新型冠状病毒奥密克戎变异株主蛋白酶的制备及其抑制剂的高通量筛选

Production of SARS-CoV-2 Omicron Variant Main Protease for Screening Approved Drugs as Its Potential Inhibitors

关键词:  新型冠状病毒  奥密克戎  主蛋白酶抑制剂  荧光共振能量转移  西吡氯铵

Key words:  SARS-CoV-2  Omicron  main protease inhibitor  fluorescence resonance energy transfer  cetylpyridinium chloride

摘要:

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OM-Mpro抑制剂。方法 利用大肠杆菌进行OM-Mpro原核表达，再以HisTrap^TM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8.76 μmol·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。

Abstract:

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 HisTrap^TM 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.