(fluorescence resonance energy transfer,FRET)原理,建立奥密克戎变异株主蛋白酶(Omicron variant main protease,OM-Mpro)抑制剂高通量筛选模型,筛选新型O M-Mpro抑制剂.方法 利用大肠杆菌进行OM-Mpro原核表达,再以HisTrapTM亲和层析柱进行分离纯化.以FRET法进行OM-Mpro与野生型新型冠状病毒主蛋白酶(wild-type main protease,WT-Mpro)的酶活性测定并评价奈玛特韦的酶抑制活性.利用OM-Mpro抑制剂FRET高通量筛选模型对上市药物库进行高通量筛选.结果 利用大肠杆菌成功进行了 OM-Mpro原核表达与分离纯化,其与WT-Mpro 酶活性无显著差异.奈玛特韦对OM-Mpro和WT-Mpro具有等同的酶抑制活性,说明奈玛特韦对奥密克戎变异株依然有效.通过对上市药物库进行高通量筛选,发现西吡氯铵是混合型OM-Mpro抑制剂,其半数抑制浓度值为8.76μmol·L-1.结论 成功制备了高活性OM-Mpro并建立了 OM-Mpro抑制剂FRET高通量筛选模型,初步证实了西吡氯铵是混合型OM-Mpro抑制剂,为广谱抗冠状病毒药物先导化合物的筛选与发现奠定了基础.
Production of SARS-CoV-2 Omicron Variant Main Protease for Screening Approved Drugs as Its Potential Inhibitors
OBJECTIVE To develop a high-throughput screening assay for the discovery of Omicron variant main protease(OM-Mpro)inhibitors based on the principle of fluorescence resonance energy transfer(FRET).METHODS The recombinant OM-Mpro enzyme was expressed in Escherichia coli Rosetta(DE3)cells,and further purified by a HisTrapTM chelating column.Subsequently,the enzymatic activity of OM-Mpro and wild type main protease(WT-Mpro)enzymes and inhibition of nirmatrelvir against both proteases were measured using FERT assay.With the FRET assay,OM-Mpro inhibitors were identified via high-throughput screening of an approved drug library.RESULTS The active OM-Mpro enzyme was successfully prepared from E.coli cells.OM-Mpro and WT-Mpro enzymes possessed the same enzymatic activity,and OM-Mpro remained susceptible to nirmatrelvir in vitro.Through high-throughput screening of the marketed drug library,it was found that cetylpyridinium chloride(CPC)is a mixed-type OM-Mpro inhibitor in vitro with an IC50 value of 8.76 μmol·L-1.CONCLUSION A robust FRET assay has been successfully developed based on the production of active OM-Mpro enzyme for screening of its inhibitors,and CPC is identified as a potential lead compound against OM-Mpro in vitro.This study provides a promising avenue for rapid discovery of broad-spectrum antivirals against coronavirus protease.
SARS-CoV-2Omicronmain protease inhibitorfluorescence resonance energy transfercetylpyridinium chloride
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维普
