Small Molecule Transcriptional Activator-Coactivator Interactions

描述（由申请人提供）：能够通过与转录机制相互作用复制天然转录激活因子功能的小分子是研究基因转录的有力工具，并且正在成为对抗疾病的新策略。然而，用小分子模拟天然活化剂的功能已被证明具有挑战性。只有两类小分子被设计为上调细胞中的转录，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。KIX结构域的一个重要的辅激活剂，环AMP反应元件结合（CREB）蛋白（CBP）最近被确定为一个细胞间的目标异恶唑烷能够上调转录。与许多共激活剂相反，KIX是一个良好折叠的蛋白质结构域，具有生物学特征，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性小分子的主要靶标。该提案使用KIX作为充分表征的多功能靶标来开发用于设计重建天然活化剂功能的分子的通用平台，并将通过三个特定目标来实现：1）天然活化剂的结构复制2）异恶唑烷：共活化剂复合物的结合分析和3）天然活化剂的功能复制。计划的实验，以满足这些目标将使用基于荧光的结合和2D-NMR实验，以评估的结合亲和力和异恶唑烷相互作用的结合概况，以及无细胞和基于细胞的报告分析，以确定异恶唑烷的功能作用，调节转录。结构信息将另外用于设计异恶唑烷，其利用KIX的可塑性通过KIX结构域的变构调节实现第二结合位点的前所未有的蛋白质样增强（增强的结合）。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：失调基因的转录是各种不同疾病状态的标志。小分子重组的功能，调节转录的天然激活剂提供了一个令人兴奋的策略，研究疾病和基因通路。本研究的结果将用于开发使用人工转录激活因子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.