Cell-permeable high-affinity tracers for Gq proteins provide structural insights, reveal distinct binding kinetics and identify small molecule inhibitors
Cell-permeable high-affinity tracers for Gq proteins provide structural insights, reveal distinct binding kinetics and identify small molecule inhibitors
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DOI：
10.1111/bph.14960
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发表时间：
2020-02-11
影响因子：
7.3
通讯作者：
Mueller, Christa E.
中科院分区：
文献类型：
--
作者：
Kuschak, Markus;Namasivayam, Vigneshwaran;Mueller, Christa E.
背景与目的G蛋白是一种细胞内开关，能够放大和放大来自GPCR的细胞外信号。G（q）蛋白亚型与PLC激活偶联，可作为癌基因，并且据报道它们的表达在癌症和炎性疾病中上调。G（q）抑制可能是一种有效的治疗策略，构成了一个新的干预水平。实验方法我们现在已经开发了基于大环缩肽FR 900359（FR）和YM-254890（YM）的G（q）特异性的、细胞可渗透的H-3标记的高亲和力探针。FR和YM对CRISPR/Cas9 G alpha（q）敲除细胞中表达的G alpha（q）、G alpha（11）和G alpha（14）显示出低纳摩尔亲和力，但对G alpha（15）没有亲和力。这两种结构非常相似的示踪剂显示出明显不同的解离动力学，预计这将导致不同的生物学效应。计算研究表明，“榫钉”效应的pseudoirreversible绑定FR。高通量结合试验导致发现新的G（q）抑制剂，抑制G（q）信号在重组细胞和原代小鼠棕色脂肪细胞，导致在增强differentiation.Conclusions和影响的Gq蛋白抑制剂YM和FR是不同的，尽管类似的结构。新的多功能工具和强大的分析将有助于G蛋白研究的新兴领域的进步。
Background and Purpose G proteins are intracellular switches that transduce and amplify extracellular signals from GPCRs. The G(q) protein subtypes, which are coupled to PLC activation, can act as oncogenes, and their expression was reported to be up-regulated in cancer and inflammatory diseases. G(q) inhibition may be an efficient therapeutic strategy constituting a new level of intervention. However, diagnostic tools and therapeutic drugs for G(q) proteins are lacking.Experimental Approach We have now developed G(q)-specific, cell-permeable H-3-labelled high-affinity probes based on the macrocyclic depsipeptides FR900359 (FR) and YM-254890 (YM). The tracers served to specifically label and quantify G(q) proteins in their native conformation in cells and tissues with high accuracy.Key Results FR and YM displayed low nanomolar affinity for G alpha(q), G alpha(11) and G alpha(14) expressed in CRISPR/Cas9 G alpha(q)-knockout cells, but not for G alpha(15). The two structurally very similar tracers showed strikingly different dissociation kinetics, which is predicted to result in divergent biological effects. Computational studies suggested a "dowel" effect of the pseudoirreversibly binding FR. A high-throughput binding assay led to the discovery of novel G(q) inhibitors, which inhibited G(q) signalling in recombinant cells and primary murine brown adipocytes, resulting in enhanced differentiation.Conclusions and Implications The Gq protein inhibitors YM and FR are pharmacologically different despite similar structures. The new versatile tools and powerful assays will contribute to the advancement of the rising field of G protein research.