Chemical Probe Development for Epigenetic Complexes Enabled by Protein-Observed 19F NMR

项目总结 我们的长期目标是开发新的化学探针来剖析与 表观遗传复合体与转录调控。建立详细的动态机制 基因表达的调控仍然是表观遗传学领域的一个重大挑战。我们的研究 计划正在通过开发选择性化学探针，发现新的蛋白质-来应对这些挑战 蛋白质相互作用，以及改进的结构生物学和生物物理方法来量化这些动态 多价相互作用。这些方法有助于创建不同分子的详细图景 表观遗传复合体的组装和促进新表观遗传疗法的开发。 含溴结构域蛋白是表观遗传阅读器蛋白的一个子集，是一种新兴的蛋白质-- 表观基因组治疗的药物类别。我的研究计划是开发化学生物学方法来研究 两个特定的含溴结构域的蛋白，BPTF和BRD4。我们方法背后的一项创新使用了 19F核磁共振蛋白质为基础的方法学发现异构型选择性溴域抑制剂。由于 氟对配体结合和简化光谱的高响应性，这种结构生物学工具是理想的 适用于快速筛选小分子，表征与天然分子相关的瞬时相互作用 转录因子-蛋白质相互作用，并量化相互作用的动力学从微观到 毫秒级的时间尺度。在接下来的五年里，我们将进一步发展这一方法，使小型 分子发现，表征大的(&gt；50 kDa)蛋白质，并量化亲和力和动力学 与合成核小体相关的多结构域蛋白。随着他们通过19F核磁共振的发现，我们的 BPTF阅读器结构域的新化学探针有可能成为抑制BPTF阅读器结构域的化学工具 癌症进展和调节自噬，而更具体的BRD4化学探针将有助于揭示 癌症和炎症的新生物学被不太具体的工具化合物所掩盖。我们将使用这些工具来 剖析这些溴结构域在转录中的功能作用，包括基因调控、染色质 参与，以及新的蛋白质-蛋白质相互作用。下一代化学探测器将进一步 发展包括双功能分子作为多结构域蛋白的抑制剂，PROTACs具有改进的 选择性图谱，以及通过与DNA靶向聚酰胺偶联合成转录因子。 这一建议对铅的早期发现和表观遗传读者的研究具有广泛的生物医学意义 在人类健康和疾病方面很重要的领域。鉴于表观遗传蛋白代表了一大类 潜在的药物靶点，我们新的19F核磁共振小分子发现方法和基于细胞的方法 这里描述的表观遗传阅读器结构域可以显著增加靶标的保留范围，从而 开辟药物发现的新途径。

PROJECT SUMMARY Our long-term goal is to develop new chemical probes to dissect the molecular mechanisms associated with epigenetic-complexes and transcriptional regulation. Establishing detailed mechanisms for the dynamic regulation of gene expression remains a significant challenge for the field of epigenetics. Our research program is addressing these challenges by developing selective chemical probes, discovery of new protein- protein interactions, and improving structural biology and biophysical approaches to quantify these dynamic multivalent interactions. These approaches are helping to create a detailed picture of diverse molecular assemblies of epigenetic complexes and facilitating development of new epigenetic therapies. Bromodomain-containing proteins are a subset of epigenetic reader protein, and are an emerging protein- drug class for epigenome therapy. My research program is developing chemical biology approaches to study two specific bromodomain-containing proteins, BPTF and BRD4. One innovation behind our approach uses a 19F NMR protein-based methodology for the discovery of isoform selective bromodomain inhibitors. Due to the hyper-responsive nature of fluorine to ligand binding and simplified spectra, this structural biology tool is ideally suited to rapidly screen small molecules, characterize the transient interactions associated with native transcription factor-protein interactions, and to quantify the dynamics of the interactions from micro to millisecond timescales. Over the next five years, we will further develop this approach for enabling small molecule discovery, characterizing large (> 50 kDa) proteins, and quantifying the affinity and dynamics of multidomain proteins associated with synthetic nucleosomes. With their discovery enabled by 19F NMR, our new chemical probes for BPTF reader domains have potential for high impact as chemical tools for inhibiting cancer progression and regulating autophagy, while more specific BRD4 chemical probes will help uncover new biology in cancer and inflammation obscured by less specific tool compounds. We will use these tools to dissect the functional roles of these bromodomains on transcription, including gene regulation, chromatin engagement, and novel-protein-protein interactions. Next generation chemical probes will be further developed including bifunctional molecules as inhibitors of multi-domain proteins, PROTACs with improved selectivity profiles, and synthetic transcription factors through conjugation to DNA-targeting polyamides. This proposal has broad biomedical significance for early lead discovery and studies of epigenetic reader domains important in human health and disease. Given that epigenetic proteins represent a major class of potential drug targets, our new 19F NMR small molecule discovery method and cell-based approaches for epigenetic reader domains described here could significantly increase the repertoire of targets and thereby open up new avenues for drug discovery.