Chemoproteomic mapping of the ligandable ribonucleoproteome using phloroglucinol meroterpenoids

使用间苯三酚类萜类化合物对可配体核糖核蛋白质组进行化学蛋白质组学作图

• 批准号: 10434114
• 负责人: Mikail E Abbasov
• 金额: $ 38.6万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10434114
• 关键词: Address; Autoimmunity; Biochemistry; Cell physiology; Cells; Chemicals; Code; Complement; Complex; Defect; Development; Drug Targeting; Epitopes; Gene Expression; Goals; Human; Image; Immune response; Immunofluorescence Immunologic; Lysine; Malignant Neoplasms; Maps; Methods; Modification; Molecular; Molecular Biology; National Institute of General Medical Sciences; Natural Products; Nerve Degeneration; Phloroglucinol; Physiology; Property; Proteomics; RNA; RNA Binding; RNA-Binding Proteins; RNA-Protein Interaction; Research; Ribonucleoproteins; Role; Site; Specificity; Technology; Therapeutic; Therapeutic Agents; Therapeutic Effect; Transcript; Untranslated RNA; chemoproteomics; drug discovery; human disease; innovation; programs; protein function; success; therapeutic candidate; tool

The long-term goal of the PI’s research program is to understand how RNAs exert their regulatory effects through interaction with RNA-binding proteins (RBPs) and identify the basis for their therapeutic effects in human diseases. RBPs interact with RNAs to form ribonucleoprotein complexes that that control the fate of nearly every transcript. These roles are essential for normal human physiology, as defects in RBP function are implicated in autoimmunity, neurodegeneration, cancer, and innate antiviral immune response. The molecular mechanisms of RNA-protein interactions (RPIs) that underpin gene expression and form the basis of numerous human diseases remain obscure. Consequently, RBPs are rapidly emerging as promising targets for the development of chemical probes and candidate therapeutic agents. The discovery of therapeutically tractable RPIs, however, remains a formidable challenge. This proposal describes an interdisciplinary program leveraging quantitative chemoproteomics, natural product synthesis, molecular biology, and biochemistry to exploit reactivity of proteinaceous lysines at the RPI interface that modulate the structural properties of ribonucleoprotein complexes. Preliminary findings from the PI demonstrate that phloroglucinol meroterpenoid natural products modulate RPIs through reversible-covalent modification of RNA-binding lysines in RBPs. The central hypothesis of this proposal is that high occurrence of reactive lysines at the RPI interface may provide an unprecedented opportunity in the development of precision chemical tools with specificity for RBPs and on-target potency gained through reversible-covalent modification. The proposed research program embodies three integrated projects that address specific challenges in targeting RBPs in human cells. First, the lack of traditional enzymatic pockets or functional epitopes renders RBPs as canonically intractable targets. This proposal aims to overcome this challenge through reversible-covalent modification of RNA-binding lysines at the RPI interface using phloroglucinol meroterpenoids. Second, the full complement of lysines with heightened reactivity at the RPI interface remains unexplored. This proposal aims to overcome this limitation by using innovative chemoproteomic methods to globally map lysine reactivity and ligandability directly in human cells. Last, the functional consequences of meroterpenoid-lysine interactions remain obscure. By leveraging integrative RNA pulldown and extraction methods with RNA-interactome capture and immunofluorescence imaging, the proposal aims to identify advanced tool compounds that perturb RPIs through site-selective modification of lysines in RBPs. Overall, the proposed research program is significant because over the next five years it will deliver innovative chemical tools and proteomic methods that enrich understanding of the pathophysiological functions of RPIs and identify the basis for their therapeutic effects in human diseases. This proposal offers progress in a number of NIGMS major research programs, including fundamentals of molecular properties and interactions (BBCB), coding and noncoding RNA mechanisms of action and function (GMCDB), and new methods and targets for drug discovery (PPBC).

PI研究计划的长期目标是了解RNA如何通过以下途径发挥其调节作用： 研究RNA结合蛋白（RBP）的相互作用，并确定其在人类疾病中的治疗作用的基础。 RBP与RNA相互作用形成核糖核蛋白复合物，控制几乎所有转录物的命运。 这些作用对于正常的人体生理是必不可少的，因为RBP功能的缺陷与自身免疫有关， 神经变性、癌症和先天性抗病毒免疫反应。 RNA-蛋白质的分子机制 支持基因表达并形成许多人类疾病基础的RPI仍然存在 晦涩难懂。因此，RBPs正迅速成为开发化学探针的有前途的目标 和候选治疗剂。然而，发现治疗上易处理的RPI仍然是一个艰巨的任务。 挑战.这项建议描述了一个跨学科的计划，利用定量化学蛋白质组学，自然 产物合成、分子生物学和生物化学，以利用蛋白质赖氨酸在RPI处的反应性 界面，调节核糖核蛋白复合物的结构特性。PI的初步结果 证明间苯三酚类单萜天然产物通过可逆-共价 RBP中RNA结合赖氨酸的修饰。这一建议的核心假设是， RPI界面上的反应性赖氨酸可能为精密制造提供前所未有的机会。 具有RBP特异性和通过可逆共价修饰获得的靶向效力的化学工具。 拟议的研究计划体现了三个综合项目，解决具体的挑战， 靶向人体细胞中的RBPs。首先，由于缺乏传统的酶口袋或功能表位， 难以对付的目标该提案旨在通过可逆-共价结合克服这一挑战。 使用间苯三酚部分萜类修饰RPI界面处的RNA结合赖氨酸。第二，全 在RPI界面处具有提高的反应性的赖氨酸的补体仍然未被探索。这项建议旨在 通过使用创新的化学蛋白质组学方法来克服这一限制，以全面绘制赖氨酸反应性， 直接在人类细胞中进行配体化。最后，类萜-赖氨酸相互作用的功能性后果仍然存在 晦涩难懂。通过利用整合RNA下拉和提取方法与RNA-相互作用体捕获， 免疫荧光成像，该提案旨在确定先进的工具化合物，通过 RBP中赖氨酸的位点选择性修饰。总的来说，拟议的研究计划是重要的，因为超过 未来五年，它将提供创新的化学工具和蛋白质组学方法，以丰富对蛋白质组学的理解。 RPI的病理生理功能，并确定其在人类疾病中的治疗效果的基础。 该提案提供了一些NIGMS主要研究计划的进展，包括 分子特性和相互作用（BBCB），编码和非编码RNA的作用和功能机制 （GMCDB）和药物发现新方法和靶点（PPBC）。