Chemoproteomic mapping of the ligandable ribonucleoproteome using phloroglucinol meroterpenoids

PI研究计划的长期目标是了解RNAs如何通过 与RNA结合蛋白(RBPs)的相互作用，并确定其在人类疾病中的治疗效果的基础。 限制性商业惯例与RNA相互作用，形成核糖核蛋白复合体，控制着几乎每一种转录本的命运。 这些角色对正常的人类生理是必不可少的，因为RBP功能缺陷与自身免疫有关， 神经变性、癌症和先天抗病毒免疫反应。 RNA-蛋白质的分子机制 支撑基因表达并形成许多人类疾病基础的相互作用(RPI)仍然存在 默默无闻。因此，限制性商业惯例正在迅速成为开发化学探针的有希望的目标。 和候选治疗剂。然而，治疗上容易处理的RPI的发现仍然是一个令人敬畏的问题。 挑战。这项提案描述了一个利用定量化学蛋白质组学的跨学科计划，自然 产品合成、分子生物学和生物化学，以开发蛋白质赖氨酸在RPI的反应性 调节核糖核蛋白复合体结构性质的界面。PI的初步发现 证明间苯三酚类化合物通过可逆共价调节RPI 限制性商业惯例中RNA结合赖氨酸的修饰。这项提议的中心假设是 RPI界面上的反应性赖氨酸可能在发展精确度方面提供前所未有的机会 通过可逆共价修饰获得具有限制性商业惯例专一性和靶标效力的化学工具。 拟议的研究方案包含三个综合项目，以应对 靶向人类细胞中的限制性商业惯例。首先，缺乏传统的酶口袋或功能表位使限制性商业惯例 作为典型的难以对付的目标。这一提议旨在通过可逆共价来克服这一挑战 使用间苯三酚类化合物修饰RPI界面上的RNA结合赖氨酸。第二，充分利用 在RPI界面上具有高反应性的赖氨酸的补充仍未被发现。这项建议旨在 通过使用创新的化学蛋白质组学方法来全球定位赖氨酸的反应性和 在人体细胞中的直接配基能力。最后，硫代萜类化合物-赖氨酸相互作用的功能后果仍然存在。 默默无闻。通过利用整合的RNA下拉和提取方法以及RNA相互作用组捕获和 免疫荧光成像，该提案旨在识别通过干扰RPI的高级工具化合物 限制性商业惯例中赖氨酸的选择性修饰。总体而言，拟议的研究计划意义重大，因为 未来五年，它将提供创新的化学工具和蛋白质组学方法，丰富对 RPI的病理生理学功能，并确定其在人类疾病中的治疗效果的基础。 这项提案提供了一些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)，以及药物发现的新方法和目标(PPBC)。

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).