A novel assay for inhibitors of influenza A virus polymerase complex assembly

甲型流感病毒聚合酶复合物组装抑制剂的新测定法

• 批准号: 7533709
• 负责人: FENG LI
• 金额: $ 17.52万
• 依托单位: SOUTH DAKOTA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-25 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7533709
• 关键词: Antiviral Agents; Biological Assay; C-terminal; Categories; Cell Density; Cell Line; Cells; Class; Complex; Condition; DNA-Directed RNA Polymerase; Detection; Development; Disease Outcome; Disruption; Drug Delivery Systems; Drug resistance; Fluorescence; Foundations; Generations; Genetic Transcription; Goals; Green Fluorescent Proteins; Growth; Human; Influenza; Influenza A virus; Lead; Life; Localized; Location; Mediating; Molecular; N-terminal; National Institute of Allergy and Infectious Disease; Nature; Neuraminidase inhibitor; Noise; Peptides; Polymerase; Preclinical Drug Evaluation; Process; Production; Protein Subunits; Proteins; Public Health; Purpose; RNA Polymerase I; RNA chemical synthesis; Reporting; Research; Research Project Grants; Research Proposals; Screening procedure; Signal Transduction; Site; Specificity; Staging; System; Translations; Treatment Failure; Validation; Venus; Viral; Viral Drug Resistance; Viral Physiology; Virion; Virus; Virus Diseases; Virus Inhibitors; Virus Replication; Visual; anti-influenza; anti-influenza drug; assay development; base; concept; coping; design; drug development; fluorophore; high throughput screening; improved; influenzavirus; inhibitor/antagonist; novel; pandemic influenza; pathogen; protein protein interaction; reconstitution; small molecule libraries; therapeutic target; tool; viral RNA

DESCRIPTION (provided by applicant): Although effective neuraminidase inhibitors are available to treat influenza viral infections in humans, the emergence of virus isolates resistant to these drugs can have a significant adverse impact on both treatment options and disease outcome. To better prepare us to cope with an emerging influenza pandemic and combat the rising problem of antiviral drug resistance, new classes of antiviral drugs targeted to conserved viral functions are urgently needed. One such target for anti-influenza drug development is the assembly of the viral RNA polymerase complex. The viral RNA polymerase complex (PA-PB1-PB2) is a heterotrimer composed by the three subunits, polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), and polymerase acidic protein (PA). PB1 subunit forms the core of the polymerase complex. PB1 interacts through its N-terminal region with the C- terminal region of PA, while the C-terminal region of PB1 is involved in an interaction with PB2. No interaction between PA and PB2 subunits has been reported. While the molecular mechanism of the polymerase complex formation remains unclear, the assembly of viral RNA polymerase complex during viral replication is a highly regulated and dynamic process and is essential for viral RNA synthesis and production of infectious influenza virus particles. Any disruption of the viral RNA polymerase complex formation or even inhibition of sequential nature of the assembly process profoundly impairs the transcription and translation of viral RNA segments and viral infectivity. Proof of principle for inhibition of influenza A virus RNA polymerase complex assembly has been recently demonstrated by developing a peptide-based inhibitor which potently inhibits growth of influenza A viruses through specifically blocking the PB1-PA interaction and subsequently interfering with viral RNA polymerase complex assembly. These studies support our focus on influenza A RNA polymerase formation as a target for therapeutic discovery and development. The overall goal of this research proposal is to develop a novel assay that may lead to the identification of antiviral compounds, which inhibit Influenza A virus replication by disrupting the PB1-PA subunit interaction that is critical to govern the assembly process of influenza A virus RNA polymerase complex. The identification of specific influenza virus inhibitors will serve two major purposes. First, these compounds will provide valuable tools for dissecting distinct stages of the assembly process of viral RNA polymerase complex at the cellular and molecular level. Second, some of the identified compounds may serve as lead compounds for the development of novel anti-influenza drugs. The technical foundation for the proposed assay is the use of Bimolecular Fluorescence Complementation (BiFC) which is based on the principle that N- and C-terminal fragments of fluorescent proteins (GFP and its derivatives) do not spontaneously fold and reconstitute a functional fluorophore. However, if fused to interacting proteins, the two non-functional halves of the fluorophore, following the expression in cells, are brought into close proximity as a result of the specific protein interactions. This initiates folding of the fragments into an active protein, which then can reconstitute a detectable fluorescent signal at the site of the protein-protein complex. Thus, through BiFC, the specific interaction between PB1 and PA subunits can be precisely visualized, quantified and localized within live cells. By disrupting PB1-PA interaction, compounds will cause reductions in BiFC readout, indicative of the presence of potential inhibitors targeting the assembly of PB1-PA complex. Two specific aims are proposed in the application: (1) to develop and characterize BiFC constructs which allow for visual identification of PB1-PA dimeric complex in living cells, and (2) to develop a BiFC-based prototypic influenza A virus RNA polymerase complex assembly inhibitor screening assay suitable for use in a high-throughput format. It is anticipated that this research project will establish a proof-of-concept for the BiFC approach to identifying assembly inhibitors of influenza A virus RNA polymerase complex, which will provide the basis for the development of a high-throughput screening assay. Importantly, successful accomplishment of this project will lead to the validation of a BiFC-based approach to drug screening for viral pathogens. This could then be applied to NIAID category A, B or C viral agents, which could not be carried out in most locations because of biosafety concerns. PUBLIC HEALTH RELEVANCE Drug resistance poses a great challenge for anti-influenza therapy and contributes to influenza treatment failure. Successful completion of the proposed research will help identify and design additional anti-influenza inhibitors that will provide additional treatment options and improve disease outcome.

