Revisiting long-held tenets in GPCR and G protein signal transduction with macrocyclic inhibitors of heterotrimeric Gq/11 proteins and state-of-the-art genome editing

G蛋白偶联受体及其相关的G蛋白直接或间接参与人体几乎所有的生理过程。尽管约25年前发现了G蛋白及其与许多细胞外信号相关的应答和维持稳态的相关性，但用细胞渗透性抑制剂选择性抑制该蛋白家族仍然是一个根本性的挑战。我们最近在这一领域取得了两项重要进展：（1）鉴定了一种特异性干扰G蛋白功能的新分子机制：GTP进入抑制剂。这些分子通过将G α捕获在空口袋构象中来阻断G蛋白信号传导。(2)深入研究植物源缩酚肽FR 900359的选择性和作用模式。我们发现，FR是非常有选择性的抑制Gq介导的信号转导，涉及Galphaq，Galpha11，和Galpha14蛋白，但绝对惰性的所有其他哺乳动物Galpha亚型。鉴于其作为分子探针通过抑制GDP释放特异性阻断Gq信号传导的突出价值，我们现在想利用FR来（i）研究GPCR在不存在活化G蛋白的情况下发出信号的一般能力，（ii）探测其作用模式的结构和分子基础以及抑制Gq家族蛋白的精确选择性，（iii）应用（ii）中的知识来促进针对那些仍然缺乏选择性抑制剂的Gala亚基的新型抑制剂的设计。此外，我们打算应用和扩展广泛的生物学，药理学和生物化学检测的“机械指纹”的新的抑制剂在我们的财团内合成。通过现有的和新开发的Galalpha工具，我们希望显着推进和完善我们对细胞感知外部刺激并将其转化为特定指令以调节细胞功能的基本原理的理解。

G protein-coupled receptors and their associated G proteins are involved, directly or indirectly, in virtually every physiological process in the human body. Despite the discovery of G proteins about 25 years ago and their relevance for responding and maintaining homeostasis in relation to numerous extracellular cues, selective inhibition of this protein family with cell-permeable inhibitors remains a fundamental challenge. We recently contributed two significant advances to this field: (1) identification of a novel molecular mechanism to specifically interfere with G protein function: GTP-entry inhibitors. These are molecules that interdict G protein signaling by trapping Galpha in the empty pocket conformation. (2) in-depth investigations into selectivity and mode of action of the plant-derived depsipeptide FR900359. We found that FR is exceptionally selective for inhibition of Gq-mediated signal transduction involving Galphaq, Galpha11, and Galpha14 proteins, but absolutely inert on all other mammalian Galpha isoforms. Given its outstanding value as molecular probe to specifically interdict Gq signaling via inhibition of GDP release, we now want to take advantage of FR to (i) investigate the general capacity of GPCRs to signal in the absence of activated G proteins, (ii) probe the structural and molecular basis for its mode of action and exquisite selectivity for inhibition of Gq family proteins, (iii) apply the knowledge from (ii) to foster design of novel inhibitors targeting those Galpha subunits for which selective inhibitors are still lacking. Moreover, we intend to apply and extend a broad range of biological, pharmacological and biochemical assays for 'mechanistic fingerprinting' of novel inhibitors synthesized within our consortium. With existing and newly developed Galpha tools we expect to significantly advance and refine our understanding of basic principles utilized by cells to perceive external stimuli and translate these into specific instructions to modulate cell function.