Inhibitors of SARS-CoV-2 Polymerase

总结 SARS-CoV-2 RNA依赖性RNA聚合酶（RDRP），即非结构蛋白12（NSP12），在 与病毒蛋白nsp7和nsp8的复合物进行必要的RNA合成反应，是一种有吸引力的， 抗病毒药物的有效靶点迄今为止，抑制RDRP活性的核苷类似物显示出作为 COVID-19治疗其中有瑞德西韦（RDV），腺苷类似物GS-441524的前药，和腺苷类似物GS-441524的前药。 FDA批准的第一种用于治疗COVID-19的抗病毒药物。莫努匹拉韦的最新3期临床数据， β-D-N4-羟基胞苷（NHC）的前药，鼓励默克寻求紧急授权使用。 鉴于RDV缺乏口服生物利用度，限制了其门诊使用，而莫努匹拉韦是口服的， 但需要频繁的高剂量。此外，没有一种最先进的核苷抑制剂是 专门用于治疗冠状病毒感染。在这里，我们提出了三种并行的方法来提供 针对SARS-CoV-2 RDRP和治疗冠状病毒感染的新型药物。在第一种方法中， 我们在非人灵长类动物中建立了有希望的初步数据，因为我们的目标是开发一种前药， 口服暴露与RDV，以便能够在门诊环境中使用口服给药，优选一次一次 日给药方案。我们的第二种方法旨在鉴定和优化新型核苷类似物， SARS-CoV-2 RDRP。这种方法的关键将是筛选一个核苷文库，其中包括167个新的 在空气-液体中分化的人支气管上皮细胞中抗SARS-CoV-2活性的类似物 接口.第三，我们将使用基于细胞的SARS-CoV-2 RDRP分析来筛选结构上 多样的"完全功能化片段"和半胱氨酸和赖氨酸反应性化合物的第二文库， 鉴别别构抑制剂。有希望的RDRP抑制剂将通过测试来测试泛冠状病毒的潜力 针对SARS-CoV、MERS-CoV和季节性冠状病毒。因为RDRP共享保守结构， 广谱活性是可能的并且是期望的。因此，我们将评估来自 本项目以及项目5和6（侧重于虫媒病毒和出血热病毒）， 为一组新出现的病毒建立了生化RDRP测定。这些测定将提供机械的 深入了解广谱活性的基础。同时，深度测序方法将用于定义 感染细胞和动物中的MOA。对于这些研究，复合进展将由迭代驱动 药物化学核心（核心B）优化，针对冠状病毒复制试验的生物学特征分析 在HTS核心（核心A）中，药理学核心（核心C）的ADME、PK和毒理学特征，动物 与类器官和动物模型核心（Core D）合作进行的疗效研究，以及基于结构的药物 与结构和建模核心（核心E）的发现，以提供临床前的泛活性冠状病毒 具有优于RDV和莫努匹拉韦的特征上级的候选物。

SUMMARY The SARS-CoV-2 RNA-dependent RNA polymerase (RDRP), non-structural protein 12 (NSP12), which in complex with viral proteins nsp7 and nsp8 carries out essential RNA synthesis reactions, is an attractive and well-validated target for antivirals. To date, nucleoside analogs that inhibit RDRP activity show promise as COVID-19 treatments. Among these is remdesivir (RDV), a prodrug of the adenosine analog GS-441524 and the first FDA-approved antiviral for the treatment of COVID-19. Recent phase 3 clinical data for molnupiravir, a prodrug of β-D-N4-hydroxycytidine (NHC), has encouraged Merck to seek Emergency Authorization Use. Whereas RDV suffers from lack of oral bioavailability, limiting its outpatient use, molnupiravir is orally administered but requires frequent high doses. Further, none of the most advanced nucleoside inhibitors were developed specifically to treat coronavirus infections. Here, we propose three parallel approaches to provide novel drugs optimized to target the SARS-CoV-2 RDRP and treat coronavirus infections. In the first approach, we build on promising preliminary data in non-human primates as we aim to develop a prodrug with improved oral exposure vs. RDV so as to enable oral administration for use in the outpatient setting, preferably a once a day dosing regimen. Our second approach seeks to identify and optimize novel nucleoside analogs against SARS-CoV-2 RDRP. Key to this approach will be the screening of a nucleoside library that includes 167 novel analogs for activity against SARS-CoV-2 in human bronchial epithelial cells differentiated in an air-liquid interface. Third, we will use a cell-based assay of SARS-CoV-2 RDRP to screen a novel library of structurally diverse “fully functionalized fragments” and a second library of cysteine- and lysine-reactive compounds to identify allosteric inhibitors. Promising RDRP inhibitors will be tested for pan-coronavirus potential by testing against SARS-CoV, MERS-CoV and seasonal coronaviruses. Because RDRPs share conserved structures, broad-spectrum activity is possible and would be desirable. Therefore, we will evaluate top RDRP inhibitors from this Project and from Projects 5 and 6, which will focus on arboviruses and hemorrhagic fever viruses, in established biochemical RDRP assays for a panel of emerging viruses. These assays will provide mechanistic insight into the basis for broad-spectrum activity. In parallel, deep-sequencing approaches will be used to define MOA in infected cells and animals. For these studies, compound progression will be driven by iterative optimization by the Medicinal Chemistry Core (Core B), biological profiling against coronavirus replication assays in the HTS Core (Core A), ADME, PK and toxicology profiling from the Pharmacology Core (Core C), animal efficacy studies in collaboration with the Organoid and Animal Model Core (Core D), and structure-based drug discovery with the Structural and Modeling Core (Core E) to deliver a pan-active coronavirus preclinical candidate with a profile superior to RDV and molnupiravir.