组织蛋白酶L小分子抑制剂的抗SARS-CoV-2活性研究

Anti-SARS-CoV-2 activity of small molecule inhibitors of cathepsin L

新冠肺炎(coronavirus disease 2019,COVID-19)是一种由新型严重急性呼吸系统综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)感染引起的急性传染性疾病,已给社会经济造成了沉重负担。由于突变体的不断出现,疫苗和单克隆抗体仅对SARS-CoV-2部分毒株导致的感染有效,因此寻找广谱高效的小分子药物以应对SARS-CoV-2感染及未来可能暴发的疫情依然具有重要意义。组织蛋白酶L(cathepsin L,CatL)切割SARS-CoV-2刺突蛋白(spike glycoprotein,S),在病毒进入宿主细胞的过程中发挥着不可或缺的作用,因此CatL是广谱抗冠状病毒药物开发的理想靶标之一。在本研究中,以荧光标记的底物建立CatL酶抑制剂筛选模型,通过高通量筛选获得两个具有CatL抑制活性的化合物IMB 6290和IMB 8014,其半数抑制浓度(half inhibitory concentration,IC50)分别为11.53±0.68和1.56±1.10μmol·L-1,且细胞毒性低;聚丙烯酰胺凝胶电泳和细胞−细胞融合实验均证实了化合物浓度依赖性地抑制CatL对S蛋白的水解作用;表面等离子体共振(surface plasmon resonance,SPR)检测表明两个化合物与CatL均具有中等强度的结合力;分子对接揭示了化合物与CatL活性口袋的结合方式;假病毒实验进一步确证了化合物IMB 8014对S蛋白介导的进入过程的抑制活性;体外药代动力学评估发现,化合物可能具有良好的成药性。所有研究结果提示,这两个化合物具有治疗SARS-CoV-2感染的潜力。

The coronavirus disease 2019(COVID-19)is an acute infectious disease caused by the new severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection,which has led to serious worldwide economic burden.Due to the continuous emergence of variants,vaccines and monoclonal antibodies are only partial effective against infections caused by distinct strains of SARS-CoV-2.Therefore,it is still of great importance to call for the development of broad-spectrum and effective small molecule drugs to combat both current and future outbreaks triggered by SARS-CoV-2.Cathepsin L(CatL)cleaves the spike glycoprotein(S)of SARS-CoV-2,playing an indispensable role in enhancing virus entry into host cells.Therefore CatL is one of the ideal targets for the development of pan-coronavirus inhibitor-based drugs.In this study,a CatL enzyme inhibitor screening model was established based on fluorescein labeled substrate.Two CatL inhibitors IMB 6290 and IMB 8014 with low cytotoxicity were obtained through high-throughput screening,the half inhibition concentrations(IC50)of which were 11.53±0.68 and 1.56±1.10μmol·L-1,respectively.SDS-PAGE and cell-cell fusion experiments confirmed that the compounds inhibited the hydrolysis of S protein by CatL in a concentration-dependent manner.Surface plasmon resonance(SPR)detection showed that both compounds exhibited moderate binding affinity with CatL.Molecular docking revealed the binding mode between the compound and the CatL active pocket.The pseudovirus experiment further confirmed the inhibitory effects of IMB 8014 on the S protein mediated entry process.In vitro pharmacokinetic evaluation indicated that the compounds had relatively good drug-likeness properties.Our research suggested that these two compounds have the potential to be further developed as antiviral drugs for COVID-19 treatment.