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: 8740512
• 负责人: Harold C Smith
• 金额: $ 43.92万
• 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/8740512
• 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; Stem cells; 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; 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）：题为“CBF 分子伴侣功能拮抗剂的识别？”的提案？已针对题为“开发用于探针和治疗前发现的高通量筛选测定法”(R01) 的计划公告 PA-10-213 编写。我们实施了一种新型活细胞猝灭 FRET (FqRET) 报告基因测定法，用于检测细胞转录因子 CBF 的分子相互作用？和 HIV Vif 蛋白，以发现新的分子探针来研究 CBF？结合并稳定 Vif。我们计划进行目标偏向的初级测定以及次级测定和计数器筛选的开发和优化，以识别一种或多种抑制 CBF 的细胞渗透性、无毒分子探针？与 Vif 结合。四个具体目标是： (1) 开发和优化细胞内淬灭 FRET (FqRET) 测定，该测定将用于 HTS 中，以鉴定抑制 CBF 相互作用的化合物？与维夫。 (2) 开发和优化正交二次筛选，验证 CBF 拮抗剂？与 Vif 结合在机制上与减少 Vi 细胞丰度以及减少 Vif 依赖性 A3G 降解的能力相关。 (3) 反筛选影响CBF能力的脱靶和细胞毒性命中？与 RUNX1 结合并充当细胞转录因子。 (4) 通过功能终点测定和结合蛋白研究来验证选定的命中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，即对主要测定和补充测定系统进行优化和验证，以便在 NIH MLPCN 内的设施中进行转移和筛选。