Department of Chemical Biology and Bioimaging, Wroclaw University of Science and Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland.
Department of Biochemistry and Molecular Biology and Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425, USA.
Sci Adv. 2020 Oct 16;6(42). doi: 10.1126/sciadv.abd4596. Print 2020 Oct.
Viral papain-like cysteine protease (PLpro, NSP3) is essential for SARS-CoV-2 replication and represents a promising target for the development of antiviral drugs. Here, we used a combinatorial substrate library and performed comprehensive activity profiling of SARS-CoV-2 PLpro. On the scaffold of the best hits from positional scanning, we designed optimal fluorogenic substrates and irreversible inhibitors with a high degree of selectivity for SARS PLpro. We determined crystal structures of two of these inhibitors in complex with SARS-CoV-2 PLpro that reveals their inhibitory mechanisms and provides a molecular basis for the observed substrate specificity profiles. Last, we demonstrate that SARS-CoV-2 PLpro harbors deISGylating activity similar to SARSCoV-1 PLpro but its ability to hydrolyze K48-linked Ub chains is diminished, which our sequence and structure analysis provides a basis for. Together, this work has revealed the molecular rules governing PLpro substrate specificity and provides a framework for development of inhibitors with potential therapeutic value or drug repurposing.
病毒木瓜蛋白酶样半胱氨酸蛋白酶 (PLpro，NSP3) 是 SARS-CoV-2 复制所必需的，是开发抗病毒药物的有希望的靶点。在这里，我们使用组合底物文库对 SARS-CoV-2 PLpro 进行了全面的活性分析。在位置扫描最佳命中的支架上，我们设计了最佳荧光底物和对 SARS PLpro 具有高选择性的不可逆抑制剂。我们确定了其中两种抑制剂与 SARS-CoV-2 PLpro 复合物的晶体结构，揭示了它们的抑制机制，并为观察到的底物特异性提供了分子基础。最后，我们证明 SARS-CoV-2 PLpro 具有与 SARS-CoV-1 PLpro 相似的去泛素化酶 (deISGylating) 活性，但水解 K48 连接的 Ub 链的能力降低，我们的序列和结构分析为此提供了依据。总之，这项工作揭示了调控 PLpro 底物特异性的分子规则，并为具有潜在治疗价值或药物再利用的抑制剂的开发提供了框架。
