The discovery of small molecules to dissect the individual roles of activator-coactivator complexes

发现小分子来剖析激活剂-共激活剂复合物的各个作用

• 批准号: 8987175
• 负责人: Jean Marie Lodge
• 金额: $ 3.5万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8987175
• 关键词: Activator Appliances; Alzheimer' s Disease; Binding; Binding Sites; Biological Process; CREB1 gene; Cells; Chemicals; Complex; Coupled; Cysteine; Disease; Disulfides; Docking; EP300 gene; Event; Fluorescence Polarization; Gene Expression; Genes; Hematopoiesis; Homeostasis; Homologous Gene; Huntington Disease; In Vitro; Individual; Ligands; Link; Lipids; MYB gene; Metabolism; Modeling; Neuronal Plasticity; Physiological; Play; Positioning Attribute; Proteins; Proto-Oncogene Proteins c-myb; Regulation; Research; Role; SREBP-1a; Site; Specificity; Surface; Synaptic plasticity; Therapeutic; Transcription Coactivator; Transcriptional Activation; Western Blotting; Work; analog; cell growth; design; disulfide bond; inhibitor/antagonist; interest; leukemia; liquid chromatography mass spectrometry; mutant; novel; public health relevance; research study; small molecule; tool

 DESCRIPTION (provided by applicant): In the activation of transcription the GACKIX motif, a small conserved domain plays an important role with two potential docking sites for activators and has been found in CBP and its homolog p300 as well as the unrelated coactivator ARC105. The functions of activator*GACKIX domain complexes are diverse and include cellular growth, hematopoiesis, synaptic plasticity, and lipid homeostasis; thus, dysregulation of these complexes are also linked to many diseases from leukemia to Alzheimer's and Huntington's disease. Chemical tools are sought to dissect the individual functions of activator*GACKIX domain complexes and to provide a potential therapeutic model. However small molecules have yet to be designed that can distinguish between the GACKIX domain of a particular coativator such as p300 versus CBP. In this research plan the site-directed strategy known as Tethering is applied to match orthogonal disulfide-containing fragments with cysteine-modified GACKIX (cysGACKIX) domain. First a liquid chromatography-mass spectrometry Tethering screen was used to identify disulfide fragments that target the MLL-binding site of the cysGACKIX domain. A novel fluorescence polarization Tethering screen streamlined the identification of small molecules inhibitors directed at the c-Myb and CREB- binding site of the cysGACKIX domain. The disulfide fragments will be converted into covalent irreversible probes. Tethering will be extended using the most selective and potent small molecules inhibitors to assess the function of activators such as CREB, and MLL interactions with GACKIX domain of CBP, p300, and ARC105 in cells.

 描述（由申请人提供）：在 GACKIX 基序的转录激活中，一个小的保守结构域发挥着重要作用，具有两个潜在的激活剂对接位点，并且已在 CBP 及其同源物 p300 以及不相关的共激活剂 ARC105 功能中发现。激活剂*GACKIX 结构域复合物的功能多种多样，包括细胞生长、造血、突触可塑性和脂质稳态；因此，这些功能的失调；复合物还与白血病、阿尔茨海默病和亨廷顿病等多种疾病有关。人们正在寻求化学工具来剖析激活剂*GACKIX 结构域复合物的各个功能，并提供潜在的治疗模型，但尚未设计出能够区分这些疾病的小分子。特定共激活因子（例如 p300 与 CBP）的 GACKIX 结构域在本研究计划中，应用称为“束缚”的定点策略来匹配含有正交二硫键的片段。首先，使用液相色谱-质谱联用筛选来鉴定针对 cysGACKIX 结构域的 MLL 结合位点的二硫键片段。一种新型荧光偏振联用筛选简化了小分子抑制剂的鉴定。针对 cysGACKIX 结构域的 c-Myb 和 CREB ​​结合位点，二硫键片段将被转化为共价键。不可逆探针将使用最具选择性和最有效的小分子抑制剂来扩展，以评估激活剂（例如 CREB）的功能，以及 MLL 与细胞中 CBP、p300 和 ARC105 的 GACKIX 结构域的相互作用。