Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies.
Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies.
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DOI：
10.1371/journal.pone.0253364
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发表时间：
2021
期刊：
影响因子：
3.7
通讯作者：
Kulathu Y
中科院分区：
文献类型：
--
作者：
Armstrong LA;Lange SM;Dee Cesare V;Matthews SP;Nirujogi RS;Cole I;Hope A;Cunningham F;Toth R;Mukherjee R;Bojkova D;Gruber F;Gray D;Wyatt PG;Cinatl J;Dikic I;Davies P;Kulathu Y
在SARS CoV-2编码的16种非结构蛋白中，Nsp 3是最大的一种，在病毒的生命周期中起着重要的作用。作为一个大的，多结构域，跨膜蛋白，Nsp 3一直是最具挑战性的Nsp的特点。Nsp 3内编码的是木瓜蛋白酶样蛋白酶结构域（PLpro），其不仅切割病毒多肽，而且切割来自宿主细胞蛋白的K48连接的多聚泛素和泛素样修饰物ISG 15。我们在这里比较PLpro和Nsp 3的相互作用，并发现一个很大程度上重叠的相互作用组。有趣的是，我们发现，近全长Nsp 3是一个更有活性的蛋白酶相比，PLpro的最小催化结构域。使用基于MALDI-TOF的测定，我们筛选了1971种批准的临床化合物，并鉴定了5种化合物，其抑制PLpro的IC 50在低微摩尔范围内，但与其他人去泛素化酶显示交叉反应性，并且在细胞SARS-CoV-2感染测定中没有显着的抗病毒活性。因此，我们寻找阻断PLpro活性的替代方法，并设计了在底物结合位点以纳摩尔亲和力结合PLpro的竞争性纳米抗体，从而抑制酶。我们的工作突出了研究Nsp 3的重要性，并为研究病毒感染周期中的Nsp 3生物学提供了工具和有价值的见解。
Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease domain (PLpro) that cleaves not only the viral polypeptide but also K48-linked polyubiquitin and the ubiquitin-like modifier, ISG15, from host cell proteins. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.