Department of Pharmacology and Toxicology and Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, Texas, United States of America.
PLoS One. 2012;7(1):e30441. doi: 10.1371/journal.pone.0030441. Epub 2012 Jan 19.
The discovery, more than ten years ago, of exchange proteins directly activated by cAMP (EPAC) as a new family of intracellular cAMP receptors revolutionized the cAMP signaling research field. Extensive studies have revealed that the cAMP signaling network is much more complex and dynamic as many cAMP-related cellular processes, previously thought to be controlled by protein kinase A, are found to be also mediated by EPAC proteins. Although there have been many important discoveries in the roles of EPACs greater understanding of their physiological function in cAMP-mediated signaling is impeded by the absence of EPAC-specific antagonist.
METHODOLOGY/PRINCIPAL FINDINGS: To overcome this deficit, we have developed a fluorescence-based high throughput assay for screening EPAC specific antagonists. Our assay is highly reproducible and simple to perform using the "mix and measure" format. A pilot screening using the NCI-DTP diversity set library led to the identification of small chemical compounds capable of specifically inhibiting cAMP-induced EPAC activation while not affecting PKA activity.
CONCLUSIONS/SIGNIFICANCE: Our study establishes a robust high throughput screening assay that can be effectively applied for the discovery of EPAC-specific antagonists, which may provide valuable pharmacological tools for elucidating the biological functions of EPAC and for promoting an understanding of disease mechanisms related to EPAC/cAMP signaling.
十多年前，人们发现了 cAMP（环磷酸腺苷）直接激活交换蛋白（EPAC），这是一种新的细胞内 cAMP 受体家族，这彻底改变了 cAMP 信号研究领域。广泛的研究表明，cAMP 信号网络更加复杂和动态，因为许多以前被认为受蛋白激酶 A 控制的与 cAMP 相关的细胞过程，现在发现也由 EPAC 蛋白介导。尽管 EPAC 发挥了许多重要作用，但由于缺乏 EPAC 特异性拮抗剂，对其生理功能的理解仍受到阻碍。
方法/主要发现：为了克服这一缺陷，我们开发了一种基于荧光的高通量筛选 EPAC 特异性拮抗剂的方法。我们的测定方法具有高度的重现性，使用“混合和测量”格式非常简单。使用 NCI-DTP 多样性文库进行的初步筛选导致发现了能够特异性抑制 cAMP 诱导的 EPAC 激活而不影响 PKA 活性的小分子化合物。
结论/意义：我们的研究建立了一种稳健的高通量筛选测定方法，可有效用于发现 EPAC 特异性拮抗剂，这可能为阐明 EPAC 的生物学功能以及促进对与 EPAC/cAMP 信号相关的疾病机制的理解提供有价值的药理学工具。
