High-throughput screen for inhibitors of Marburg virus VP24-Keap1

 描述(申请人提供)：马尔堡病毒(Marburg Virus，MARV)可引起人类病毒性出血热暴发，死亡率高达90%，是A类优先病原体。然而，丝状病毒抗病毒药物和分子探针的发现因活体病毒研究需要生物安全水平(BSL)4而受到阻碍。在这类研究中使用活病毒的另一种选择是开发概括致病所需的特定宿主-病毒相互作用的分析方法。这些相互作用可以在不需要活病毒的情况下表征并针对治疗开发，从而减轻对BSL4遏制的需求。此外，这样的筛查将处于有利地位，以利用复杂的HTS基础设施，这将使许多非BSL4设施能够进行抗丝状病毒识别。这项提案侧重于Marv生物学的一个独特方面，最近由参与小组实验确定了这一方面的特征。我们发现Marv VP24(MVP24)和Kelch样ECH相关蛋白1(Keap1)之间的相互作用通过转录因子核因子(红系衍生2)样蛋白2(Nrf2)的稳定/核定位而激活细胞保护性抗氧化反应。在核中，Nrf2结合 促进细胞保护性基因表达程序转录的抗氧化反应元件(ARE)序列。Keap1是CUL3泛素连接酶的特异性因子，调节细胞内Nrf2的稳定性和水平。我们证明Keap1Kelch结构域与mVP24高亲和力结合，释放Nrf2激活基因表达。生物物理和建模研究表明，mVP24以一种独特的方式与Keap1相互作用，不同于包括NRF在内的其他Keap1结合剂使用的结合模式。我们假设，细胞保护通路的诱导将维持感染细胞的生存能力，并促进病毒复制。因此，针对Marv复制的这一新方面可能提供对病毒复制的洞察和对策开发的机会。这项建议的目标是基于ARE报告基因和荧光偏振方法开发优化的一次和二次筛选分析，以识别阻止mVP24介导的ARE反应激活的小分子，并进行小规模中试筛选，使我们能够建立命中选择的标准。这些努力将为更大规模的全尺寸MALL分子筛查铺平道路，这些分子筛查有可能识别出新型的抗马尔堡病毒化合物，这些化合物可以抑制mVP24激活ARE反应，包括破坏mVP24-Keap1相互作用的分子。

 DESCRIPTION (provided by applicant): Marburg viruses (MARV), which cause outbreaks of viral hemorrhagic fever in humans with mortality rates of up to 90%, are category A priority pathogens. However, the discovery of filovirus antivirals and molecular probes has been hampered by the need for biosafety level (BSL) 4 containment for live virus studies. An alternative to the use of live virus for such studies is to develop assays that recapitulate specifc host-viral interactions that are required for pathogenesis. These interactions can be characterized and targeted for therapeutic development without the need for live virus, alleviating the need for BSL4 containment. Additionally, such screens will be well-positioned to take advantage of sophisticated HTS infrastructure that would enable antifiloviral identification a many non-BSL4 facilities. This proposal focuses on a unique aspect of MARV biology that was recently experimentally characterized by the participating groups. We showed that an interaction between MARV VP24 (mVP24) and Kelch-like ECH-associated protein 1 (Keap1) results in activation of cytoprotective antioxidant responses through the stabilization/nuclear localization o transcription factor Nuclear factor (erythroid-derived 2)-like 2 (Nrf2). In the nucleus, Nrf2 binds antioxidant response element (ARE) sequences to promote transcription of a cytoprotective gene expression program. Keap1 is a specificity factor for Cul3 ubiquitin ligase that regulates the stability and the level of Nrf2 in cells. We demonstrated that the Keap1 Kelch domain binds mVP24 with high affinity, freeing Nrf2 to activate gene expression. Biophysical and modeling studies indicate that mVP24 interacts with Keap1 in a manner that is unique and distinct from the binding mode used by other Keap1 binders, including Nrf. We hypothesize that induction of the cytoprotective pathways will sustain the viability of infected cells and facilitate virus replication. Thus, targeting this novel aspect of MARV replication may provide insight into viral replication and opportunities for countermeasure development. The goals of this proposal are to develop optimized primary and secondary screening assays, based on ARE reporter genes and on fluorescence polarization approaches, to identify small molecules that prevent mVP24-mediated activation of an ARE response, and to perform a small-scale pilot screen that allows us to establish criteria for hit selection. These efforts will pave the way for larger, full-scale mall molecule screens with the potential to identify novel anti-Marburg virus compounds that inhibit mVP24 activation of an ARE response, including molecules that disrupt mVP24-Keap1 interaction.