Illuminating Druggable Targets via Interrogation of Direct GPCR-kinase Interactions

通过检测直接 GPCR-激酶相互作用来阐明可药物靶标

• 批准号: 10217863
• 负责人: Benjamin Myers
• 金额: $ 15.25万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-15 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10217863
• 关键词: ARNT gene; Address; Affect; Arrestins; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Biological Process; Biology; Bioluminescence; Cancer Biology; Catalytic Domain; Cell physiology; Cells; Cilia; Clinical; Communication; Complex; Cultured Cells; Cyclic AMP-Dependent Protein Kinases; Databases; Development; Developmental Biology; Disease; Drug Screening; Drug Targeting; Endocrine Gland Neoplasms; Energy Transfer; Event; FDA approved; Foundations; Future; G protein coupled receptor kinase; G-Protein-Coupled Receptors; Goals; Grant; Health; Heterotrimeric GTP-Binding Proteins; Human; Knowledge; Learning; Ligands; Lighting; Link; Malignant Neoplasms; Membrane; Membrane Proteins; Metabolic; Metabolic Diseases; Minor; Modernization; Modification; Natural regeneration; Nervous System Physiology; PRKCA gene; Pathway interactions; Pharmaceutical Preparations; Pharmacopoeias; Phosphorylation; Phosphotransferases; Physiological; Procedures; Process; Protein Isoforms; Proteins; Reagent; Reporting; Research; Resources; Signal Transduction; System; T2R taste receptors; Testing; Therapeutic; Tissues; Tracer; Work; base; cell motility; cilium motility; design; drug use screening; druggable target; high throughput screening; human disease; insight; interest; luminescence; neurotransmission; novel therapeutics; prevent; receptor; screening; smoothened signaling pathway; therapeutic protein; therapeutic target; tool

Illuminating Druggable Targets via Interrogation of Direct GPCR-kinase Interactions G protein-coupled receptors (GPCRs) and kinases represent two of the most highly druggable classes of membrane proteins, serving as targets for over one-third of FDA-approved drugs. Nevertheless, both of these protein superfamilies remain therapeutically underexploited, as many GPCRs and kinases still lack clinically useful ligands. A significant number of these understudied GPCRs and kinases may communicate with one another via noncanonical mechanisms not captured by existing functional assays. Our goal is to identify these noncanonical mechanisms, which will not only teach us how understudied GPCRs affect cell physiology, but also help us develop new assays to identify therapeutically beneficial ligands. We have discovered a new GPCR- kinase communication mechanism that does not conform to traditional signaling paradigms in the GPCR field. Instead, active GPCRs directly bind to and sequesters protein kinase A catalytic ɑ (PKA-Cɑ) subunits at the membrane, blocking phosphorylation of soluble PKA substrates to ultimately affect a plethora of cellular signaling processes. We hypothesize that a significant number of understudied GPCRs in the IDG portfolio control their respective intracellular signaling mechanisms via sequestration of PKA-C. This new idea likely applies not just to the heavily studied PKA-C isoform, but also to the understudied PKA-C and PKA-C isoforms that are designated as IDG targets and implicated in cancer and metabolic disorders. In this R03 grant we critically evaluate the above hypothesis, in the process developing concepts and generating reagents to interrogate understudied GPCRs and kinases more generally. Our strategy is to design a high-throughput assay for GPCR / PKA-C interactions, and then use this assay to systematically evaluate interactions between the GPCRs and/or PKA-C isoforms in IDG’s portfolio. By identifying and characterizing GPCR / PKA-C interactions on a broad scale, our work will reveal the extent to which GPCR / PKA-C interactions represent a general theme in receptor biology. The proposed studies will provide a foundation for at least one future R01 grant focusing on the specific GPCR / PKA-C interactions we identify, particularly those involved in developmental and cancer biology, nervous system function, and the biology of motile and primary cilia. The proposed research capitalizes on existing IDG- generated resources, including the PRESTO-Tango system and nanoBRET kinase target engagement assays, both of which we can use following relatively minor modifications, along with the AMIS database. The availability of these key reagents, combined with our expertise in cultured cell functional assays, will allow us to complete the project within one year. Our work will establish a new paradigm to understand and therapeutically target understudied GPCRs and kinases. This will provide major insights into these proteins’ biological functions and regulatory mechanisms and enable efforts to identify drugs that modulate these interactions. Given the ubiquity of GPCRs and PKA in biology, our studies are likely to be relevant to many aspects of human health and disease.

通过询问直接GPCR-激酶相互作用来照明可药物的目标 G蛋白偶联受体（GPCR）和激酶代表了两种最可吸毒的类别 膜蛋白，作为FDA批准药物中三分之一以上的靶标。但是，这两个 蛋白质超家族保持热不足，因为许多GPCR和激酶在临床上仍然缺乏 有用的配体。其中大量理解的GPCR和激酶可能与一个 另一种通过现有功能测定未捕获的非规范机制。我们的目标是确定这些 非规范机制，这不仅会教会我们理解GPCR如何影响细胞生理，而且还会教会 帮助我们开发新的测定法，以识别治疗效果的配体。我们发现了一个新的GPCR- 激酶通信机制不符合GPCR领域中传统的信号范式。 取而代之的是，主动GPCR直接与蛋白激酶结合并隔离蛋白激酶在 膜，阻断固体PKA底物的磷酸化，最终影响过多的细胞信号传导 过程。我们假设IDG投资组合中的大量理解的GPCR控制着他们 通过PKA-C隔离的一级细胞内信号传导机制。这个新想法不仅适用 对大量研究的PKA-C同工型，但也可以理解为PKA-C和PKA-C同工型 被指定为IDG靶标，并暗示在癌症和代谢障碍中。在这笔R03赠款中，我们批判性 评估上述假设，在开发概念并生成试剂询问的过程中 更广泛地研究了GPCR和激酶。我们的策略是为GPCR设计高通量测定法 / PKA-C相互作用，然后使用此测定法系统地评估GPCR和/或之间的相互作用 IDG投资组合中的PKA-C亚型。通过识别和表征GPCR / PKA-C的相互作用 规模，我们的工作将揭示GPCR / PKA-C交互代表接收器中的一般主题的程度 生物学。拟议的研究将为至少一项将来的R01赠款提供基础，重点是特定的 GPCR / PKA-C相互作用我们识别出，特别是参与发育和癌症生物学的人，神经 系统功能以及母亲和原发性纤毛的生物学。拟议的研究资本利用了现有的IDG- 生成的资源，包括Presto-Tango系统和纳米型激酶目标参与分析， 我们都可以与AMIS数据库一起使用以下相对较小的修改。可用性 在这些关键试剂中，结合我们在培养的细胞功能测定方面的专业知识，将使我们能够完成 该项目在一年内。我们的工作将建立一个新的范式来理解和治疗目标 研究了GPCR和激酶。这将为这些蛋白质的生物学功能和 调节机制和能够努力确定调节这些相互作用的药物。给定无处不在 在生物学中的GPCR和PKA中，我们的研究可能与人类健康和疾病的许多方面有关。