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

项目总结/摘要 目前由SARS-CoV-2引起的COVID-19大流行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。但现有 COVID-19疫苗靶向SARS-CoV-2的高度可变的膜刺突蛋白。新的病毒株， Spike中的关键突变已经出现，如α，β，δ和γ变体，可以使电流 疫苗效果不佳。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的抗病毒药物至关重要， 在未来对抗新型冠状病毒（CoV）。为了满足这一迫切需要，本提案旨在 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CoV PROTAC的技术。PLpro是两种重要的SARS-CoV之一， 2蛋白酶，在促进病毒转录和复制以及拮抗宿主先天性 免疫反应。PLpro由非结构蛋白3（nsp3）编码，该蛋白相对保守。 各种冠状病毒。因此，PLpro是开发广谱抗CoV药物的有吸引力的靶标。的 所提出的小分子PROTAC是含有两个连接的活性配体的双功能小分子 通过化学连接剂。一种配体特异性结合病毒蛋白靶PLpro，而另一种配体选择性结合病毒蛋白靶PLpro。 接合E3泛素连接酶。E3连接酶向PLpro的募集促进了三聚体的形成。 复杂，导致泛素化和最终降解的PLpro的泛素化蛋白酶体途径。 由于PROTAC通过降解靶蛋白而不是抑制它们来调节蛋白质功能，因此它们提供了许多 与传统的基于占据性的抑制剂相比的优点，包括（i）亚化学计量活性，（ii）高阻隔性 抗性，（iii）改进的目标选择性，和（iv）弱结合剂（不需要抑制）可以成为 强力降解剂在此基础上，本申请的总体目标是验证PLpro作为 开发广谱抗病毒疗法以对抗CoV的新策略。目标1： 通过连接VHL E3化学优化非共价、可逆共价和多重结合模式PROTAC 配体和PLpro抑制剂/结合剂，并评估它们在基于细胞的测定中对PLpro的降解效力。 在目的2中，确定开发的PROTAC分子的抗SARS-CoV-2活性。强效PROTAC（EC50 <500 nM）将进一步测试其对其他CoV的抗病毒活性。体外DMPK和细胞毒性 将进行强效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， 未来的冠状病毒爆发。

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.