Identification of Antagonists of the Molecular Chaperone Function of CBF beta

CBFβ分子伴侣功能拮抗剂的鉴定

• 批准号: 8550192
• 负责人: Harold C Smith
• 金额: $ 46.85万
• 依托单位: OYAGEN, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-30 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8550192
• 关键词: Affect; Antigen-Presenting Cells; Antineoplastic Agents; Antiviral Agents; Basic Science; Binding; Biochemical; Biological Assay; Boxing; Cell physiology; Cells; Chemicals; Co-Immunoprecipitations; Core-Binding Factor; DNA Binding; DNA-Binding Proteins; Dimerization; Dose; Embryo; Enabling Factors; End Point Assay; Firefly Luciferases; Future; Gene Expression; Gene Expression Regulation; Genetic Transcription; Goals; HIV; HIV Infections; Hematopoiesis; Host Defense; Libraries; Life; Molecular Chaperones; Molecular Probes; Mutation; NIH Program Announcements; Performance; Powder dose form; Promega; Proteins; RNA Interference; RUNX1 gene; Renilla Luciferases; Reporter; Research; Resistance; Role; Signal Transduction; Specificity; Staging; System; Testing; Therapeutic; Transcriptional Activation; Triage; Ubiquitination; United States National Institutes of Health; Validation; Viral; Viral Genome; Writing; analog; assay development; bindin; biochemical model; cancer cell; cell type; cofactor; cytotoxic; cytotoxicity; elongin C; high throughput screening; human CBFB protein; inhibitor/antagonist; novel; protein degradation; public health relevance; response; scaffold; screening; small molecule libraries; statistics; stem; stem cells; therapeutic development; transcription factor

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.

描述（由申请人提供）：标题为CBF分子伴侣功能拮抗剂的鉴定？为响应PA-10-213项目公告，标题为开发用于探针和治疗前发现的高通量筛选试验（R 01）。我们实施了一种新型活细胞猝灭FRET（FqRET）报告基因测定，用于细胞转录因子CBF？的分子相互作用。和HIV Vif蛋白，以发现新的分子探针，用于研究CBF？结合并稳定Vif。我们的目标偏置的主要分析和开发和优化的二次检测和计数器屏幕计划确定一个或多个细胞可渗透的，无毒的分子探针，抑制CBF？绑定到Vif。四个具体目标将是：（1）开发和优化细胞内淬灭FRET（FqRET）测定，将用于HTS，以确定抑制CBF？关于VIF (2)开发和优化正交二次筛选，验证CBF拮抗剂？与Vif的结合在其降低Vi的细胞丰度以及降低A3 G的Vif依赖性降解的能力方面是机械相关的。(3)对影响CBF能力的脱靶和细胞毒性命中进行计数器筛选？与RUNX 1结合并作为细胞转录因子发挥作用。和（4）通过功能终点测定和结合蛋白研究验证所选命中物和市售化学类似物的抗病毒活性和靶特异性。在此阶段，我们将实现PA的目标，即在NIH MLPCN内的设施中优化和验证主要测定和补充测定系统的转移和筛选。