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: 7921295
• 负责人: FENG LI
• 金额: $ 10万
• 依托单位: SOUTH DAKOTA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-22 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7921295
• 关键词: Antiviral Agents; Biological Assay; C-terminal; Categories; Cell Density; Cell Line; Cells; Complex; DNA-Directed RNA Polymerase; Detection; Development; Disease Outcome; Drug Delivery Systems; Drug resistance; Fluorescence; Foundations; Generations; Genetic Transcription; Goals; Growth; Human; Influenza; Influenza A virus; Lead; Life; 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; 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; combat; coping; design; drug development; fluorophore; high throughput screening; improved; influenzavirus; inhibitor/antagonist; novel; pandemic influenza; pathogen; protein complex; protein protein interaction; public health relevance; 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（由申请方提供）：尽管有效的神经氨酸酶抑制剂可用于治疗人类流感病毒感染，但出现对这些药物耐药的病毒分离株可能对治疗选择和疾病结局产生显著不良影响。为了更好地科普新出现的流感大流行并对抗日益严重的抗病毒药物耐药性问题，迫切需要靶向保守病毒功能的新型抗病毒药物。抗流感药物开发的一个这样的靶标是病毒RNA聚合酶复合物的组装。病毒RNA聚合酶复合物（PA-PB 1-PB 2）是由聚合酶碱性蛋白1（PB 1）、聚合酶碱性蛋白2（PB 2）和聚合酶酸性蛋白（PA）三个亚基组成的异源三聚体。PB 1亚基形成聚合酶复合物的核心。PB 1通过其N-末端区域与PA的C-末端区域相互作用，而PB 1的C-末端区域参与与PB 2的相互作用。PA和PB 2亚基之间没有相互作用的报道。虽然聚合酶复合物形成的分子机制仍不清楚，但病毒复制期间病毒RNA聚合酶复合物的组装是一个高度调节和动态的过程，对于病毒RNA合成和感染性流感病毒颗粒的产生至关重要。病毒RNA聚合酶复合物形成的任何破坏或甚至组装过程的顺序性质的抑制都会深刻地损害病毒RNA片段的转录和翻译以及病毒感染性。最近通过开发一种基于肽的抑制剂证明了抑制甲型流感病毒RNA聚合酶复合物组装的原理，该抑制剂通过特异性阻断PB 1-PA相互作用并随后干扰病毒RNA聚合酶复合物组装而有效抑制甲型流感病毒的生长。这些研究支持我们将甲型流感RNA聚合酶形成作为治疗发现和开发的靶点。本研究提案的总体目标是开发一种新的检测方法，该方法可能导致抗病毒化合物的鉴定，该化合物通过破坏PB 1-PA亚基相互作用来抑制甲型流感病毒复制，该相互作用对控制甲型流感病毒RNA聚合酶复合物的组装过程至关重要。特异性流感病毒抑制剂的鉴定将用于两个主要目的。首先，这些化合物将为在细胞和分子水平上剖析病毒RNA聚合酶复合物组装过程的不同阶段提供有价值的工具。第二，一些已鉴定的化合物可作为开发新型抗流感药物的先导化合物。所提出的测定的技术基础是使用双分子荧光互补（BiFC），其基于荧光蛋白（GFP及其衍生物）的N-和C-末端片段不会自发折叠和重建功能性荧光团的原理。然而，如果与相互作用的蛋白质融合，则在细胞中表达后，荧光团的两个非功能性半部分由于特异性蛋白质相互作用而变得非常接近。这引发了片段折叠成活性蛋白，然后可以在蛋白质-蛋白质复合物的位点重建可检测的荧光信号。因此，通过BiFC，PB 1和PA亚基之间的特异性相互作用可以精确地可视化，定量和定位在活细胞内。通过破坏PB 1-PA相互作用，化合物将引起BiFC读数的降低，表明存在靶向PB 1-PA复合物组装的潜在抑制剂。本申请提出了两个具体目标：（1）开发和表征BiFC构建体，其允许在活细胞中目视鉴别PB 1-PA二聚体复合物，以及（2）开发适用于高通量形式的基于BiFC的原型甲型流感病毒RNA聚合酶复合物组装抑制剂筛选试验。预计该研究项目将建立BiFC方法的概念验证，以识别甲型流感病毒RNA聚合酶复合物的组装抑制剂，这将为开发高通量筛选试验提供基础。重要的是，该项目的成功完成将导致基于BiFC的病毒病原体药物筛选方法的验证。然后，这可适用于NIAID A、B或C类病毒剂，由于生物安全问题，在大多数地方无法进行。公共卫生相关性耐药性对抗流感治疗构成巨大挑战，并导致流感治疗失败。成功完成拟议的研究将有助于确定和设计额外的抗流感抑制剂，这将提供额外的治疗选择和改善疾病的结果。