Restraining cAMP signaling via EPAC2 allosteric modulation: Conformational transitions and dynamic residue network analysis

Exchange Protein Activated by cAMP (EPAC) is a key cAMP-responsive signaling protein that regulates intracellular processes through activation of Rap-family small GTPases and plays important roles in cellular proliferation, metabolism, inflammation, and cardiovascular and neurological functions. Dysregulated EPAC signaling has been implicated in several pathological conditions, including metabolic disorders, cardiovascular diseases, cancer, and neurological dysfunction, making EPAC an increasingly attractive therapeutic target. EPAC2, one of the two major EPAC isoforms, is characterized by its distinct multi-domain architecture and its ability to switch its conformational state upon binding an analogue of cAMP. This conformational plasticity makes EPAC particularly attractive for allosteric inhibition. Drugs targeting EPACs' regulatory mechanisms, including their modulation with RAP-like proteins and associated pathologies, have therefore gained increasing attention. In this context, allosteric inhibitors represent a promising strategy for controlling EPAC activity without directly competing for cAMP binding. Here, we utilize an extensive 1.2 μs atomistic molecular dynamics simulation ensemble to elucidate the structural thermodynamics governing the transitions between the auto-inhibited (closed) and catalytically competent (open) states of EPAC2, specifically focusing on the mechanism of the natural allosteric inhibitor dimethylocta-dienyl methoxy flavone (AAD026). While the exogenous activator, Sp-cAMP, narrows conformational variance (43.5% captured by PC1 alone) to prime the protein for structural opening, the reference inhibitor ESI-05 achieves inhibition purely via localized suppression confined to the cyclic nucleotide-binding domain (cNBD-B) and adjacent hinge regions. In contrast, AAD026 acts as a genuine conformational reverser, broadening structural heterogeneity across a diverse, inactive-like free-energy landscape (Silhouette score 0.69). K- means clustering analysis and dynamic residue network analysis revealed similar regions of intra-protein communications and identified several motifs that specifically mediate the inhibitor-driven reversion of the open conformation. This global change is driven by a prominent topological reachability (ΔLi) spike localized at the cNBD-B lid region, which effectively re-sequesters the catalytic residue Arg886 back into the functional ionic latch (IL). Our findings provide quantitative, network-level evidence that allosteric inhibition can extend beyond simple binding-site obstruction, operating via an active, state-reverting mechanism that overrides active ensembles even in the presence of triggering ligands.