Inhibiting Viral Macrodomains Using Structure-Based Design

项目5：使用基于结构的设计抑制病毒巨噬细胞 摘要 病毒大结构域通过去除宿主的ADP-核糖化修饰来对抗宿主的免疫反应 蛋白质，对干扰素和许多其他信号通路有重要影响。以前的实验 在使用野生型和催化死亡的SARS-CoV和基孔肯雅病毒(CHIKV)的细胞和动物模型中 大结构域使这些蛋白质成为潜在的药物靶点。然而，目前还没有针对病毒的抑制剂。 宏域。使用基于高通量X射线的碎片筛选和对接的集成，我们拥有 确定了200多个SARS-CoV-2宏结构域(MAC1)的结合子。我们随后的化验结果指导了 开发第一个结构表征的比底物ADP更有效的工具化合物- 核糖。此外，我们还利用化学知识通过对接发现了更多的脚手架。在……里面 在这项建议中，我们通过基于结构的设计方法，使用生化分析来推进化合物 具有抗肽结合和催化作用。为了确定CHIKV宏域的起点，我们 将进行新的基于X射线晶体的碎片筛查，并执行超大型对接活动 (筛选核心)。我们的计划是设计针对人类大分子结构域的低效性化合物，并 利用药物化学来提高针对病毒大域的效力。我们的早期活动将确定 在体外具有高效率的多种替代支架，以进展到细胞中的靶向参与。我们以细胞为基础的 AIMS将使用干扰素处理细胞的细胞热移和免疫荧光作为靶标的早期标记 参与(蛋白质组学核心、体外病毒学核心)。我们将通过比较RNAseq， 复合处理细胞对突变体的蛋白质组学、磷酸化蛋白质组学和ADP-核糖基化AP-MS 大结构域(作为转基因和在病毒的背景下)。在这些活动期间，我们将继续 与药物化学核心解决渗透性、偏离目标的影响和其他责任方面的问题。 我们将通过验证靶点来研究具有细胞效应的化合物对复制的动物模型 订婚。具有有效靶向参与和最小药代动力学风险的铅分子将允许 美国将在SARS-CoV-2和CHIKV(体内)动物模型中测试候选分子的有效性 病毒学核心)。基于先前对具有催化失活大结构域突变的动物模型的研究 对于病毒，我们将优先考虑将病毒载量降低至少100倍的分子。我们的工作将产生一个目标 由我们的工业合作伙伴罗氏公司进一步开发的包。针对SARS-CoV-2和 CHIKV大结构域将适用于开发未来针对其他病毒和 那些与人类疾病有关的人。

PROJECT 5: INHIBITING VIRAL MACRODOMAINS USING STRUCTURE-BASED DESIGN SUMMARY Viral macrodomains counter the host immune response by removing ADP-ribosylation modifications from host proteins, with important consequences for interferon and many other signaling pathways. Previous experiments in cell and animal models that use wild type and catalytically dead SARS-CoV and chikungunya virus (CHIKV) macrodomains validate these proteins as potential drug targets. However, no inhibitors exist for viral macrodomains. Using an integration of high throughput X-ray based fragment screen and docking, we have identified over 200 binders to the SARS-CoV-2 macrodomain (Mac1). Our subsequent assays guided the development of the first structurally characterized tool compounds that are more potent than the substrate ADP- ribose. In addition, we have leveraged the chemical knowledge to uncover additional scaffolds by docking. In this proposal, we advance compounds through a structure-based design approach, using biochemical assays against peptide binding and catalytic function. To identify starting points against the CHIKV macrodomain, we will perform a new X-ray crystallography-based fragment screen and perform an Ultra-large docking campaign (Screening Core). Our plan is to design compounds with low potency against human macrodomains and to leverage medicinal chemistry to drive potency against viral macrodomains. Our early activities will identify multiple alternative scaffolds with high potency in vitro to progress to target engagement in cells. Our cell-based aims will use cellular thermal shift and immunofluorescence of interferon treated cells as early markers of target engagement (Proteomics Core, In Vitro Virology Core). We will identify biomarkers by comparing RNAseq, proteomics, phosphoproteomics and ADP-Ribosylation AP-MS of compound treated cells to mutant macrodomains (both as a transgene and in the context of virus). During these activities, we will continue addressing aspects of permeability, off target effects, and other liabilities with the Medicinal Chemistry Core. We will progress to animal models compounds with cellular effects on replication with validated target engagement. Lead molecules with validated target engagement and minimal pharmacokinetic liabilities will allow us to test the effectiveness of candidate molecules in animal models of SARS-CoV-2 and CHIKV (In Vivo Virology Core). Based on previous studies in animal models with catalytically inactive macrodomain mutant viruses, we will prioritize molecules that drop viral load by at least 100-fold. Our work will generate a target package for further development by our industrial partner, Roche. The lessons of targeting SARS-CoV-2 and CHIKV macrodomains will be applicable to developing future agents against macrodomains in other viruses and those implicated in human disease.