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Biochemical and Pharmacological Characterizations of ESI‐09 based EPAC inhibitors
Author(s) -
Zhu Yingmin,
Chen Haijun,
Mei Fang,
Zhou Jia,
Cheng Xiaodong
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.1022.4
Subject(s) - guanine nucleotide exchange factor , chemistry , antagonist , moiety , second messenger system , nucleotide , intracellular , guanine , ic50 , biochemistry , stereochemistry , receptor , in vitro , signal transduction , gene
Exchange protein directly activated by cAMP (EPAC) is one of two intracellular receptors that mediate the effects of the prototypic second messenger cAMP. Identifying pharmacological probes for selectively modulating EPAC activity represents a significant unmet need within the research field. In this study, we present a thorough biochemical and pharmacological characterization of ESI‐09 based EPAC specific inhibitors, provide convincing evidence that ESI‐09 acts as an EPAC selective antagonist by directly competing with cAMP binding, and argue against the notion that ESI‐09′s effect on EPAC proteins is due to its non‐specific protein denaturing property. Our data show that ESI‐09 dose‐dependently inhibits cAMP‐mediated guanine nucleotide exchange activity in both EPAC1 and EPAC2 with apparent IC50 values well below the concentrations shown to induce “thermal unfolding shifts” reported by Rehmann. Furthermore, structure‐activity relationship analysis reveals that the exact position and number of the chloro‐substituents on the chlorophenyl moiety are important for the potency of ESI‐09 analogs in competing with 8‐NBD‐cAMP for EPAC2 binding and HJC0726 with 3,5‐dichloro‐substituent is five‐fold more potent than ESI‐09 in inhibiting both EPACs. These results suggest that ESI‐09′s action towards EPAC proteins is specific as it is highly sensitive to minor modifications of the 3‐chlorophenyl moiety. In summary, our current study validates our previous publications by demonstrating that ESI‐09 is a specific EPAC antagonist with providing convincing evidence that it acts as a competitive inhibitor.