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)

MUÑOZ MARCOS,; GUTIERREZ, LUCAS ; DELIGNAT, SANDRINE; ; RUSSICK, JULES ; GOMEZ MEJIBA, SANDRA;; DANIEL R ENRIZ; DARIO C. RAMIREZ

Congreso; FREE RADICAL B AND MEDICINE; 2018

Inflammatory activation of macrophages throughout toll-like Receptors(TLRs) is a fundamental step in the development of immune response triggeredby bacterial and fungal infections. TLR deregulated signaling has beenimplicated in a great number of inflammatory diseases, thus their inhibitionhas become a therapeutic target. Our group has studied the effects of thenitrone spin trap 5,5-dimethyl-1-pirroline N-oxide (DMPO) on lipopolysaccharide(LPS)-primed RAW264.7 cells at transcriptomic and functionallevels. Using microarray technology we found that the dampening effects ofDMPO could be explained by impaired signaling of TLR-2 and -4. BecauseTLRs have structural similarities in their intracellular domain responsible forsignal transduction called TIR, we hypothesize that the effects of DMPO inthese receptors are caused by a direct binding of the spin trap to their TIRdomain. Currently, TLR2-TIR domain is the only one crystallized, thereforewe use combined techniques of docking, molecular dynamics simulationsand QTAIM (Quantum Theory of Atoms In Molecules) calculations to determinethe interaction between DMPO and this receptor. Our results showthat DMPO could bind to four specific residues in a key region pointed asresponsible for signal transduction known as BB-loop. To functionally corroboratethese 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 proteinMyD88 we used co-immunoprecipitation to test whether DMPO can preventTLR2-MyD88 binding and found no effect. Taking together our results showthat DMPO blocks TLR2 signaling without preventing completely the couplingof MyD88 to its TLR2-TIR domain. This phenomena could be due toDMPO disrupting proper coupling of TLRs with MyD88 by direct binding tothe BB-loop region responsible for signal transduction. These data encourageDMPO derivatives as mechanism-based TLR inhibitors. Supported byPICT3369, PIP916 and PROICO-2018 to DCR and SEGMhttps://doi.org/10.1016/j.freeradbiomed.2018.10.260