描述（由申请人提供）：尽管有效的神经氨酸酶抑制剂可用于治疗人类的流感病毒感染，但对这些药物抗药性的病毒分离株的出现可能会对治疗方案和疾病结果产生重大不利影响。为了更好地准备我们应对新兴的流感大流行并打击抗病毒药耐药性的不断增长的问题，紧急需要针对保守病毒功能的新型抗病毒药物。这种抗激素药物开发的一个靶标是病毒RNA聚合酶复合物的组装。病毒RNA聚合酶复合物（PA-PB1-PB2）是由三个亚基组成的异三聚体，聚合酶碱性蛋白1（PB1），聚合酶碱性蛋白2（PB2）和聚合酶酸性蛋白（PA）。 PB1亚基构成聚合酶复合物的核心。 PB1通过其N末端区域与PA的C末端区域相互作用，而PB1的C末端区域参与与PB2的相互作用。 PA和PB2亚基之间没有相互作用。虽然聚合酶复合物的分子机制尚不清楚，但病毒复制过程中病毒RNA聚合酶复合酶复合物的组装是一个高度调节的动态过程，对于病毒RNA的合成和感染性流感病毒颗粒的产生至关重要。病毒RNA聚合酶复合酶复合物的任何破坏，甚至抑制了组装过程的顺序性质，都会严重损害病毒RNA片段和病毒感染性的转录和翻译。最近，通过开发基于肽的抑制剂来证明了抑制流感病毒RNA聚合酶复合酶复合物的原理证明，该抑制剂通过特异性阻断PB1-PA相互作用并随后干扰病毒RNA聚合酶复合酶复合物的干扰来抑制流感病毒的生长。这些研究支持我们关注流感的RNA聚合酶形成，作为治疗发现和发育的靶标。该研究提案的总体目标是开发一种新的测定方法，该测定法可能导致抗病毒药化合物的鉴定，该化合物通过破坏PB1-PA亚基相互作用来抑制流感病毒复制，这对于控制流感A A A型流感病毒RNA RNA聚合酶复合物的组装过程至关重要。特定流感病毒抑制剂的鉴定将有两个主要目的。首先，这些化合物将提供有价值的工具，以剖析细胞和分子水平的病毒RNA聚合酶复合物的组装过程的不同阶段。其次，某些已鉴定的化合物可以用作开发新型抗激素药物的铅化合物。该提议的测定的技术基础是使用双分子荧光互补（BIFC），该原理是基于荧光蛋白（GFP及其衍生物）的N和C末端片段的原理，并不会自发地折叠并重新构成功能性荧光团。但是，如果与相互作用的蛋白质融合在一起，由于特定的蛋白质相互作用，荧光团表达后的两个非功能性一半在细胞中的表达近距离接近。这将片段折叠成活性蛋白，然后可以在蛋白质蛋白质复合物位点重建可检测的荧光信号。因此，通过BIFC，可以精确地将PB1与PA亚基之间的特异性相互作用在活细胞内进行可视化，量化和局部定位。通过破坏PB1-PA相互作用，化合物将导致BIFC读数的减少，这表明存在针对PB1-PA复合物组装的潜在抑制剂。应用程序中提出了两个具体的目的：（1）开发和表征BIFC构建体，这些构建体可以直观地识别活细胞中PB1-PA二聚体复合物，（2）开发基于BIFC的原型型原型A a病毒RNA聚合酶RNA聚合酶复合酶复合物抑制剂抑制剂抑制剂抑制剂，适用于高率进行高发行格式。可以预料，该研究项目将为BIFC方法建立概念概念，以识别流感型A型病毒RNA聚合酶复合物的组装抑制剂，这将为开发高通量筛查测定法提供基础。重要的是，该项目的成功完成将导致验证基于BIFC的病毒病原体药物筛查方法。然后可以将其应用于NIAID类别A，B或C病毒剂，由于生物安全问题，该剂在大多数位置无法进行。公共卫生相关性耐药性对抗激素疗法构成了巨大挑战，并导致流感治疗衰竭。成功完成拟议的研究将有助于识别和设计其他抗激素抑制剂，这些抑制剂将提供额外的治疗选择并改善疾病结果。