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.

通过直接GPCR-激酶相互作用的询问照亮可药用靶标 G蛋白偶联受体（GPCR）和激酶代表了两种最高度可药用的类 膜蛋白，作为超过三分之一的FDA批准的药物的目标。然而，这两个 蛋白质超家族在治疗上仍然没有得到充分利用，因为许多GPCR和激酶在临床上仍然缺乏 有用的配体。这些未充分研究的GPCR和激酶中的相当数量可能与一个 另一种是通过现有功能测定法未捕获的非规范机制。我们的目标是找出这些 非经典机制，这不仅会告诉我们如何研究不足的GPCR影响细胞生理学，而且 帮助我们开发新的检测方法来鉴定治疗上有益的配体。我们发现了一种新的气相聚合反应- 激酶通讯机制，不符合GPCR领域的传统信号传导模式。 相反，活性GPCR直接结合并隔离蛋白激酶A催化激酶（PKA-C）亚基， 膜，阻断可溶性PKA底物的磷酸化，最终影响过多的细胞信号传导 流程.我们假设IDG投资组合中大量未充分研究的GPCR控制着它们的 通过PKA-C的螯合的各自的细胞内信号传导机制。这个新的想法可能不仅适用于 大量研究的PKA-C β亚型，但也研究不足的PKA-C β和PKA-C β亚型， 被指定为IDG靶点并与癌症和代谢紊乱有关。在R 03赠款中，我们批判性地 评估上述假设，在这个过程中发展概念和生成试剂来询问 GPCR和激酶的研究更普遍。我们的策略是设计一种高通量的GPCR检测方法 / PKA-C相互作用，然后使用该测定系统地评价GPCR和/或 IDG产品组合中的PKA-C亚型。通过鉴定和表征GPCR / PKA-C相互作用， 规模，我们的工作将揭示GPCR / PKA-C相互作用在多大程度上代表了受体的一般主题， 生物学拟议的研究将为未来至少一个R 01赠款提供基础，重点是具体的 我们鉴定了GPCR / PKA-C相互作用，特别是那些涉及发育和癌症生物学、神经系统和神经系统的相互作用。 系统功能，以及能动纤毛和初级纤毛的生物学。这项研究利用了现有的IDG- 产生的资源，包括PRESTO-Tango系统和nanoBRET激酶靶向接合测定， 这两个数据库我们都可以在经过相对较小的修改后与AMIS数据库一起使用。的可用性 结合我们在培养细胞功能检测方面的专业知识，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 未充分研究的GPCR和激酶。这将为这些蛋白质的生物学功能提供重要的见解， 调节机制，并使努力确定药物调节这些相互作用。考虑到 我们的研究可能与人类健康和疾病的许多方面有关。