Identification of a Tetrahydroquinoline Analog as a Pharmacological Inhibitor of the cAMP-binding Protein Epac
Identification of a Tetrahydroquinoline Analog as a Pharmacological Inhibitor of the cAMP-binding Protein Epac
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DOI：
10.1074/jbc.m112.422956
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发表时间：
2012-12-28
影响因子：
4.8
通讯作者：
Lezoualc'h, Frank
中科院分区：
文献类型：
--
作者：
Courilleau, Delphine;Bisserier, Malik;Lezoualc'h, Frank
camp结合蛋白Epac是治疗心脏肥厚、肿瘤侵袭等多种疾病的治疗靶点。这指出了开发Epac抑制剂以更好地了解这些cAMP传感器在生理和病理生理中的作用的重要性。在这里，我们开发了一种基于功能性荧光的高通量检测方法，其Z值约为0.7，用于筛选epac特异性拮抗剂。我们发现了一种名为CE3F4的Epac1抑制剂化合物，它可以在无细胞系统和完整细胞中阻断Epac1与其效应物Rap1的鸟嘌呤核苷酸交换活性。CE3F4是一种不影响蛋白激酶a全酶活性的四氢喹啉类似物。CE3F4既不抑制Rap1与Epac1的相互作用，也不直接抑制Rap1上的GDP交换。CE3F4的抑制动力学表明，该化合物不竞争激动剂与Epac1的结合，表明该化合物对Epac1激动剂具有非竞争抑制机制。结构活性研究表明，CE3F4在四氢喹啉骨架上1位的甲酰基和5位的溴原子是发挥其抑制活性的重要因素。最后，在Epac选择性激动剂8-（4-氯苯基硫）-2′- o -甲基- camp或非选择性β -肾上腺素受体激动剂异丙肾上腺素激活Epac后，CE3F4抑制了活培养细胞中Rap1的激活。我们的研究表明，CE3F4及其相关化合物可以作为开发新的治疗药物的基础。
The cAMP-binding protein Epac is a therapeutic target for the treatment of various diseases such as cardiac hypertrophy and tumor invasion. This points out the importance to develop Epac inhibitors to better understand the involvement of these cAMP sensors in physiology and pathophysiology. Here, we have developed a functional fluorescence-based high-throughput assay with a Z' value around 0.7 for screening Epac-specific antagonists. We identified an Epac1 inhibitor compound named CE3F4 that blocked Epac1 guanine nucleotide exchange activity toward its effector Rap1 both in cell-free systems and in intact cells. CE3F4 is a tetrahydroquinoline analog that fails to influence protein kinase A holoenzyme activity. CE3F4 inhibited neither the interaction of Rap1 with Epac1 nor directly the GDP exchange on Rap1. The kinetics of inhibition by CE3F4 indicated that this compound did not compete for binding of agonists to Epac1 and suggested an uncompetitive inhibition mechanism with respect to Epac1 agonists. A structure-activity study showed that the formyl group on position 1 and the bromine atom on position 5 of the tetrahydroquinoline skeleton were important for CE3F4 to exert its inhibitory activity. Finally, CE3F4 inhibited Rap1 activation in living cultured cells, following Epac activation by either 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, an Epac-selective agonist, or isoprenaline, a non-selective beta-adrenergic receptor agonist. Our study shows that CE3F4 and related compounds may serve as a basis for the development of new therapeutic drugs.