253 - Inhibition of toll-like receptor (TLR-2) activity by the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) through direct interaction with the toll/interleukin-1 receptor domain (TIR)

Abstract

Inflammatory activation of macrophages throughout toll-like Receptors (TLRs) is a fundamental step in the development of immune response triggered by bacterial and fungal infections. TLR deregulated signaling has been implicated in a great number of inflammatory diseases, thus their inhibition has become a therapeutic target. Our group has studied the effects of the nitrone spin trap 5,5-dimethyl-1-pirroline N-oxide (DMPO) on lipopolysaccharide (LPS)-primed RAW264.7 cells at transcriptomic and functional levels. Using microarray technology we found that the dampening effects of DMPO could be explained by impaired signaling of TLR-2 and -4. Because TLRs have structural similarities in their intracellular domain responsible for signal transduction called TIR, we hypothesize that the effects of DMPO in these receptors are caused by a direct binding of the spin trap to their TIR domain. Currently, TLR2-TIR domain is the only one crystallized, therefore we use combined techniques of docking, molecular dynamics simulations and QTAIM (Quantum Theory of Atoms In Molecules) calculations to determine the interaction between DMPO and this receptor. Our results show that DMPO could bind to four specific residues in a key region pointed as responsible for signal transduction known as BB-loop. To functionally corroborate these results we used an experimental model based on hTLR2.6-expressing HEKs cells and observed that DMPO can block zymozan-triggered-TLR2-mediated NF-kb activation. Because TLRs bind to adaptor protein MyD88 we used co-immunoprecipitation to test whether DMPO can prevent TLR2-MyD88 binding and found no effect. Taking together our results show that DMPO blocks TLR2 signaling without preventing completely the coupling of MyD88 to its TLR2-TIR domain. This phenomena could be due to DMPO disrupting proper coupling of TLRs with MyD88 by direct binding to the BB-loop region responsible for signal transduction. These data encourage DMPO derivatives as mechanism-based TLR inhibitors. Supported by PICT3369, PIP916 and PROICO-2018 to DCR and SEGM