Small Molecule Transcriptional Activator-Coactivator Interactions

小分子转录激活剂-辅激活剂相互作用

• 批准号: 7806222
• 负责人: William Charles Krause Pomerantz
• 金额: $ 4.76万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7806222
• 关键词: Activities of Daily Living; Affinity; Allosteric Regulation; Binding; Binding Sites; Biological Assay; CREB1 gene; Cells; Cellular Assay; Chemosensitization; Complex; Cyclic AMP Response Element; Cyclic AMP-Responsive DNA-Binding Protein; DNA; DNA Binding; DNA Binding Domain; Data; Development; Disease; Disease Pathway; Docking; Figs - dietary; Fluorescence; Fluorescence Polarization; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Human T-lymphotropic virus 1; Image; Individual; JUN gene; Lead; Ligands; MYB gene; Molecular; Output; Pathway interactions; Pattern; Phosphotransferases; Proteins; Proto-Oncogene Proteins c-myb; Relaxation; Reporter; Research; Research Design; Resolution; Role; Side; Structure; TP53 gene; Taxes; Tertiary Protein Structure; Transcription Coactivator; Transcriptional Activation; Transcriptional Activation Domain; analog; base; cellular targeting; combat; design; functional outcomes; improved; insight; meetings; model design; next generation; public health relevance; quantum; reconstitution; research study; small molecule; tool

DESCRIPTION (provided by applicant): Small molecules capable of replicating the function of natural transcriptional activators through interactions with the transcriptional machinery are powerful tools for studying gene transcription and are emerging as a new strategy for combating disease. However, mimicking the function of natural activators with small molecules has proven challenging. Only two classes of small molecules have been designed that upregulate transcription in cells, whereas only amphiphatic isoxazolidines display potent activity at nanomolar concentrations. The KIX domain of an essential co-activator, cyclic-AMP response element-binding (CREB)-protein (CBP) was recently identified as one intercellular target for isoxazolidines capable of upregulating transcription. In contrast to many co-activators, KIX is a well-folded protein domain, biophysically characterized, and is allosterically controlled through two binding sites. KIX is therefore a prime target for studying structure and binding for the design of new functional small molecules capable of regulating transcription. This proposal uses KIX as a well-characterized, multi-functional target to develop a general platform for designing molecules that reconstitute the function of natural activators and will be accomplished through three specific aims: 1) Structural replication of natural activators 2) Binding analysis of an isoxazolidine:co-activator complex and 3) Functional replication of natural activators. Planned experiments to meet these goals will use fluorescence-based binding and 2D-NMR experiments to assess the binding affinity and binding profiles of isoxazolidine interactions, as well as cell-free and cell-based reporter assays to determine the functional role of isoxazolidines for regulating transcription. Structural information will additionally be used for designing isoxazolidines that exploit the plasticity of KIX to achieve unprecedented protein-like potentiation (enhanced binding) of a second binding site through allosteric regulation of the KIX domain. Finally, binding and structural analysis of isoxazolidine:KIX interactions will be compared with isoxazolidine functional activity in cellular assays. PUBLIC HEALTH RELEVANCE: Transcription of misregulated genes is a hallmark for a variety of different disease states. Small molecules that reconstitute the function of natural activators for regulating transcription offer an exciting strategy for studying disease and gene pathways. Results from this study will be used to develop general strategies for controlling transcription using artificial transcriptional activators.

描述（由申请人提供）：能够通过与转录机制相互作用来复制天然转录激活剂功能的小分子是研究基因转录的强大工具，并且正在成为对抗疾病的新策略。然而，用小分子模拟天然激活剂的功能已被证明具有挑战性。仅设计了两类小分子来上调细胞中的转录，而只有两亲性异恶唑烷在纳摩尔浓度下显示出有效的活性。一种重要的共激活剂环AMP反应元件结合（CREB）蛋白（CBP）的KIX结构域最近被确定为能够上调转录的异恶唑烷的一个细胞间靶标。与许多共激活剂相比，KIX 是一个折叠良好的蛋白质结构域，具有生物物理特征，并通过两个结合位点进行变构控制。因此，KIX 是研究结构和结合以设计能够调节转录的新功能小分子的主要目标。该提案使用 KIX 作为特征明确的多功能靶点，开发一个通用平台，用于设计重建天然激活剂功能的分子，并将通过三个具体目标来实现：1）天然激活剂的结构复制，2）异恶唑烷：共激活剂复合物的结合分析，3）天然激活剂的功能复制。为实现这些目标而计划的实验将使用基于荧光的结合和 2D-​​NMR 实验来评估异恶唑烷相互作用的结合亲和力和结合概况，以及无细胞和基于细胞的报告基因测定来确定异恶唑烷调节转录的功能作用。结构信息还将用于设计异恶唑烷，利用 KIX 的可塑性，通过 KIX 结构域的变构调节，实现第二个结合位点前所未有的类蛋白质增强（增强结合）。最后，异恶唑烷：KIX 相互作用的结合和结构分析将与细胞测定中的异恶唑烷功能活性进行比较。 公共卫生相关性：失调基因的转录是多种不同疾病状态的标志。重建天然激活剂调节转录功能的小分子为研究疾病和基因途径提供了令人兴奋的策略。这项研究的结果将用于开发使用人工转录激活剂控制转录的一般策略。