Biochemical and pharmacological characterizations of ESI-09 based EPAC inhibitors: defining the ESI-09 "therapeutic window".

Yingmin Zhu,Giuseppe Melacini,Jia Zhou,Stephen Boulton,Fang Mei,Na Ye,Haijun Chen,Xiaodong Cheng

doi:10.1038/srep09344

Abstract

The cAMP signaling cascade is one of the most frequently targeted pathways for the development of pharmaceutics. A plethora of recent genetic and pharmacological studies suggest that exchange proteins directly activated by cAMP (EPACs) are implicated in multiple pathologies. Selective EPAC inhibitors have been recently developed. One specific inhibitor, ESI-09, has been shown to block EPAC activity and functions, as well as to recapitulate genetic phenotypes of EPAC knockout mice when applied in vivo. However, a recent study raised concern that ESI-09 might act as a non-specific protein denaturant. Herein, we present a detailed biochemical and pharmacological characterization, as well as a structure-activity relationship (SAR) analysis of ESI-09. Our studies show that ESI-09 dose-dependently inhibits activity of both EPAC1 and EPAC2 with apparent IC50 values well below the concentrations shown to induce “protein denaturation”. Moreover, the ESI-09's action towards EPAC proteins is highly sensitive to minor modifications of the 3-chlorophenyl moiety. Taken together, these results demonstrate that ESI-09 indeed acts as an EPAC specific antagonist and does not significantly destabilize/denature proteins at pharmacological effective concentrations. This conclusion is further supported by NMR data showing that ESI-09 induces residue-dependent chemical shift changes at low concentrations, while preserving well dispersed peaks.

Highlights

The cAMP signaling cascade is one of the most frequently targeted pathways for the development of pharmaceutics
EPAC specific inhibitors (ESIs)-09, 3-[5-(tert-butyl)isoxazol-3-yl]-2-[2-(3-chlorophenyl) hydrazono] -3-oxopropanenitrile, was initially identified from a high throughput screening (HTS) of the Maybridge HitFinder diversity library along with several other compounds that were able to compete with 8-NBD-cAMP (8-(2-[7-Nitro-4-benzofurazanyl] aminoethylthio) adenosine-39, 59-cyclic monophosphate) in binding to EPAC2 and inhibit both EPAC2 and EPAC1’s guanine nucleotide exchange factor (GEF) activity[18,19]
In this study, we present a thorough biochemical and pharmacological characterization of ESI-09 based EPAC specific inhibitors, provide solid evidence that ESI-09 acts as an EPAC selective antagonist by directly competing with cAMP binding, and argue against the notion that the ESI-09’s effect on EPAC proteins is fully accounted for by ‘‘a non-specific protein denaturing property’’22

Summary

Introduction

The cAMP signaling cascade is one of the most frequently targeted pathways for the development of pharmaceutics. A plethora of recent genetic and pharmacological studies suggest that exchange proteins directly activated by cAMP (EPACs) are implicated in multiple pathologies. A recent study raised concern that ESI-09 might act as a non-specific protein denaturant. The ESI-09’s action towards EPAC proteins is highly sensitive to minor modifications of the 3-chlorophenyl moiety Taken together, these results demonstrate that ESI-09 acts as an EPAC specific antagonist and does not significantly destabilize/denature proteins at pharmacological effective concentrations. These results demonstrate that ESI-09 acts as an EPAC specific antagonist and does not significantly destabilize/denature proteins at pharmacological effective concentrations This conclusion is further supported by NMR data showing that ESI-09 induces residue-dependent chemical shift changes at low concentrations, while preserving well dispersed peaks. Our results demonstrate that ESI-09 and its close analogs act as EPAC specific antagonists by competing with cAMP binding to EPAC

Methods

Results

Conclusion