Development of PROTACs Targeting Papain-like Protease as Broad-Spectrum Anti-Coronavirus Therapeutics

开发针对木瓜蛋白酶的 PROTAC 作为广谱抗冠状病毒治疗药物

• 批准号: 10527571
• 负责人: Shiqing Xu
• 金额: $ 22.18万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-26 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10527571
• 关键词: 2019-nCoV; Active Sites; Address; Antiviral Agents; Binding; Binding Sites; Biological Assay; COVID-19; COVID-19 pandemic; COVID-19 therapeutics; COVID-19 vaccine; Cells; Cessation of life; Chemicals; Complex; Coronavirus; Development; Disadvantaged; Disease; Disease Outbreaks; Drug Targeting; Event; Fingers; Future; Genetic Transcription; Goals; Health Promotion; Human; Immunization; In Vitro; Infection; Innate Immune Response; Knowledge; Lead; Length; Ligand Binding; Ligands; Link; Mass Spectrum Analysis; Membrane; Mission; Mutation; Nature; Nonstructural Protein; Papain; Paralysed; Pathway interactions; Peptide Hydrolases; Pharmaceutical Preparations; Play; Property; Protac; Protease Inhibitor; Proteins; Public Health; RNA Viruses; Research; Resistance; Role; SARS-CoV-2 B.1.617.2; SARS-CoV-2 P.1; SARS-CoV-2 protease; Site; System; Technology; Testing; Thumb structure; Ubiquitination; United States; United States National Institutes of Health; Vaccines; Variant; Viral; Viral Proteins; Virus Replication; antiviral drug development; base; combat; coronavirus therapeutics; cytotoxicity; drug development; drug discovery; global health emergency; human coronavirus; improved; inhibitor; insight; multicatalytic endopeptidase complex; new technology; novel coronavirus; pathogen; protein function; protein protein interaction; recruit; small molecule; targeted treatment; technology development; tool; ubiquitin-protein ligase; vaccine development; variants of concern

PROJECT SUMMARY/ABSTRACT The current COVID-19 pandemic caused by SARS-CoV-2 has paralyzed much of the world. Encouragingly, a few COVID-19 vaccines have been developed and approved for human immunization. However, existing COVID-19 vaccines target the highly mutable membrane Spike protein of SARS-CoV-2. New viral strains with critical mutations in Spike have emerged such as alpha, beta, delta, and gamma variants that could make current vaccines less effective. To truly overcome the threat posed by SARS-CoV-2 and its emerging variants of concern, it is paramount to develop antiviral drugs that can combat COVID-19 and also be potentially repurposed to combat novel coronaviruses (CoVs) in the future. To address this urgent need, this proposal aims to interface papain-like protease (PLpro) inhibitors/binders and the emerging proteolysis targeting chimera (PROTAC) technology for the development of broad-spectrum anti-CoV PROTACs. PLpro, one of two essential SARS-CoV- 2 proteases, plays a dual role in promoting viral transcription and replication, and antagonizing host innate immune responses. PLpro is encoded by nonstructural protein 3 (nsp3) which is relatively conserved across various CoVs. Thus, PLpro is an attractive target for the development of broad-spectrum anti-CoV drugs. The proposed small-molecule PROTACs are bifunctional small molecules containing two active ligands connected via a chemical linker. One ligand binds specifically to a viral protein target PLpro while the other ligand selectively engages an E3 ubiquitin ligase. The recruitment of the E3 ligase to PLpro facilitates the formation of a ternary complex, leading to ubiquitination and ultimate degradation of PLpro by the ubiquitination-proteasome pathway. As PROTACs regulate protein function by degrading target proteins instead of inhibiting them, they offer many advantages over traditional occupancy-based inhibitors, including (i) sub-stoichiometric activity, (ii) high barrier to resistance, (iii) improved target selectivity, and (iv) that weak binders (no inhibition required) can become potent degraders. On this basis, the overall goal of this application is to validate the degradation of PLpro as a new strategy for the development of broad-spectrum antiviral therapeutics to combat CoVs. In Aim 1, to chemically optimize non-covalent, reversible covalent, and multiple binding-mode PROTACs by linking VHL E3 ligands and PLpro inhibitors/binders, and to evaluate their degradation potency of PLpro in cell-based assays. In Aim 2, to determine the anti-SARS-CoV-2 activity of developed PROTAC molecules. Potent PROTACs (EC50 < 500 nM) will be further tested for their antiviral activities against other CoVs. In vitro DMPK and cytotoxicity studies of potent PROTACs will be conducted. The successful completion of the proposed study will lead to potent anti-CoV PROTACs against COVID-19 that the mechanism of action is fundamentally different from existing antivirals. The developed PROTAC molecules will also have the potential to be repurposed to contain future coronavirus outbreaks.

