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Isoform-specific antagonists of exchange proteins directly activated by cAMP

机译:cAMP直接激活的交换蛋白的同工型特异性拮抗剂

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摘要

The major physiological effects of cAMP in mammalian cells are transduced by two ubiquitously expressed intracellular cAMP receptors, protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC), as well as cyclic nucleotide-gated ion channels in certain tissues. Although a large number of PKA inhibitors are available, there are no reported EPAC-specific antagonists, despite extensive research efforts. Here we report the identification and characterization of noncyclic nucleotide EPAC antagonists that are exclusively specific for the EPAC2 isoform. These EAPC2-specific antagonists, designated as ESI-05 and ESI-07, inhibit Rap1 activation mediated by EAPC2, but not EPAC1, with high potency in vitro. Moreover, ESI-05 and ESI-07 are capable of suppressing the cAMP-mediated activation of EPAC2, but not EPAC1 and PKA, as monitored in living cells through the use of EPAC- and PKA-based FRET reporters, or by the use of Rap1-GTP pull-down assays. Deuterium exchange mass spectroscopy analysis further reveals that EPAC2-specific inhibitors exert their isoform selectivity through a unique mechanism by binding to a previously undescribed allosteric site: the interface of the two cAMP binding domains, which is not present in the EPAC1 isoform. Isoform-specific EPAC pharmacological probes are highly desired and will be valuable tools for dissecting the biological functions of EPAC proteins and their roles in various disease states.
机译:cAMP在哺乳动物细胞中的主要生理作用是通过两个普遍表达的细胞内cAMP受体,蛋白激酶A(PKA)和被cAMP直接激活的交换蛋白(EPAC)以及某些组织中的环核苷酸门控离子通道来转导的。尽管有大量的PKA抑制剂,但尽管有大量的研究工作,也没有报道过的EPAC特异性拮抗剂。在这里,我们报告鉴定和表征非环核苷酸的EPAC拮抗剂,它们专门对EPAC2同工型具有特异性。这些EAPC2特异性拮抗剂(分别命名为ESI-05和ESI-07)在体外具有很高的抑制作用,可抑制EAPC2介导的Rap1激活,而不抑制EPAC1。此外,ESI-05和ESI-07能够抑制cAMP介导的EPAC2激活,但不能抑制EPAC1和PKA,如在活细胞中通过使用基于EPAC和PKA的FRET报告子或通过使用Rap1-GTP下拉测定。氘交换质谱分析进一步揭示,EPAC2特异性抑制剂通过与先前未描述的变构位点结合而通过独特的机制发挥其同工型选择性:两个cAMP结合域的界面,这在EPAC1同工型中不存在。非常需要同工型的EPAC药理探针,它将成为剖析EPAC蛋白质的生物学功能及其在各种疾病状态中的作用的有价值的工具。

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    Department of Pharmacology and Toxicology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555;

    Department of Pharmacology and Toxicology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555;

    Department of Medicine and Biomedical Sciences Graduate Program, University of California at San Diego, La Jolla, CA 92093;

    Departments of Medicine State University of New York, Upstate Medical University, Syracuse, NY 13210;

    Departments of Medicine State University of New York, Upstate Medical University, Syracuse, NY 13210;

    Department of Medicine and Biomedical Sciences Graduate Program, University of California at San Diego, La Jolla, CA 92093;

    Departments of Medicine State University of New York, Upstate Medical University, Syracuse, NY 13210,Departments of Pharmacology, State University of New York, Upstate Medical University, Syracuse, NY 13210;

    Department of Medicine and Biomedical Sciences Graduate Program, University of California at San Diego, La Jolla, CA 92093;

    Department of Pharmacology and Toxicology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    cAMP-regulated guanine nucleotide exchange factor; high-throughput screening; hydrogen/deuterium exchange mass spectrometry;

    机译:cAMP调节的鸟嘌呤核苷酸交换因子;高通量筛选氢/氘交换质谱;

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