Illuminating Druggable Targets via Interrogation of Direct GPCR-kinase Interactions

通过询问GPCRK的直接相互作用来照亮可药物靶点 G蛋白偶联受体(GPCRs)和激酶代表了两类最易被药物抑制的 膜蛋白，作为FDA批准的三分之一以上药物的靶标。尽管如此，这两个 蛋白质超家族在治疗上仍未得到充分利用，因为许多gpcr和激酶在临床上仍然缺乏。 有用的配体。大量这些未被研究的gpcr和激酶可能与一种 另一种是通过现有功能分析没有捕捉到的非规范机制。我们的目标是找出这些 非规范机制，这不仅将告诉我们未被充分研究的GPCRs如何影响细胞生理学，而且 帮助我们开发新的分析方法来识别对治疗有益的配体。我们发现了一种新的gpr- 在GPCR领域，不符合传统的信号传递范式的蛋白激酶通讯机制。 相反，活性的GPCRs直接结合和隔离蛋白激酶A催化的ɑ(PKA-Cɑ)亚单位。 膜，阻止可溶性PKA底物的磷酸化最终影响过多的细胞信号 流程。我们假设，IDG投资组合中相当数量的未被研究的GPCR控制着他们的 通过隔离PKA-C的不同细胞内信号机制。这一新想法可能不仅适用于 对于研究较多的PKA-C亚型，也涉及研究较少的PKA-C和PKA-C亚型，这两种亚型 被指定为IDG靶标，并与癌症和代谢紊乱有关。在这份R03拨款中，我们严格地 评估上述假设，在开发概念和生成试剂的过程中进行讯问 对GPCRs和激酶的研究还不够广泛。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，然后利用本实验系统地评价GPCRs和/或 在IDG的投资组合中，PKA-C亚型。通过鉴定和表征广泛的 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受体的总体主题 生物学。拟议的研究将为未来至少一笔R01赠款提供基础，重点放在特定的 我们确定的GPCR/PKA-C相互作用，特别是那些涉及发育和癌症生物学的相互作用，神经 系统功能，以及运动纤毛和初级纤毛的生物学。这项拟议的研究利用了现有的IDG- 生成的资源，包括Presto-Tango系统和NanBRET激酶靶向参与分析， 这两个都可以使用以下相对较小的修改，连同非盟驻苏特派团数据库。可得性 这些关键试剂，结合我们在培养细胞功能分析方面的专业知识，将使我们能够完成 该项目在一年内完成。我们的工作将建立一种新的范式来理解和治疗目标 未被充分研究的GPCRs和激酶。这将为了解这些蛋白质的生物学功能和 监管机制，并使确定调节这些相互作用的药物的努力成为可能。鉴于无处不在的 对于生物学中的GPCRs和PKA，我们的研究可能与人类健康和疾病的许多方面相关。

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.