Inhibitors of SARS-CoV-2 Polymerase

摘要 SARS-CoV-2 RNA依赖RNA聚合酶(RDRP)，非结构蛋白12(NSP12)，它在 与病毒蛋白nsp7和nsp8的复合体进行必要的RNA合成反应，是一种诱人的 抗病毒药物的有效靶点。到目前为止，抑制RDRP活性的核苷类似物显示出如下前景 新冠肺炎治疗。其中包括腺苷类似物GS-441524的前体药物雷米西韦(Rdv)和 首个获得美国食品和药物管理局批准的治疗新冠肺炎的抗病毒药物。莫努比拉韦最近的3期临床数据，a β-D-N4-羟基胞苷(NHC)的前药，鼓励默克寻求紧急授权使用。 虽然RDV缺乏口服生物利用度，限制了其门诊使用，但Molnupiravir是口服的 给药，但需要经常服用高剂量。此外，最先进的核苷抑制剂中没有一种是 专为治疗冠状病毒感染而开发。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而优化的新药。在第一种方法中， 我们在非人类灵长类动物身上建立了有希望的初步数据，因为我们的目标是开发一种经过改进的前药 口腔暴露与RDV相比，以便能够在门诊环境中使用口服给药，最好是每年一次 日间给药方案。我们的第二种方法试图识别和优化新的核苷类似物，以对抗 SARS-CoV-2 RDRP。这一方法的关键将是筛选包含167个新的 气-液分化的人支气管上皮细胞抗SARS-CoV-2活性的类似物 界面。第三，我们将使用基于细胞的SARS-CoV-2 RDRP分析来筛选一个新的结构上的文库 和第二个半胱氨酸和赖氨酸反应性化合物文库，以 识别变构抑制剂。有希望的RDRP抑制剂将通过测试来测试泛冠状病毒的潜力 对抗SARS冠状病毒、MERS冠状病毒和季节性冠状病毒。因为RDRP共享保守的结构， 广谱活动是可能的，也是可取的。因此，我们将从以下方面评估顶级RDRP抑制剂 本项目和项目5和项目6将侧重于虫媒病毒和出血热病毒，#年 建立了一组新出现病毒的生化RDRP检测方法。这些化验结果将提供机械性的 洞察广谱活动的基础。同时，将使用深度测序方法来确定 在受感染的细胞和动物中存在MOA。对于这些研究，复合进程将由迭代驱动 通过药物化学核心(核心B)、针对冠状病毒复制的生物图谱分析进行优化 在HTS核心(核心A)中，ADME、PK和来自药理学核心(核心C)的毒理学描述，动物 与有机化合物和动物模型核心(核心D)和基于结构的药物合作进行的疗效研究 具有结构和建模核心(核心E)的发现提供泛活性临床前冠状病毒 资料优于RDV和Molnupiravir的候选人。

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.