Small Molecule Transcriptional Activator-Coactivator Interactions

描述(申请人提供)：小分子能够通过与转录机制的相互作用复制天然转录激活剂的功能，是研究基因转录的有力工具，并正在成为抗击疾病的新策略。然而，事实证明，用小分子模拟天然激活剂的功能是具有挑战性的。只有两类小分子被设计成在细胞中上调转录，而只有两亲性异恶唑烷在纳摩尔浓度下显示出强大的活性。最近发现了一种重要的共激活因子--环腺苷反应元件结合蛋白(CREB)-蛋白(CBP)的KIX结构域，它是异恶唑烷类化合物上调转录的细胞间靶标之一。与许多共激活子不同的是，KIX是一个折叠良好的蛋白结构域，具有生物物理学特征，并通过两个结合位点受变构控制。因此，KIX是研究结构和结合的主要目标，用于设计能够调节转录的新的功能小分子。这项建议使用KIX作为一个表征良好的多功能靶来开发一个通用的平台，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：共激活剂复合体的结合分析；3)天然激活剂的功能复制。为实现这些目标，计划中的实验将使用基于荧光的结合和2D-核磁共振实验来评估异恶唑烷相互作用的结合亲和力和结合图谱，以及无细胞和基于细胞的报告分析，以确定异恶唑烷在调节转录方面的功能作用。结构信息还将被用于设计异恶唑烷类化合物，利用KIX的可塑性，通过对KIX结构域的变构调节，实现前所未有的第二结合位点的蛋白样增强(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异恶唑烷在细胞检测中的功能活性进行比较。 与公共卫生相关：调控不当的基因转录是各种不同疾病状态的标志。小分子重建调节转录的天然激活剂的功能，为研究疾病和基因途径提供了一种令人兴奋的策略。这项研究的结果将被用来开发使用人工转录激活剂控制转录的一般策略。

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.