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

描述(由申请人提供)：虽然有效的神经氨酸酶抑制剂可用于治疗人类流感病毒感染，但对这些药物具有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。为了更好地为应对新出现的流感大流行做好准备，并应对日益严重的抗病毒药物耐药性问题，迫切需要针对保守的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目标是组装病毒RNA聚合酶复合体。病毒RNA聚合酶复合体(PA-PB1-PB2)是由聚合酶碱性蛋白1(PB1)、聚合酶碱性蛋白2(PB2)和聚合酶酸性蛋白(PA)三个亚基组成的异源三聚体。PB1亚基形成聚合酶复合体的核心。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亚基之间的相互作用尚未见报道。虽然聚合酶复合体形成的分子机制尚不清楚，但病毒RNA聚合酶复合体在病毒复制过程中的组装是一个高度调控的动态过程，对于病毒RNA的合成和传染性流感病毒颗粒的产生是必不可少的。病毒RNA聚合酶复合体形成的任何破坏，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，以及病毒的感染性。最近，通过开发一种基于多肽的抑制剂，通过特异性地阻断PB1-PA相互作用，从而干扰病毒RNA聚合酶复合体组装，有效地抑制了甲型流感病毒的生长，证明了抑制甲型流感病毒RNA聚合酶复合体组装的原理。这些研究支持我们将甲型流感RNA聚合酶的形成作为治疗发现和开发的目标。这项研究计划的总体目标是开发一种新的检测方法，可能导致鉴定抗病毒化合物，这些化合物通过破坏PB1-PA亚单位的相互作用来抑制甲型流感病毒的复制，而PB1-PA亚单位的相互作用对控制甲型流感病毒RNA聚合酶复合体的组装过程至关重要。识别特定的流感病毒抑制剂将有两个主要目的。首先，这些化合物将为在细胞和分子水平上剖析病毒RNA聚合酶复合体组装过程的不同阶段提供有价值的工具。其次，一些已鉴定的化合物可能作为开发新型抗流感药物的先导化合物。该分析的技术基础是使用双分子荧光互补技术(BIFC)，该技术基于荧光蛋白(GFP及其衍生物)的N-端和C-端片段不会自发折叠和重建功能荧光团的原理。然而，如果与相互作用的蛋白质融合，随着细胞中的表达，荧光团的两个非功能部分由于特定的蛋白质相互作用而变得非常接近。这启动了片段的折叠成活性蛋白质，然后可以在蛋白质-蛋白质复合体的位置重建可检测到的荧光信号。因此，通过BIFC，PB1和PA亚基之间的特定相互作用可以在活细胞内精确地可视化、量化和定位。通过破坏PB1-PA相互作用，化合物将导致BIFC读数减少，这表明存在针对PB1-PA复合体组装的潜在抑制剂。在应用中提出了两个特定的目标：(1)开发和表征能够在活细胞中直观地鉴定PB1-PA二聚体复合体的BIFC结构；(2)建立一种基于BIFC的适合于高通量形式使用的甲型流感病毒RNA聚合酶复合体组装抑制物筛选方法。预计这项研究项目将为BIFC方法鉴定甲型流感病毒RNA聚合酶复合体的组装抑制剂建立概念验证，这将为高通量筛选试验的发展提供基础。重要的是，该项目的成功完成将导致对基于BIFC的病毒病原体药物筛选方法的验证。然后，这可能适用于NIAID A、B或C类病毒制剂，由于生物安全方面的考虑，这些病毒制剂不能在大多数地点进行。与公共卫生相关的耐药性给抗流感治疗带来了巨大的挑战，并导致流感治疗失败。这项拟议研究的成功完成将有助于确定和设计更多的抗流感抑制剂，这些药物将提供更多的治疗选择并改善疾病结果。

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.