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

 描述（由申请人提供）：在转录激活中，GACKIX基序，一个小的保守结构域发挥重要作用，具有两个潜在的激活剂对接位点，并已在CBP及其同源物p300以及不相关的辅激活剂ARC 105中发现。激活剂 *GACKIX结构域复合物的功能是多样的，包括细胞生长、造血、突触可塑性和脂质稳态;因此，这些复合物的失调也与从白血病到阿尔茨海默病和亨廷顿病的许多疾病有关。寻求化学工具来剖析激活剂 *GACKIX结构域复合物的个体功能，并提供潜在的治疗模型。然而，小分子还没有被设计成可以区分特定共活化因子（例如p300）的GACKIX结构域与CBP。在本研究计划中，被称为Tethering的定点策略被应用于匹配正交的含二硫键的片段与半胱氨酸修饰的GACKIX（cysGACKIX）结构域。首先，使用液相色谱-质谱系链筛选来鉴定靶向cysGACKIX结构域的ML L结合位点的二硫键片段。一种新的荧光偏振栓系筛选简化了针对c-Myb和CREB结合位点的cysGACKIX结构域的小分子抑制剂的鉴定。二硫键片段将转化为共价不可逆探针。将使用最具选择性和有效的小分子抑制剂来扩展束缚，以评估激活剂如CREB和MLL与细胞中CBP、p300和ARC 105的GACKIX结构域的相互作用的功能。

 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.