Novel Inhibitors Targeting Early Steps of Human Cytomegalovirus Replication

针对人类巨细胞病毒复制早期步骤的新型抑制剂

• 批准号: 8847279
• 负责人: Terry L. Bowlin
• 金额: $ 28.96万
• 依托单位: MICROBIOTIX, INC
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-08 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8847279
• 关键词: Adverse effects; Animals; Antiviral Agents; Benign; Binding; Biological Assay; Biological Availability; Bone Marrow Transplantation; Capsid; Cell Nucleus; Cell Surface Proteins; Cell Survival; Cell membrane; Cells; Cidofovir; Clinical; Collaborations; Cytomegalovirus; Cytomegalovirus Infections; Cytosol; DNA Polymerase Inhibitor; DNA-Directed DNA Polymerase; Deposition; Development; Disease; Dose; Drug resistance; Event; Family; Fluorescence; Foscarnet; Future; Ganciclovir; Gene Expression; Genes; Genetic Transcription; Genome; Goals; Health; Herpesviridae; Human; Immediate-Early Proteins; Immunocompetent; Immunocompromised Host; Individual; Infection; Laboratories; Licensing; Life Cycle Stages; Luciferases; Maintenance Therapy; Measures; Medical; Mental Retardation; Modeling; Morbidity - disease rate; Neutropenia; Open Reading Frames; Oral; Organ; Patients; Pattern; Pharmaceutical Preparations; Phase; Population; Process; Prodrugs; Protein Kinase; Proteins; Reporter Genes; Resistance; Resistance development; Solid; Staging; Toxic effect; Transcript; United States Food and Drug Administration; Valganciclovir; Variant; Viral; Viral Genes; Viral Proteins; Virus; Virus Diseases; Work; base; cytotoxic; deafness; drug development; drug resistant virus; fomivirsen; high risk; high throughput screening; improved; in vivo; inhibitor/antagonist; member; mortality; mutant; neonate; novel; nucleoside analog; pathogen; patient population; recombinant virus; resistance mutation; screening; small molecule; trafficking; transcription factor; viral DNA

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.

描述(申请人提供)：人类巨细胞病毒(HCMV)感染是免疫抑制患者，特别是实体器官或骨髓移植受者发病率和死亡率的主要原因。新生儿巨细胞病毒感染与耳聋、智力低下和死亡率有关。尽管五种抗病毒药物(GCV、Val-GCV、CDV、PFA、Foscarnet)已被FDA批准和许可用于HCMV感染患者，但所有这些药物都有限制其长期使用的限制，包括口服生物利用度差、剂量相关毒性和耐药性。此外，还观察到病毒DNA聚合酶抑制剂(GCV、Val-GCV、CDV、PFA)之间存在交叉耐药性，因此，针对病毒复制周期中不同步骤的化合物至关重要。我们开发一种改进的治疗方法的策略是针对病毒感染的初始阶段，包括病毒附着、融合和小分子的即刻早期病毒基因表达。HCMV进入是一个多步骤的过程，首先与细胞表面蛋白结合，然后发生融合事件。病毒衣壳被释放到细胞质中，并运输到细胞核，在那里它没有被包裹，将基因组存放在细胞核中。病毒基因表达具有即刻-早期(IE)、早期(E)和晚期(L)基因表达的时间模式。IE1和IE2是病毒复制和控制下游转录因子所必需的两种IE蛋白。在初步研究中，我们开发并试行了一种高含量的巨细胞病毒复制早期事件抑制物筛选。这一筛选验证了使用融合到IE2的报告基因(YFP)作为读数来测量对HCMV感染早期步骤的抑制。然而，筛查的局限性包括使用实验室的AD169巨细胞病毒株，而且该检测受到荧光猝灭和化合物干扰的影响。因此，我们计划通过构建HCMV临床株TB40/E的变体来重新设计筛选，该变体表达IE2蛋白产物与荧光素酶的嵌合融合。这种重组病毒将用于基于细胞的高通量筛查，该筛查将在同一个测试中很好地测量细胞活力和荧光素酶的表达。这一筛选策略有可能识别针对细胞和病毒蛋白的小分子抑制物，这些蛋白对病毒感染的初始阶段至关重要。这项提案的目标是确定， 验证和鉴定HCMV感染早期阶段的小分子抑制剂。在第一阶段，我们将筛选e20万个离散的小分子。我们将应用几种基于二次细胞的分析来消除非特异性抑制物和细胞毒性化合物。优先命中药物的抗病毒活性将得到确认。我们还将研究早期先导的作用和抵抗机制。这项工作将是MicroBiotix和西奈山的Tortorella实验室之间的合作。在第二阶段，我们将对最佳候选药物进行化学优化，并在动物感染模型中展示体内活性。该项目的最终目标是将这些优化的抑制剂开发成临床候选药物，用于治疗巨细胞病毒病的高危患者。