Protein interaction, cytotoxic, transcriptomic and proteomic responses to structurally distinct EPAC1 activators in...

Area of Science:

• Pharmacology
• Molecular Biology
• Endothelial Cell Biology

Background:

• EPAC1 (Exchange protein directly activated by cAMP 1) is a key signaling molecule in endothelial cells.
• Development of specific EPAC1 activators is crucial for understanding and modulating endothelial function.
• Previous research identified initial EPAC1 activators, necessitating further characterization of novel compounds.

Purpose of the Study:

• To evaluate the efficacy, safety, and molecular effects of novel EPAC1 activators, PWO577 and SY007.
• To investigate the impact of these compounds on gene expression and protein levels in endothelial cells.
• To assess the potential of PWO577 and SY007 in modulating inflammatory signaling pathways.

Main Methods:

• In vitro assays for protein interaction, cytotoxicity, and EPAC1 activation.
• RNA sequencing (RNAseq) in Human Umbilical Vein Endothelial Cells (HUVECs).
• LC-MS-based proteomics and Western blotting to analyze protein expression.
• Assessment of STAT3 activation and downstream gene expression.

Main Results:

• PWO577 and SY007 are potent EPAC1 binders, well-tolerated in HUVECs at high concentrations and long incubation times.
• These compounds activate EPAC1 in U2OS cells and regulate ~11,000 shared genes in HUVECs with minimal off-target effects.
• Upregulation of endothelial activation markers (ICAM1, MMP1, CCL2) at the gene level, with only MMP1 increased at the protein level.
• Suppression of IL-6-induced STAT3 activation and downstream gene expression, including SOCS3, STAT3, IL6ST, and JAK3.

Conclusions:

• PWO577 and SY007 are effective, specific, and non-toxic EPAC1 activators with potential therapeutic applications.
• These compounds modulate key endothelial cell functions and inflammatory responses.
• Further optimization may reduce any remaining off-target effects for enhanced therapeutic utility.