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蛋白酶的小分子抑制剂和降解剂 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;解离 药物的作用是立即恢复功能。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抗体。 阻力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发挥作用。在本项目中，我们将采用两种创新方法， 开发靶向肠道病毒（EV）2A蛋白酶的DAA，该蛋白酶是一种具有至少两种基本功能的病毒蛋白：（1） 它催化所需的病毒多蛋白裂解，和（2）它介导蛋白-蛋白相互作用， 对于复制至关重要。首先，我们将寻求针对2A的靶向蛋白降解（TPD）作为抗病毒药物。 战略TPD是药物开发中的一种新范式，其中结合感兴趣的靶标的小分子 与E3连接酶配体缀合，导致蛋白质降解。这种方法允许更广泛的 蛋白靶点，因为其事件驱动的药理学不需要化学计量抑制病毒 这是因为蛋白质的有效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telaprevir，一种FDA批准的 丙型肝炎病毒NS 3 -4A蛋白酶与CRBNCRL E3泛素连接酶的配体的结合。由此产生的NS 3降解剂 体外抑制HCV，包括已知对特拉普韦耐药的点突变体。由于特拉匹韦最近 显示对肠道病毒2A蛋白具有广谱活性，我们将在先前工作的基础上， 开发基于替拉普利韦的2A降解剂，从细胞中去除蛋白质并消除2A的所有功能。 第二，我们将进行高通量筛选，寻找与2A结合并抑制其介导功能的DAA 重要的蛋白质相互作用。在这些筛选中发现的化合物将被验证为2A配体 并测试了抗病毒活性，并将经验证的配体作为降解剂开发的先导， 具有抗病毒活性的化合物作为开发DAA的先导化合物。我们还将承担 筛选DNA编码的文库以更广泛地对化学空间进行采样并发现另外的化学物质 适合于抑制剂和降解剂开发的物质。我们还将确定针对活性位点的DAA (Aim 1）和2A相互作用位点（Aim 2）可以组合以获得上级功效和抗性特征。这 工作利用了我们的实验室在TPD和2A生物学领域的创新发现，沿着 我们的团队和AViDD在病毒学，药物化学，化学生物学生物化学， 结构生物学和药物发现。我们的工作是开发针对多功能EV 2A蛋白酶的DAA 将通过验证两种针对2A的新抗病毒机制和证明 TPD作为抗病毒策略的效用。

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.