Development of Broad-Spectrum Antiviral Therapeutics by Destabilizing the Main Protease of Coronaviruses

摘要 目前，2019年冠状病毒病（COVID-19）在全球范围内爆发，这是由 严重急性呼吸综合征冠状病毒2（SARS-CoV-2）感染。我们迫切需要有效的 针对SARS-CoV-2和相关冠状病毒的抗病毒治疗，以避免未来潜在的爆发。这 该提案寻求试点资金，以支持药物化学家和 病毒学家开发新的小分子，可以使3-糜蛋白酶样蛋白酶（3CLpro）不稳定， SARS-CoV-2和许多相关冠状病毒复制所必需的主要病毒蛋白酶。的 提出的小分子能够通过宿主细胞的泛素催化3CLpro的降解， 蛋白酶体系统，其常规地去除受损或未折叠的蛋白质。 所提出的降解剂是在两个配体之间具有短接头的双功能分子。一个配体结合 具有对E3泛素连接酶的高选择性，而另一种配体同时接合病毒蛋白 目标当结合事件发生时，病毒蛋白与E3泛素连接酶紧密接触 复合物，并被聚泛素化以在蛋白酶体中降解。然后释放降解剂， 继续其降解病毒蛋白的催化活性。降解剂只需要瞬时结合 靶病毒蛋白诱导其泛素化，这提供了许多优势，比传统的小 分子抑制剂。 基于3CLpro在病毒复制中的关键作用，我们选择3CLpro作为选择性降解的主要靶标， 选择性抑制剂的可用性，其将作为与3CLPro结合的配体。虽然抑制剂 3CLpro存在，降解剂的催化性能将使它们更有效。通过破坏稳定， 破坏病毒蛋白而不是化学计量地结合病毒蛋白，降解剂也应该起作用， 比抑制剂更快。3CLpro在许多冠状病毒中是保守的，包括SARS-CoV、MERS-CoV和 SARS-CoV-2.所提出的小分子降解剂可以是针对广泛的抗病毒治疗剂。 冠状病毒谱，包括对抗病毒抑制剂耐药的病毒株。在目标1中， 利用我们最近的研究成果，通过连接E3连接酶和3CLPro的配体来制备双功能小分子降解剂， 制定了两阶段战略。在目的2中，我们将评估3CLPro的降解和抗病毒活性 降解物在基于细胞的测定中。 PI和合作研究者的研究小组在双功能小分子降解剂方面经验丰富 开发和抗病毒研究。拟议的研究将产生重要的概念验证 为开发针对SARS-CoV-2和相关冠状病毒的新型抗病毒疗法提供数据。

Abstract At the moment, there is a worldwide outbreak of coronavirus disease 2019 (COVID-19) that is caused by the infection from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We urgently need effective anti-viral therapeutics against SARS-CoV-2 and related coronaviruses to avoid potential future outbreaks. This proposal seeks pilot funding to support the interdisciplinary collaboration between medicinal chemists and virologists to develop novel small molecules that can destabilize 3-chymotrypsin like protease (3CLpro), the main viral protease that is essential for the replication of SARS-CoV-2 and many related coronaviruses. The proposed small molecules are able to catalyze the degradation of 3CLpro through the host cell’s ubiquitin- proteasome-system, which routinely removes damaged or unfolded proteins. The proposed degraders are bifunctional molecules with a short linker between two ligands. One ligand binds with high selectivity to an E3 ubiquitin ligase while the other ligand simultaneously engages the viral protein target. As the binding event occurs, the viral protein is brought in close contact with the E3 ubiquitin ligase complex and is poly-ubiquitinated for degradation in the proteasome. The degrader is then released to continue its catalytic activity for the degradation of the viral protein. The degrader only needs to bind transiently to the target viral protein to induce its ubiquitination, which offers many advantages over traditional small molecule inhibitors. We selected 3CLpro as the primary target for selective degradation based on its key role in viral replication and the availability of selective inhibitors, which will serve as the ligand that binds to 3CLPro. Although inhibitors for 3CLpro exist, degraders’ catalytic properties will render them much more potent. By destabilizing and destroying the viral protein instead of stoichiometrically binding to the viral protein, degraders should also act faster than inhibitors. 3CLpro is conserved among many coronaviruses including SARS-CoV, MERS-CoV, and SARS-CoV-2. The proposed small molecule degraders can be potent antiviral therapeutics against a broad spectrum of coronaviruses, including viral strains that are resistant to antiviral inhibitors. In aim 1, we will prepare bifunctional small molecule degraders by linking ligands of E3 ligase and 3CLPro using our recently developed two-stage strategy. In aim 2, we will evaluate the degradation and anti-viral activities of 3CLPro degraders in cell-based assays. The research groups of PI and Co-Investigator are highly experienced in bifunctional small molecule degrader development and anti-viral research, respectively. The proposed study will produce important proof-of-concept data for the development of novel antiviral therapeutics against SARS-CoV-2 and related coronaviruses.