High Throughput Screen Development for Modulators of PAS/Coactivator Interactions

描述（由申请人提供）：真核生物bHLH/PAS（碱性螺旋-环-螺旋/Per-ARNT-Sim）转录因子在调节细胞对缺氧、异生物质化合物和几种其他环境条件的反应中起关键作用。这些途径的失调与几种形式的癌症高度相关，包括实体瘤的发作和进展，使其控制特别感兴趣。为此，PAS结构域内的这些因素代表特别有吸引力的小分子调控的目标，这些领域发挥的作用，作为辅因子调节的蛋白质/蛋白质相互作用域在整个生物学范围广泛的感觉蛋白。在这里，我们提出了一个HTS兼容的蛋白质/蛋白质相互作用屏幕的发展，以确定化合物，可以破坏的PAS-B结构域的ARNT（芳烃核受体转运蛋白），几个bHLH/PAS异二聚体复合物的共同元素，和卷曲螺旋段的几个辅激活蛋白，是必不可少的ARNT功能之间的复合物。初步的低通量分析表明，这种方法是可行的转换为HTS格式，并在体外生物物理和基于细胞的生化分析的组合已经到位，以验证以这种方式发现的化合物。 公共卫生相关性：我们建议开发一种用于高通量筛选ARNT/AINT转录激活因子/共激活因子对之间相互作用的小分子干扰物的测定法。该途径的不适当的失调与多种癌症相关，并且使用该测定来鉴定可以破坏该复合物的人工化合物将为该复合物的研究和操作提供新的研究工具。

DESCRIPTION (provided by applicant): Eukaryotic bHLH/PAS (basic helix-loop-helix/Per-ARNT-Sim) transcription factors play a critical role in regulating cellular responses to hypoxia, xenobiotic compounds, and several other environmental conditions. Deregulation of these pathways is highly correlated with several forms of cancer, including solid tumor onset and progression, making their control of particular interest. To this end, the PAS domains within these factors represent particularly attractive targets for small molecule regulation given the role that these domains play as cofactor-regulated protein/protein interaction domains in a wide range of sensory proteins throughout biology. Here we propose the development of an HTS-compatible protein/protein interaction screen to identify compounds that can disrupt the complex between the PAS-B domain of ARNT (aryl hydrocarbon nuclear receptor translocator), a common element of several bHLH/PAS heterodimeric complexes, and coiled-coil segments of several coactivator proteins that are essential for ARNT function. Preliminary low-throughput assays suggest that this approach is feasible for conversion to HTS format, and a combination of in vitro biophysical and cell-based biochemical assays are already in place to validate compounds found in this manner. PUBLIC HEALTH RELEVANCE: We propose to develop an assay for high-throughput screening of small molecule disruptors of interactions between the ARNT/AINT transcriptional activator/coactivator pair. Inappropriate deregulation of this pathway is correlated with a variety of cancers, and the use of this assay to identify artificial compounds which can disrupt this complex will provide novel research tools for the study and manipulation of this complex.