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？已针对PA-10-213题为《用于探针和治疗前发现的高通量筛查分析的开发》(R01)的计划公告进行了编写。我们实现了一种新的活细胞，猝灭FRET(FqRET)报告分析细胞转录因子CBF？和HIV Vif蛋白，以发现新的分子探针来研究CBF？绑定并稳定VIF。我们的靶标偏向初步分析以及二次分析和计数器筛选的开发和优化计划是为了识别一个或多个细胞可渗透的、无毒的抑制CBF？的分子探针。绑定到Vif。这四个具体目标将是：(1)建立和优化细胞内猝灭FRET(FqRET)分析方法，该方法将用于HTS，以确定抑制CBF？和Vif在一起。(2)建立和优化正交二次筛选，验证CBF拮抗剂？与Vif的结合在机制上与它们降低Vi的细胞丰度以及减少A3G的Vif依赖的降解的能力有关。(3)对影响CBF能力的非靶点和细胞毒作用进行反筛选？与RUNX1结合并作为细胞转录因子发挥作用。以及(4)通过功能终点分析和结合研究，验证选定的HITS和商业上可获得的化学类似物的抗病毒活性和靶向性。在这一阶段，我们将实现PA的目标，即在NIH MLPCN内的设施中优化和验证初级检测和补充检测系统，以进行转移和筛选。

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.