项目概要/摘要 当前由 SARS-CoV-2 引起的 COVID-19 大流行已使世界大部分地区陷入瘫痪。令人鼓舞的是， 一些 COVID-19 疫苗已被开发并批准用于人类免疫。然而，现有的 COVID-19 疫苗针对 SARS-CoV-2 高度可变的膜刺突蛋白。新病毒株 Spike 中的关键突变已经出现，例如 alpha、beta、delta 和 gamma 变体，这些突变可能使当前的 疫苗效果较差。为了真正克服 SARS-CoV-2 及其新出现的令人担忧的变种所构成的威胁， 开发能够对抗 COVID-19 并有可能被重新利用的抗病毒药物至关重要 未来对抗新型冠状病毒（CoV）。为了解决这一迫切需求，该提案旨在将 木瓜蛋白酶样蛋白酶 (PLpro) 抑制剂/结合剂和新兴的蛋白水解靶向嵌合体 (PROTAC) 开发广谱抗冠状病毒 PROTAC 的技术。 PLpro，两种重要的 SARS-CoV 之一 2种蛋白酶，在促进病毒转录和复制以及拮抗宿主先天性方面发挥双重作用 免疫反应。 PLpro 由非结构蛋白 3 (nsp3) 编码，该蛋白在整个过程中相对保守 各种冠状病毒。因此，PLpro是开发广谱抗冠状病毒药物的一个有吸引力的靶点。这 提出的小分子 PROTAC 是双功能小分子，含有两个连接的活性配体 通过化学接头。一种配体特异性结合病毒蛋白靶标 PLpro，而另一种配体选择性结合 参与 E3 泛素连接酶。 E3 连接酶向 PLpro 的募集促进了三元结构的形成 复合物，导致 PLpro 通过泛素化-蛋白酶体途径泛素化并最终降解。 由于 PROTAC 通过降解而不是抑制目标蛋白质来调节蛋白质功能，因此它们提供了许多 与传统基于占用的抑制剂相比的优势，包括（i）亚化学计量活性，（ii）高阻隔 抗性，（iii）提高靶标选择性，（iv）弱结合物（不需要抑制）可以变成 强效降解剂。在此基础上，本应用的总体目标是验证 PLpro 作为 开发广谱抗病毒疗法来对抗冠状病毒的新策略。在目标 1 中， 通过连接 VHL E3 对非共价、可逆共价和多种结合模式 PROTAC 进行化学优化 配体和 PLpro 抑制剂/结合剂，并在基于细胞的测定中评估其 PLpro 的降解效力。 目标 2 是确定已开发的 PROTAC 分子的抗 SARS-CoV-2 活性。有效的 PROTAC（EC50 < 500 nM）将进一步测试其针对其他冠状病毒的抗病毒活性。体外 DMPK 和细胞毒性 将进行有效的 PROTAC 的研究。拟议研究的成功完成将导致 针对 COVID-19 的有效抗 CoV PROTAC，其作用机制与 现有的抗病毒药物。开发的 PROTAC 分子也将有可能被重新利用以包含 未来冠状病毒的爆发。