Identification of Antagonists of the Molecular Chaperone Function of CBF beta

描述（由申请人提供）：题为“鉴定CBF分子伴侣功能拮抗剂”的提案？已根据项目公告PA-10-213题为“开发用于探针和治疗前发现的高通量筛选测定法”（R01）撰写。我们实现了一种新的活细胞，淬灭FRET （FqRET）报告检测细胞转录因子CBF？和HIV Vif蛋白，以便发现新的分子探针来研究CBF？结合并稳定Vif。我们的目标偏向初级分析以及二级分析和反筛的开发和优化计划用于鉴定一种或多种细胞渗透性，无毒的抑制CBF？绑定到Vif。四个特定目标将是：(1)开发和优化细胞内淬灭FRET （FqRET）检测，该检测将用于HTS，以识别抑制CBF相互作用的化合物。Vif。(2)开发和优化正交二次筛选，验证CBF？与Vif的结合机制与它们降低细胞中Vi的丰度以及减少Vif依赖性的A3G降解的能力有关。(3)对影响CBF能力的脱靶和细胞毒性命中的计数器筛选？与RUNX1结合并发挥细胞转录因子的作用。(4)通过功能终点分析和结合蛋白研究，验证选定的靶点和市售化学类似物的抗病毒活性和靶标特异性。在此阶段，我们将实现PA的目标，即在NIH MLPCN内的设施中进行转移和筛选，优化和验证初级分析和补充分析系统。

DESCRIPTION (provided by applicant): The proposal entitled Identification of Antagonists of the Molecular Chaperone Function of CBF? has been written in response to the Program Announcement PA-10-213 entitled Development of Assays for High-Throughput Screening for Use in Probe and Pre-therapeutic Discovery (R01). We implement a novel live cell, quenched FRET (FqRET) reporter assay for the molecular interaction of the cellular transcription factor CBF? and the HIV Vif protein in order to discover novel molecular probes for studying the mechanism whereby CBF? binds to and stabilizes Vif. Our target-biased primary assay and the development and optimization of secondary assays and counter screens are planned to identify one or more cell permeable, nontoxic molecular probes that inhibit CBF? binding to Vif. The four Specific Aims will be: (1) Develop and optimize an in-cell quenched FRET (FqRET) assay that will be used in HTS to identify compounds that inhibit the interaction of CBF? with Vif. (2) Develop and optimize orthogonal secondary screens validating that antagonists of CBF? binding to Vif are mechanistically relevant in their ability to reduce the cellular abundance of Vi as well as reduce Vif- dependent degradation of A3G. (3) Counter screen for off-target and cytotoxic hits that affect the ability of CBF? to bind to RUNX1 and function as a cellular transcription factor. And (4) validate selected hits and commercially available chemical analogs for their antiviral activities and target-specificity through functional endpoint assays and bindin studies. At this stage we will have achieved the objective of the PA of having a primary assay and complementary assay systems optimized and validated for transfer and screening at a facility within the NIH MLPCN.