Small Molecule Transcriptional Activator-Coactivator Interactions

描述（由申请人提供）：能够通过与转录机制的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成可以上调细胞中的转录，而只有两相异恶唑烷类在纳摩尔浓度下显示出有效的活性。一种重要的共激活因子环amp反应元件结合蛋白（CREB）的KIX结构域最近被确定为能够上调转录的异恶唑烷类的一个细胞间靶点。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。本研究将KIX作为一个具有良好特征的多功能靶点来开发一个通用平台，用于设计重建天然活化剂功能的分子，并将通过三个具体目标来完成：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；3)天然活化剂的功能复制。实现这些目标的计划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谱，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。此外，结构信息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，实现前所未有的第二结合位点的蛋白样增强（增强结合）。最后，结合和结构分析异恶唑烷：KIX相互作用将与细胞分析中的异恶唑烷功能活性进行比较。

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.