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.

项目摘要/摘要 目前由SARS-CoV-2病毒引起的新冠肺炎大流行已使世界大部分地区陷入瘫痪。令人鼓舞的是， 一些新冠肺炎疫苗已经被开发出来，并被批准用于人类免疫。然而，现有的 新冠肺炎疫苗针对的是SARS-CoV-2高度突变的膜刺突蛋白。新的病毒株具有 SPEKE的关键突变已经出现，如阿尔法、贝塔、德尔塔和伽马变异，这些变异可能会使电流 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构成的威胁， 最重要的是开发能够对抗新冠肺炎并有可能被重新用于 未来抗击新型冠状病毒(CoV)。为了解决这一紧迫需求，本提案旨在将 类木瓜蛋白水解酶(PLPro)抑制剂/结合物与新出现的靶向嵌合体蛋白水解酶(PROTAC) 开发广谱抗冠状病毒PROTAC的技术。PLPro，两种基本的SARS-CoV之一- 2蛋白水解酶，具有促进病毒转录复制和拮抗宿主先天的双重作用。 免疫反应。PLPro是由非结构蛋白3(Nsp3)编码的，它在 各式各样的Cov。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。这个 提出的小分子PROTAC是含有两个连接的活性配体的双功能小分子 通过化学链接物。一个配体与针对PLPro的病毒蛋白特异性结合，而另一个配体选择性地与病毒蛋白结合 与E3泛素连接酶结合。E3连接酶对PLPro的募集有助于三元体系的形成 复合体，导致泛素化并最终通过泛素化-蛋白酶体途径降解PLPro。 由于PROTAC通过降解而不是抑制目标蛋白来调节蛋白质的功能，它们提供了许多 与传统的以占有率为基础的抑制剂相比的优势，包括(一)亚化学计量比活性，(二)高阻隔性 对于抗药性，(Iii)改进了靶标选择性，以及(Iv)弱结合物(不需要抑制)可以成为 强有力的降解者。在此基础上，本应用程序的总体目标是验证PLPro作为一种 开发广谱抗病毒治疗药物以对抗冠状病毒的新战略。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的PROTAC进行化学优化 配体和PLPro抑制剂/结合剂，并在基于细胞的检测中评估它们对PLPro的降解能力。 在目的2中，测定已开发的PROTAC分子的抗SARS-CoV-2活性。强大的PROTAC(EC50 &lt；500 NM)将进一步测试其对其他CoV的抗病毒活性。体外DMPK及其细胞毒作用 将对有效的PROTAC进行研究。拟议研究的成功完成将导致 针对新冠肺炎的强效抗冠状病毒PROTAC，其作用机制与 现有的抗病毒药物。开发的PROTAC分子也将有可能被改变用途，以包含 未来冠状病毒暴发。