a fluorescence-based high-throughput assay for the discovery of exchange protein directly activated by cyclic amp (epac) antagonists的荧光技术高通量分析的发现直接交换蛋白激活环腺苷酸(epac)拮抗剂.pdfVIP

A Fluorescence-Based High-Throughput Assay for the Discovery of Exchange Protein Directly Activated by Cyclic AMP (EPAC) Antagonists Tamara Tsalkova, Fang C. Mei, Xiaodong Cheng* 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 Abstract Background: 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 signal