CIO-IAA is a dual ETA/ETB receptor antagonist that suppresses endothelin-1-induced macrophage activation and inflammatory signaling.

Elevation of endothelin-1 (ET-1), a potent vasoactive and inflammatory peptide, occurs in several cardiovascular and metabolic disorders associated with macrophage activation and inflammation. Using integrated pharmacological, molecular dynamics simulation (MDS), functional, and cellular approach, we investigated the effects of 2-(6-chloro-1H-indol-3-yl)-2-oxoacetic acid (CIO-IAA) on ET receptor function and ET-1-induced macrophage activation. CIO-IAA inhibited ET-1-induced reactive oxygen species (ROS) production, NADPH oxidase 2 (Nox2) expression, TNF-α and IL-6 production, Mac-1 (CD11β) expression, macrophage adhesion to endothelial cells, and NF-κB p65 activation and nuclear translocation. Notably, conventional ETA/ETB receptor antagonism did not fully suppress several ET-1-induced inflammatory responses, revealing an antagonist-insensitive component under the experimental conditions used. CIO-IAA inhibited these residual responses, suggesting that its effects on macrophage activation extend beyond those reproduced by conventional ET receptor antagonists. In heterologous systems expressing ETAR or ETBR, CIO-IAA inhibited ET-1-evoked increases in intracellular Ca2+, while pressure myography demonstrated rightward shifts in ET-1 concentration-response curves without reducing maximal constriction, consistent with ETR antagonist activity. MDS supported stable CIO-IAA interactions within the orthosteric binding pockets of both ETRs. Collectively, these findings support dual ETAR/ETBR antagonist activity of CIO-IAA while identifying additional suppression of antagonist-insensitive ET-1-induced macrophage responses. The present data do not establish whether these residual responses are molecularly independent of ETAR/ETBR or whether NF-κB is a direct target of CIO-IAA. Rather, reduced NF-κB activation accompanies the broader anti-inflammatory effects of CIO-IAA and may represent a converging pathway downstream of ETR. These findings warrant further investigation of the mechanisms underlying this additional anti-inflammatory activity.