Novel TLR4-antagonizing peptides inhibit LPS-induced release of inflammatory mediators by monocytes.

Toll-like receptor 4 (TLR4) has become a new target for combating Gram-negative bacterium-induced sepsis. In this study, we screened peptides that can interact with TLR4 from a random 16-peptide library using yeast two-hybrid system and performed functional identification for the obtained peptides. We got two positive clones out of 1.28x10(7) transformants. The peptides were sequenced and synthesized. Protein sequence comparison confirmed that the two peptides had no homologous proteins. The two peptides were found to significantly inhibit LPS-induced NF-kappaB activation in HEK-293 cells that were transfected with TLR4 cDNA, LPS-induced IkappaBalpha (IkappaB alpha) phosphorylation and NF-kappaB activation in monocytes, and release of IL-1, IL-6, and TNF-alpha by monocytes. We further confirmed that the No. 9 peptide could bind to TLR4 extracellular domain, but the No. 24 peptide could not, suggesting that two novel peptides were identified as the antagonists of TLR4, which significantly inhibited the effects of endotoxin in vitro. The No. 9 peptide may function through binding to TLR4 extracellular domain. Our findings suggest a promising countermeasure against Gram-negative bacterium-induced sepsis.