Small molecule inhibitors and degraders of picornavirus 2A proteases as direct-acting antivirals

摘要-项目2：小核糖核酸病毒2A型蛋白水解酶的小分子抑制剂和降解物 传统的直接作用抗病毒药物(DAA)通过占据酶口袋来靶向病毒蛋白质；解离 这种药物的使用可立即恢复功能。因此，大多数抗病毒药物必须具有高亲和力， 化学计量结合是有效的，而降低亲和力的点突变可以迅速产生抗病毒药物 抵抗。预防耐药性的既定方法是使用直接作用的抗病毒药物的组合。 通过独立的目标和机制采取行动。在这个项目中，我们将采用两种创新的方法来 开发针对肠道病毒(EV)2A蛋白酶的DAA，EV是一种至少具有两个基本功能的病毒蛋白：(1) 它催化病毒多蛋白的必需切割，以及(2)它介导蛋白质-蛋白质相互作用，即 对复制至关重要。首先，我们将针对2A进行靶向蛋白质降解(TPD)作为一种抗病毒药物 策略。TPD是药物开发中的一种新范式，在这种范式中，一个小分子结合目标靶标 与E3连接酶配体结合，导致蛋白质降解。这种方法允许更广泛的 蛋白质靶标，因为其事件驱动的药理不需要对病毒的化学计量比抑制 这是因为有效的蛋白质降解所需的亲和力通常较低。我们之前 开发了第一个小分子抗病毒降解剂，通过偶联美国食品和药物管理局批准的 丙型肝炎病毒NS3-4A蛋白水解酶与CRBNCRL E3泛素连接酶的配基结合。由此产生的NS3降级 在体外抑制丙型肝炎病毒，包括已知对替拉维韦耐药的点突变。因为Telapvir是最近 被证明对肠道病毒2A蛋白具有广谱活性，我们将在先前工作的基础上 开发基于telapvir的2A降解剂，将蛋白质从细胞中移除，并消除2A的所有功能。 其次，我们将对绑定到2A并抑制其中介功能的DAA执行高通量筛选 基本的蛋白质-蛋白质相互作用。在这些筛选中发现的化合物将被确认为2A型配体 并进行了抗病毒活性测试，验证的配体作为降解剂开发和开发的先导 具有抗病毒活性的化合物是开发DAAs的先导化合物。我们还将承诺 筛选DNA编码文库以更广泛地采样化学空间并发现更多的化学物质 适合缓蚀剂和降解剂开发的物质。我们还将确定DAA是否针对活动站点 (AIM 1)和2A相互作用部位(AIM 2)可以结合在一起，以获得更好的疗效和耐药性。这 Work利用了我们的实验室在TPD和2A生物学领域取得的创新发现，以及 我们团队和AViDD在病毒学、药物化学、化学生物生物化学、 结构生物学和药物发现。我们针对多功能EV-2A蛋白酶的DAAs开发工作 将产生广泛的影响，验证两种针对2A的新抗病毒机制，并通过演示 TPD作为一种抗病毒策略的效用。

ABSTRACT – Project 2: Small molecule inhibitors and degraders of picornavirus 2A proteases Traditional direct-acting antivirals (DAAs) target a viral protein by occupying an enzymatic pocket; dissociation of the drug leads to immediate regain of function. Consequently, most antivirals must have high-affinity, stoichiometric binding to be effective, and point mutations that reduce affinity can rapidly give rise to antiviral resistance. The established method to prevent resistance is the use of combinations of direct-acting antivirals that act via independent targets and mechanisms. In this project, we will pursue two innovative approaches to develop DAAs targeting the enterovirus (EV) 2A protease, a viral protein with at least two essential functions: (1) it catalyzes a required cleavage of the viral polyprotein and (2) it mediates a protein-protein interaction that is essential for replication. First, we will pursue targeted protein degradation (TPD) against 2A as an antiviral strategy. TPD is a new paradigm in drug development in which a small molecule that binds the target of interest is conjugated to an E3 ligase ligand resulting in protein degradation. This approach allows a wider range of protein targets because its event-driven pharmacology does not require stoichiometric inhibition of the viral protein and because the affinity required for effective protein degradation is generally lower. We previously developed the first small molecule antiviral degrader by coupling telaprevir, an FDA-approved inhibitor of the hepatitis C virus NS3-4A protease, to a ligand of the CRBNCRL E3 ubiquitin ligase. The resulting NS3 degraders inhibit HCV in vitro, including point mutants that are known to be telaprevir-resistant. Since telaprevir was recently shown to have broad-spectrum activity against enterovirus 2A proteins, we will build upon our prior work to develop telaprevir-based degraders of 2A that remove the protein from the cell and ablate all of 2A’s functions. Second, we will perform high-throughput screens for DAAs that bind to 2A and inhibit its function as a mediator of essential protein-protein interactions. Compounds discovered in these screens will be validated as 2A ligands and tested for antiviral activity, with validated ligands advanced as leads for degrader development and compounds with antiviral activity advanced as lead compounds for development of DAAs. We will also undertake screening of DNA-encoded libraries to more broadly sample chemical space and to discover additional chemical matter suitable for inhibitor and degrader development. We will also determine if DAAs targeting the active site (Aim 1) and the 2A interacting site (Aim 2) can be combined for superior efficacy and resistance profile. This work leverages the innovative discoveries made by our labs in the areas of TPD and 2A biology along with the collective expertise of our groups and the AViDD in virology, medicinal chemistry, chemical biology biochemistry, structural biology, and drug discovery. Our work developing DAAs targeting the multifunctional EV 2A protease will have wide-reaching impact by validating two new antiviral mechanisms for targeting 2A and by demonstrating the utility of TPD as an antiviral strategy.