P10 – The Anti-inflammatory Drug Semapimod (CNI-1493) Blocks Responses to LPS by Inhibiting the ATPase Activity of the TLR Chaperone gp96

Author(s):
Anatoly Grishin, Rudolph Davis, Mary Beth Amrine, Jin Wang, Henri Ford, University of Southern California Keck School of Medicine

Background: Semapimod, an experimental anti-inflammatory drug, is effective against a variety of inflammatory disorders including necrotizing enterocolitis. The mechanism of action of Semapimod has not been unambiguously established. We have previously demonstrated that Semapimod blocks TLR4 signaling and interferes with the ATP-binding activity of the HSP90 family chaperone gp96, which plays a critical role in the biogenesis of Toll-like receptors.

Hypothesis: Semapimod blocks TLR4 signaling by inhibiting gp96.

Methods: Activation of p38 MAPK and NF-kB was examined by Western blotting with activation-specific antibodies. ATP-binding proteins were pulled down using ATP-desthiobiotin agarose. gp96 was identified using mass spectroscopy of tryptic digest and Western blotting with anti-gp96 antibody. ATPase activity of gp96 was measured using γ-32P ATP.

Results: Semapimod blocks LPS-induced activation of both p38 and NF-kB, indicating that the blockade is upstream in the TLR4 pathway. Moreover, this blockade is partially alleviated by high concentrations of LPS, which is consistent with Semapimod targeting the interaction between LPS and its cognate receptor. Semapimod also blocks gp96 pulldown by desthiobiotin-agarose and inhibits the ATPase activity of purified gp96 in a dose-dependent manner.

Conclusions: Semapimod blocks TLR4 interaction with LPS by inhibiting gp96. gp96 is an essential component of the TLR4 signaling complex.