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的程序化反应上， 转录控制在正常稳态和疾病进展中起中心作用。因此，委员会认为， 基因调控机制的药理学扰动是一种有吸引力的方法， 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物对传统生物学的有限可及性， 药物设计方法，以及转录过程中断引起的无差别活性 在不同的细胞类型和状态中共享。在这个建议中，我们提出并建立在一个新的发现， 称为“L4”的免疫抑制亲电化学探针通过其显著的 引起多个NuRD（核小体重塑脱乙酰酶）复合物的选择性降解的能力 以T细胞限制的方式表达。得益于我们实验室最近在化学蛋白质组学方面的发展 策略，全球地图小分子蛋白质相互作用在天然生物系统中，我们提出了一个 研究策略，以研究L4前所未有的药理学特征的机制及其 对靶向转录复合物的更广泛药物设计方法的影响。此外，我们将 研究L4介导的NuRD复合物降解与化合物 阻断T细胞活化。在具体目标1中，我们将运用我们小组的化学蛋白质组学专业知识， 表征L4对NuRD的降解，并使用一套L4- 相关化合物和NuRD复合物定向富集方法。在具体目标2中，我们将评估 使用遗传和生物化学方法支持NuRD降解的候选L4靶标的作用， 研究所确定的机制如何导致多亚基降解和状态的显著特征， 依赖性（细胞类型限制）活动。在具体目标3中，我们扩展到考察L4作用的关系 研究NuRD阻断T细胞活化的能力，并评估和应用该化合物作为进一步研究的工具 T细胞生物学通过深入了解L4的独特药理活性， 研究将有助于探索靶向蛋白质的两种新机制的总体目标 基因调控蛋白的降解和状态依赖性控制。总之，这些更广泛的野心 代表了克服转录靶向化学品开发挑战的有希望的途径 探针和药物。

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.