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

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.

Figure 1. Structure-activity relationship analysis of ESI-09.

(A). Chemical structure of ESI-09 and its analogs based on the core structure of 2-(5-(tert-butyl)isoxazol-3-yl)-2-oxo-N-phenyl- acetohydrazonoyl cyanide. (B) Dose-dependent competition of cAMP and ESI-09 analogs with 8-NBD-cAMP in binding to EPAC2.

Figure 2. Inhibition of EPAC2 and EPAC1 GEF activities by ESI-09 and HJC0726.

Dose-dependent inhibition of EPAC2 GEF activity by ESI-09 (A) or HJC0726 (B) in the presence of 20 µM cAMP. (C) Relative EPAC2 GEF activity as a function of ESI-09 and HJC0726 concentration. Dose-dependent inhibition of EPAC1 GEF activity by ESI-09 (D) or HJC0726 (E) in the presence of 20 µM cAMP. (F) Relative EPAC1 GEF activity as a function of ESI-09 and HJC0726 concentration.

Figure 3. EPAC2 activation by cAMP in the absence and presence of ESI-09 or HJC0726.

Dose-dependent activation of EPAC2 by cAMP in the absence of inhibitor (A), or in the presence of 5 µM ESI-09 (B) or 1 µM HJC0726 (C). (D) Relative EPAC2 GEF activity as a function of cAMP concentration.

Figure 4. Effect of ESI-09 on thermal denaturation of EPAC2 and GST.

Thermal-induced protein denaturation of EPAC2 (A) and GST (C) in the presence of various ESI-09 concentrations. Thermal melting temperature (T_m) of EPAC2 (B) and GST (D) as a function of ESI-09 concentration.

Figure 5. Effect of ESI-09 on EPAC1_h 149-318 {¹⁵N, ¹H} NMR resonances.

{¹⁵N, ¹H}-HSQC spectra of 100 μM EPAC1_h 149-318 in the absence (A) and presence of 50 μM (B) and 500 μM (C) ESI-09. (D) Representative section from the spectral overlay of 25 μM apo EPAC (+1% DMSO) with 25 μM EPAC bound with 100 μM ESI-09 (+1% DMSO).