Proteomic discovery and characterization of chemical tools that degrade the NuRD chromatin regulatory complex

项目摘要 由于细胞行为和身份的几乎所有方面都集中在基因表达中的程序化反应上， 转录调控在正常的动态平衡和疾病进展中都起着重要的作用。因此， 基因调控机制的药理学扰动是一种有吸引力的理解和 调节复杂的细胞疾病状态。转录靶向药物必须克服关键障碍， 然而，包括DNA结合因子和多组分复合体对传统DNA的可及性有限 药物设计方法，以及转录过程中断造成的不分青红皂白的活性 在不同的细胞类型和状态之间共享。在这项提案中，我们提出并建立在一项新发现的基础上 名为L4的免疫抑制亲电化学探针通过其显著的 导致多种核小体重塑脱乙酰酶复合体选择性降解的能力 亚基以T细胞受限的方式。由我们实验室最新的化学蛋白质组学发展提供支持 为了在全球范围内绘制天然生物系统中小分子-蛋白质相互作用的图谱，我们提出了一种 研究L4前所未有的药理作用机制及其机制的研究策略 对靶向转录复合体的更广泛的药物设计方法的影响。此外，我们还将 研究L4介导的NuRD复合体降解与化合物降解的关系 阻断T细胞的激活。在具体目标1中，我们将应用我们团队的化学蛋白质组专业知识来 表征L4对NuRD的降解，并使用一套L4- 相关化合物和NuRD络合物导向的富集法。在具体目标2中，我们将评估 候选L4靶在利用遗传和生化方法支持NuRD降解方面的作用 研究已确定的机制如何导致多亚基降解和状态的显著特征 依赖(细胞类型受限)活动。在具体目标3中，我们扩展到考察L4‘S动作之间的关系 评价NuRD阻断T细胞活化的能力，并将其作为进一步研究的工具进行评估和应用 T细胞生物学的研究。通过深入了解L4的独特药理活性，建议的 研究将有助于探索靶蛋白中这两种新机制的总体目标 基因调控蛋白的降解和状态依赖控制。总而言之，这些更宏大的雄心 代表了克服转录靶向化学物质开发中的挑战的有希望的途径 探头和药物。

Project Summary As virtually all aspects of cell behavior and identity converge upon programmed responses in gene expression, transcriptional control features centrally in both normal homeostasis and progression to disease. Accordingly, pharmacologic perturbation of gene regulatory machinery is an attractive approach for understanding and modulating complex cellular disease states. Transcriptionally targeted drugs must overcome key hurdles, however, including the limited accessibility of DNA binding factors and multi-component complexes to traditional drug design approaches, as well as the indiscriminate activity caused by disruption of transcriptional processes shared across distinct cell types and states. In this proposal, we present and build on a novel discovery of an immunosuppressive electrophilic chemical probe termed "L4" that avoids these pitfalls through its remarkable ability to cause selective degradation of multiple NuRD (Nucleosome Remodeling Deacetylase) complex subunits in a T-cell-restricted manner. Empowered by our laboratory’s recent development of chemical proteomic strategies to globally map small molecule-protein interactions in native biological systems, we propose a research strategy to study the mechanism of the unprecedented pharmacological features of L4 and their implications for broader drug design approaches to target transcriptional complexes. In addition, we will investigate the relationship between L4-mediated degradation of the NuRD complex and the compound’s blockade of T-cell activation. In Specific Aim 1, we will apply our group’s chemical proteomic expertise to characterize NuRD degradation by L4 and map relevant protein targets in human T-cells using a suite of L4- related compounds and NuRD complex-directed enrichment approaches. In Specific Aim 2, we will evaluate the role of candidate L4 targets underpinning NuRD degradation using genetic and biochemical approaches and examine how the identified mechanisms result in the striking features of multi-subunit degradation and state- dependent (cell type-restricted) activity. In Specific Aim 3, we extend to examine the relationship of L4’s action on NuRD to its ability to block T-cell activation, and assess and apply this compound as a tool for further study of T-cell biology. By providing in-depth understanding of the unique pharmacological activity of L4, the proposed research will contribute to the overarching goals of exploring both novel mechanisms in targeted protein degradation and state-dependent control of gene regulatory proteins. Together, these broader ambitions represent promising avenues to overcome challenges in the development of transcriptionally targeted chemical probes and medicines.