Modeling Epac1 interactions with the allosteric inhibitor AM-001 by co-solvent molecular dynamics

Marianna Bufano,Andrea Brancale,Antonio Coluccia,Romano Silvestri,Jean-Paul Blondeau,Marianna Nalli,Frank Lezoualc’H,Marion Laudette

doi:10.1007/s10822-020-00332-y

Abstract

The exchange proteins activated by cAMP (EPAC) are implicated in a large variety of physiological processes and they are considered as promising targets for a wide range of therapeutic applications. Several recent reports provided evidence for the therapeutic effectiveness of the inhibiting EPAC1 activity cardiac diseases. In that context, we recently characterized a selective EPAC1 antagonist named AM-001. This compound was featured by a non-competitive mechanism of action but the localization of its allosteric site to EPAC1 structure has yet to be investigated. Therefore, we performed cosolvent molecular dynamics with the aim to identify a suitable allosteric binding site. Then, the docking and molecular dynamics were used to determine the binding of the AM-001 to the regions highlighted by cosolvent molecular dynamics for EPAC1. These analyses led us to the identification of a suitable allosteric AM-001 binding pocket at EPAC1. As a model validation, we also evaluated the binding poses of the available AM-001 analogues, with a different biological potency. Finally, the complex EPAC1 with AM-001 bound at the putative allosteric site was further refined by molecular dynamics. The principal component analysis led us to identify the protein motion that resulted in an inactive like conformation upon the allosteric inhibitor binding.

Highlights

The cyclic adenosine monophosphate is a universal second messenger that regulates many biological processes, including cell proliferation, differentiation, and apoptosis [1]
The superimposition of the EPAC2 crystal structure (PDB code: 4MGI) [49], bearing Rap interacting domain, with the EPAC1 model clearly showed that the areas 1 and 2 were filled by this domain
We observed that the area 1 involved 4 α helices of the CDC25-HD namely α1 (671–680), α3 (708–713), α5 (750–756) and α9 (839–844). This area bound the β4 and the loop linking β4 to α3 of the Rap domain. (Fig. S5) The area 2 involved the CDC25 Helical Hairpin (α9 and α10 from 823 to 854) that accommodated the loop between α1 and β2 of Rap [50]. (Fig. S5) The area 2 was reported to be potentially involved in an interaction between EPAC1 and RanPB2 [51]

Summary

Introduction

The cyclic adenosine monophosphate (cAMP) is a universal second messenger that regulates many biological processes, including cell proliferation, differentiation, and apoptosis [1]. The effects of cAMP in mammalian cells are mediated by at least three effector families: protein kinase A (PKA), exchange proteins activated by cAMP (EPAC) and ion channels bearing a cyclic nucleotide binding domains (CNBD). The EPAC proteins regulate a variety of physiological processes, such as calcium homeostasis in cardiomyocytes, insulin secretion from pancreatic β cells, integrin-mediated cell adhesion, and cell death [2]. Two EPAC isoforms have been identified, EPAC1 and EPAC2 which display distinct pattern of tissue expression. EPAC1 is ubiquitously expressed whereas EPAC2 and its slice variants are localized in the brain (EPAC2A), pancreatic cells (EPAC2B) testis and liver (EPAC2C) [4]

Methods

Results

Conclusion