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

 描述（由申请方提供）：马尔堡病毒（MARV）是A类优先病原体，可引起人类病毒性出血热暴发，死亡率高达90%。然而，丝状病毒抗病毒药物和分子探针的发现受到活病毒研究需要生物安全水平（BSL）4级遏制的阻碍。使用活病毒进行此类研究的替代方法是开发重现致病所需的特异性宿主-病毒相互作用的测定法。这些相互作用可以被表征和靶向用于治疗开发，而不需要活病毒，从而减轻了对BSL 4遏制的需要。此外，这种筛选将充分利用复杂的HTS基础设施，使许多非BSL 4设施能够进行抗丝状病毒鉴定。该提案集中在MARV生物学的一个独特方面，该方面最近由参与小组进行了实验表征。我们发现MARV VP 24（mVP 24）和Kelch样ECH相关蛋白1（Keap 1）之间的相互作用通过转录因子核因子（红细胞衍生2）样2（Nrf 2）的稳定/核定位导致细胞保护性抗氧化反应的激活。在细胞核中，Nrf 2结合 抗氧化反应元件（ARE）序列以促进细胞保护基因表达程序的转录。Keap 1是Cul 3泛素连接酶的特异性因子，其调节细胞中Nrf 2的稳定性和水平。我们证明了Keap 1 Kelch结构域以高亲和力结合mVP 24，释放Nrf 2以激活基因表达。生物物理和建模研究表明，mVP 24与Keap 1相互作用的方式是独特的，不同于其他Keap 1结合剂，包括Nrf使用的结合模式。我们推测，诱导的细胞保护途径将维持感染细胞的活力，并促进病毒复制。因此，靶向MARV复制的这一新方面可以提供对病毒复制的深入了解和对策开发的机会。该提案的目标是开发优化的初级和二级筛选试验，基于ARE报告基因和荧光偏振方法，以识别阻止mVP 24介导的ARE应答激活的小分子，并进行小规模的中试筛选，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小分子筛选铺平道路，这些筛选有可能鉴定出抑制mVP 24激活ARE反应的新型抗马尔堡病毒化合物，包括破坏mVP 24-Keap 1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.