Semapimod Reduces Intestinal Cell Inflammatory Signaling by Blocking ATPase Activity in Toll-like Receptor-4 Chaperone gp96 to Reduce Phosphorylation of p38 MAPKinase in a Cell Line model for Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a severe intestinal inflammatory disorder of newborns. Lipopolysaccharide-mediated pro-inflammatory signaling via p38 MAPK is one of the molecular hallmarks of intestinal inflammation in necrotizing enterocolitis. Semapimod (CNI-1493), an experimental guanylhydrazone drug, has a potent anti-inflammatory effect. Recent phase III clinical trials demonstrated effectiveness of semapimod in inflammatory conditions of the adult GI tract. Although several putative molecular targets of semapimod have been proposed, the mechanism of action of this drug remains unclear. Previously we demonstrated that semapimod blocks LPS-induced, but not IL-1b-induced activation of p38 in enterocytes. This suggested distinct upstream activation pathways for TLR ligands and inflammatory cytokines. Using an intestinal cell line model, we show that semapimod blocks TLR signaling by specifically targeting the ATPase domain of TLR4 chaperone gp96, and suppresses downstream phosphorylation of p38 MAPKinase.