Experimental Anti-Inflammatory Drug Semapimod Inhibits TLR Signaling by Targeting the TLR Chaperone gp96

Abstract

Semapimod, a tetravalent guanylhydrazone, suppresses inflammatory cytokine production and has potential in a variety of inflammatory and autoimmune disorders. The mechanism of action of Semapimod is not well understood. Here we demonstrate that in rat IEC-6 intestinal epithelioid cells, Semapimod inhibits activation of p38 MAPK, NF-kB and induction of COX-2 by TLR ligands, but not by IL-1β or stresses. Semapimod inhibits TLR4 signaling (IC50≈0.3 μM) and acts by desensitizing cells to LPS; it fails to block responses to LPS concentrations of 5 μg/ml or higher. Inhibition of TLR signaling by Semapimod is almost instantaneous: the drug is effective when applied simultaneously with LPS. Semapimod blocks cell surface recruitment of the MyD88 adapter, one of the earliest events in TLR signaling. gp96, the ER-localized chaperone of the HSP90 family critically involved in the biogenesis of TLRs, was identified as a target of Semapimod using ATP-desthiobiotin pull-down and mass spectroscopy. Semapimod inhibits ATP-binding and ATPase activities of gp96 in vitro (IC50≈0.2-0.4 μM). On prolonged exposure, Semapimod causes accumulation of TLR4 and TLR9 in perinuclear space, consistent with ER retention, an anticipated consequence of impaired gp96 chaperone function. Our data indicate that Semapimod desensitizes TLR signaling via its effect on the TLR chaperone gp96. Fast inhibition by Semapimod is consistent with gp96 participating in high affinity sensing of TLR ligands in addition to its role as a TLR chaperone.

Regulation of dendritic cell function improves survival in experimental sepsis through immune chaperone

“…Deletion of gp96 in DCs is beneficial in both LPS-induced endotoxemia and CLP-Induced polymicrobial sepsis through a variety of mechanisms including blocking TLR signaling, inhibiting Wnt signaling, and increasing systemic IgA and IgG1. Therefore, targeting gp96 in DCs either genetically or pharmacologically 29,[52][53][54] may provide a potential novel approach for the sepsis treatment. Figure 5.…”

Retention of normal glia function by an isoform-selective protein kinase inhibitor drug candidate that modulates cytokine production and cognitive outcomes

“…However, previous studies with some p38αMAPK inhibitors that hit multiple kinases, such as VX-745 and CNI-1493, report inhibition of Aβ production or amyloid plaque deposition [28–30]. Whether the effects on these inhibitors on amyloid reflect engagement of targets other than p38αMAPK [31, 32], such as Abl [33], is not known. Further, a full evaluation of the differences is limited by the type of animal model used in efficacy and pharmacodynamic endpoint analyses.…”

Advances in paediatric gastroenterology

“…Thus, TLR-4 is a promising target for therapeutic intervention in necrotising enterocolitis,67 and clinical trials are being developed. 68 In view of the importance of the microbiota in stimulating the inflammatory cascade that leads to necrotising enterocolitis, numerous bacteria-derived and host-derived biomarkers have been investigated. Faecal calprotectin,69 urine alanine,70 and urine intestinal fatty acid-binding protein71 are examples of promising biomarkers; however, they have yet to be validated.…”