The Development of Reversible Covalent PROTAC Technology as a New Anti-COVID-19 Strategy

项目总结/摘要 目前由SARS-CoV-2引起的COVID-19大流行是全球卫生紧急情况。然而，迄今为止， 目前还没有发现有效的靶向药物或疫苗。寻找有效的靶向治疗方案是 至关重要SARS-CoV-2是一种有包膜的正向RNA病毒。主要蛋白酶（Mpro），a 半胱氨酸蛋白酶是病毒复制和发病所必需的，其代表了对免疫调节剂有吸引力的靶点。 开发抗SARS-CoV-2的抗病毒药物。传统的抗病毒策略之一是开发高- 亲和配体直接结合病毒蛋白并抑制其功能，如SARS-CoV-2 Mpro（SC2Mpro）。 然而，这些占据驱动的抑制剂可能导致几个潜在的问题，如脱靶毒性， 剂量限制性毒性和耐药性。因此，迫切需要新的抗病毒策略， 通过利用替代机制来对抗现有的冠状病毒病原体（如SARS）来应对这些挑战- 二型冠状病毒蛋白水解靶向嵌合体（PROTAC）是一种新兴的靶向蛋白降解技术， 药物发现。PROTAC是事件驱动的双功能小分子， 泛素连接酶与靶蛋白形成三元复合物，导致泛素化 并最终降解靶蛋白。PROTAC具有许多潜在优势， 传统的基于占有率的抑制剂，包括（i）允许亚化学计量活性的催化性质，（ii） 增强的靶选择性，（iii）高抗性屏障;和（iv）消除靶蛋白的所有功能 及其下游蛋白质。在此基础上，该提案提供了一种创新的抗CoV策略：可逆的 通过结合超强效可逆共价SC2Mpro抑制剂的优点， 事件驱动的PROTAC技术。总体目标是验证SC2Mpro的降解作为一种新策略， 开发具有更高选择性和疗效的COVID-19药物。目前的建议是建立在 发现几种有效的可逆共价SC2Mpro抑制剂的初步工作（最低IC50 <10 nM） 和一个小分子SC2Mpro PROTAC降解剂。在这些令人兴奋的初步研究的鼓舞下， 将通过追求以下目标来实现：（1）开发用于评估降解的细胞系统 （2）开发靶向SC2Mpro的各种有效的可逆共价抗CoV PROTAC;（3） SC2Mpro降解能力与抗SARS-CoV-2活性关系的探讨 可逆共价抗CoV PROTAC。如果这项研究顺利完成， 具有良好药物样特性的强效抗CoV PROTAC，可潜在地推进至临床前 评估治疗COVID-19，但也将提供一个概念验证研究，更广泛地开发抗 针对各种冠状病毒的CoV PROTAC。

PROJECT SUMMARY/ABSTRACT The current COVID-19 pandemic caused by SARS-CoV-2 is a global health emergency. However, to date, no effective targeted drug or vaccine has been identified yet. Finding effective targeted treatment options is of paramount importance. SARS-CoV-2 is an enveloped, positive-sensed RNA virus. Main protease (Mpro), a cysteine protease, is essential for viral replication and pathogenesis which represents an attractive target for the development of antiviral drugs against SARS-CoV-2. One of the traditional antiviral strategies is to develop high- affinity ligands that bind directly to viral proteins and inhibit their functions like SARS-CoV-2 Mpro (SC2Mpro). However, these occupancy-driven inhibitors may lead to several potential problems such as off-target toxicity, dose-limiting toxicity, and drug resistance. Thus, there is an urgent need for new antiviral strategies that can address these challenges by exploiting alternative mechanisms to combat existing CoV pathogens like SARS- CoV-2. Proteolytic targeting chimaera (PROTAC) is an emerging technology for targeted protein degradation in drug discovery. PROTACs are event-driven bifunctional small molecules that simultaneously engage an E3 ubiquitin ligase and a target protein to facilitate the formation of a ternary complex, leading to the ubiquitination and ultimate degradation of the target protein. PROTACs have many potential advantages compared to traditional occupancy-based inhibitors, including (i) catalytic nature to allow for sub-stoichiometric activity, (ii) enhanced target selectivity, (iii) high barrier to resistance; and (iv) abrogating all functions of the target protein and its downstream proteins. On this basis, this proposal provides an innovative anti-CoV strategy: reversible covalent PROTACs by combination of the advantages of ultra-potent reversible covalent SC2Mpro inhibitors and event-driven PROTAC technology. The overall goal is to validate degradation of SC2Mpro as a new strategy for developing COVID-19 drugs with improved selectivity and efficacy. The current proposal is built upon the preliminary work on the discovery of several potent reversible covalent SC2Mpro inhibitors (lowest IC50 < 10 nM) and one small-molecule SC2Mpro PROTAC degrader. Encouraged by these exciting preliminary studies, the goal will be achieved by pursuing the following aims: (1) the development of cellular systems to evaluate degradation of SC2Mpro; (2) the development of various potent reversible covalent anti-CoV PROTACs targeting SC2Mpro; (3) the exploration of the relationship between SC2Mpro degradation potencies and anti-SARS-CoV-2 activities of reversible covalent anti-CoV PROTACs. The successful completion of the proposed study will not only lead to potent anti-CoV PROTACs with good drug-like properties that can be potentially advanced to pre-clinical evaluation for treating COVID-19, but also will provide a proof-of-concept study for more broadly developing anti- CoV PROTACs against various coronaviruses.