Multiplex Small Molecule Discovery to Identify Broad-Acting Viral Protease Inhibitors

摘要 病毒病原体给社会带来了严重的健康和经济负担，但对大多数病毒来说， 没有经过批准的抗病毒化合物。更糟糕的是，由于持续的 新出现的病毒病原体(例如MERS、寨卡病毒和SARS-CoV-2)。这项建议的目的是 利用高通量、多路复用的方法与一种新的方法相结合进行药物筛选 调查耐药变异，以指导长效抗病毒药物的开发，长期目标是 弥合现有的抗病毒治疗缺口。我们的建议是基于这样一个中心假设：DNA- 条形码技术与基本病毒蛋白的深度突变扫描(DMS)相结合，可以快速 在化学空间中搜索，并指导从头到尾的小分子发现过程。其基本原理是 这一提议的基础是，如果成功，我们将能够开发出积极应对的优化线索 多种病毒和强大的病毒逃逸能力，其时间、成本和工作量仅为传统方法的一小部分。 鉴于正在进行的大流行及其已证实的大流行潜力，在我们提案的初始阶段，我们 将专注于产生针对冠状病毒蛋白酶的广泛活性的抑制剂。在接下来的几年里，我们将瞄准 其他基本病毒蛋白(如甲基转移酶)和病毒家族(如黄病毒科)。我们的初步数据 支持我们的方法同时筛选数十种病毒蛋白酶抑制剂的可行性， 以及我们表征数百种突变对病毒蛋白水解酶反应的影响的能力 化学抑制剂。为了实现我们项目的目标，我们将追求以下三个目标：1)增加 要同时筛选到≥100的病毒靶点的数量，并针对它们进行小分子筛选； 2)测试我们针对活病毒的筛选命中，进化其效力和类似药物的特性，并展示其 体内疗效；以及3)使用全面的诱变来了解药物与靶点的相互作用，并指导我们的 药物开发努力。这一建议具有创新性，因为它提出了一种小分子的多重方法 筛选，增加获得的数据的数量和丰富性。它还发展了一种研究方法 数千种基本病毒蛋白的突变变体对化学抑制的反应，并使用这一方法 指导从点击到领先的优化过程的信息。这项工作意义重大，预计将有一个 通过确定一组有前景的抗人和动物病毒的宽效蛋白水解酶抑制剂产生积极影响 病原体，开发高度可扩展的药物筛选方法，并提供合并的框架 在整个药物发现过程中使用结构和药物化学进行耐药性分析。

ABSTRACT Viral pathogens present a serious health and economic burden to society, yet for the majority of viruses, there are no approved antiviral compounds. Worse still, this treatment gap continues to widen due to a continuous stream of emerging viral pathogens (e.g. MERS, Zika, and SARS-CoV-2). The objective of this proposal is to utilize a high-throughput, multiplexed approach for drug screening in combination with a novel approach to surveying drug resistant variants to guide the development of broad-acting antivirals with the long-term goal of bridging the existing antiviral therapeutic gap. Our proposal is based on the central hypothesis that DNA- barcoding technology coupled with deep mutational scans (DMS) of essential viral proteins can be used to rapidly search through chemical space and guide the hit-to-lead small molecule discovery process. The rationale underlying this proposal is that, if successful, we will be able to develop optimized leads that are active against multiple viruses and robust to viral escape at a fraction of the time, cost, and effort of traditional approaches. Given the ongoing pandemic and their proven pandemic potential, during the initial stages of our proposal, we will focus on generating broadly active inhibitors against coronavirus proteases. In later years, we will target other essential viral proteins (e.g. methyltransferase) and viral families (e.g. Flaviviridae). Our preliminary data support the feasibility of our approach for screening for inhibitors to dozens of viral proteases at the same time, along with our ability to characterize the effects of hundreds of mutations on the response of a viral protease to chemical inhibitors. To achieve our project’s goals, we will pursue the following three aims: 1) Increase the number of viral targets to be simultaneously screened to ≥100 and perform small molecule screens against them; 2) Test our screening hits against live virus, evolve their potency and drug-like properties, and demonstrate their in vivo efficacy; and 3) Use comprehensive mutagenesis to understand drug-target interactions and guide our drug development efforts. This proposal is innovative because it presents a multiplex method of small molecule screening that increases the quantity and richness of the data obtained. It also develops a method of studying the response of thousands of mutant variants of essential viral proteins to chemical inhibition, and uses this information to guide the hit-to-lead optimization process. This work is significant and is expected to have a positive impact by identifying a set of promising broad-acting protease inhibitors against human and animal viral pathogens, developing a highly-scalable approach to drug screening, and providing a framework for merging resistance profiling with structural and medicinal chemistry throughout the drug discovery process.