Novel Inhibitors Targeting Early Steps of Human Cytomegalovirus Replication

描述（由申请人提供）：人巨细胞病毒（HCMV）感染是免疫抑制患者发病和死亡的主要原因，特别是实体器官或骨髓移植的接受者。新生儿感染HCMV与耳聋、智力迟钝和死亡率有关。尽管五种抗病毒药物（GCV、val-GCV、CDV、PFA、Foscarnet）已被FDA批准并许可用于HCMV感染患者，但所有这些药物都有局限性，无法长期使用，包括口服生物利用度差、剂量相关毒性和耐药性。此外，观察到病毒DNA聚合酶抑制剂（GCV, val- GCV， CDV， PFA）之间的交叉耐药，因此靶向病毒复制周期不同步骤的化合物至关重要。我们开发改进HCMV治疗的策略是针对病毒感染的初始阶段，包括病毒附着、融合和小分子的即时早期病毒基因表达。HCMV进入是一个多步骤的过程，首先与细胞表面蛋白结合，然后是融合事件。病毒衣壳被释放到细胞质中，并运输到细胞核中，在那里它没有被包裹，将基因组沉积在细胞核中。病毒基因表达以瞬时表达模式进行，即早期（IE）、早期(E)和晚期(L)基因表达。IE1和IE2这两种特性良好的IE蛋白对病毒复制和控制下游转录因子至关重要。在初步研究中，我们开发并试点了HCMV复制早期事件抑制剂的高含量筛选。该筛选验证了使用融合IE2的报告基因（YFP）作为读数来测量HCMV感染早期步骤的抑制。然而，筛选的局限性包括使用实验室AD169 HCMV菌株，并且该检测受到荧光猝灭和化合物的干扰。因此，我们计划通过构建一种表达IE2蛋白产物与荧光素酶嵌合融合的HCMV临床菌株TB40/E变体来重新设计筛选。该重组病毒将用于基于细胞的高通量筛选，该筛选将在同一测定井中测量细胞活力和荧光素酶表达。这种筛选策略有可能识别出针对病毒感染初始阶段所必需的细胞和病毒蛋白的小分子抑制剂。本提案的目标是确定，

DESCRIPTION (provided by applicant): Human cytomegalovirus (HCMV) infection is a major cause of morbidity and mortality in immunosuppressed patients, especially recipients of solid organ or bone marrow transplants. HCMV infection of neonates is associated with deafness, mental retardation, and mortality. Although five anti-viral drugs (GCV, val-GCV, CDV, PFA, Foscarnet) have been approved and licensed by the FDA for use in patients with HCMV infection, all of them have limitations that preclude their long-term use, including poor oral bioavailability dose-related toxicity, and resistance. Furthermore, cross-resistance among the viral DNA polymerase inhibitors (GCV, val- GCV, CDV, PFA) is observed, and therefore, compounds targeting a different step in the viral replication cycle are of vital importance. Our strategy to develop an improved treatment for HCMV is to target the initial phase of virus infection, including virus attachment, fusion and immediate-early viral gene expression with small molecules. HCMV entry is a multi-step process that is initiated with its binding to cell surface proteins followed by a fusion event. The viral capsid is released into the cytosol and traffics to the nucleus where it is uncoated depositing the genome in the nucleus. Viral gene expression proceeds in a temporal expression pattern with immediate-early (IE), early (E) and late (L) gene expression. Two well-characterized IE proteins, IE1 and IE2, are essential for viral replication and for controlling downstream transcription factors. In preliminary studies, we developed and piloted a high-content screen for inhibitors of early events of HCMV replication. This screen validated the use of a reporter gene (YFP) fused to IE2 as a readout to measure inhibition of early steps in HCMV infection. However, the limitations of the screen include the use of the laboratory AD169 HCMV strain, and the assay is subject to fluorescence quenching and interference by compounds. Thus, we plan to redesign the screen by constructing a variant of the HCMV clinical strain TB40/E that expresses a chimeric fusion of the IE2 protein product with luciferase. This recombinant virus will be used in a cell-based high throughput screen that will measure both cell viability and luciferase expression in the same assay well. This screening strategy has the potential to identify small molecule inhibitors that target both cellular and vira proteins essential for initial stages of viral infection. The goal of this proposal is to identify, validate, and characterize small molecule inhibitors of the early stages of HCMV infection. In Phase I, we will screen e200,000 discrete small molecules. We will apply several secondary cell-based assays to eliminate nonspecific inhibitors and cytotoxic compounds. The antiviral activity of priority hits will be confirmed. We will also investigate the mechanisms of action and resistance of early leads. This work will be collaboration between Microbiotix and the Tortorella lab at Mount Sinai. In Phase II, we will chemically optimize the best candidates and demonstrate activity in vivo in animal infection models. The ultimate goal of this project is to develop these optimized inhibitors into clinical candidates for treating patients at high-risk for HCMV disease.