INHIBITION OF TOLL-LIKE RECEPTOR 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, ; RUSSICK, JULES ; SANDRA E GOMEZ MEJIBA; GUTIERREZ, LUCAS ; GOMEZ MEJIBA, SANDRA;; DARIO C RAMIREZ; DELIGNAT, SANDRINE; ; DANIEL R ENRIZ

Congreso; SAIC; 2018

INHIBITION OF TOLL-LIKE RECEPTOR 2 ACTIVITY BY THE NITRONE SPIN TRAP 5,5-DIMETHYL-1-PYRROLINE N-OXIDE (DMPO) THROUGH DIRECT INTERACTION WITH THE TOLL/INTERLEUKIN-1RECEPTOR DOMAIN (TIR)Muñoz Marcos, Gutierrez, Lucas Delignat, Sandrine; Russick, Jules Gomez Mejiba, Sandra; Enriz, DanielUniv Pierre et Marie Curie Paris 6 UMR S1138Universidad Nacional de San LuisInflammatory activation of macrophages throughoutToll-like Receptors (TLRs) is a fundamental step in thedevelopment of immune response triggered by bacterialand fungal infections. TLR4 and 2 deregulated signalinghas been implicated in a great number of inflammatorydiseases, thus their inhibition has become a therapeutictarget. Previously, we found that the nitrone spin trap5,5-dimethyl-1-pirroline N-oxide (DMPO) dampens lipopolysaccharide(LPS)- triggered TLR4 receptor signalingin RAW264.7 cells at transcriptomic and functionallevels. However its effect on TLR2 signaling remainsunclear. Because TLRs 4 and 2 have structural similaritiesin their intracellular domain responsible for signaltransduction called TIR, we hypothesize that the effectsof DMPO in these two receptors were caused by directbinding of the spin trap to their TIR domain. UnfortunatelyTLR4 TIR domain has not been crystallized, thereforewe use combined techniques of docking, molecular dynamicssimulations and QTAIM (Quantum Theory of AtomsIn Molecules) calculations to determine the interactionbetween DMPO and TLR2 TIR domain. Our resultsshow that DMPO could bind to four specific residues ina key region implicated in signal transduction known asBB-loop. To corroborate these results we used an experimentalmodel based on hTLR2.6-expressing HEKscells and determine that DMPO can block zymozan-triggered-TLR2-mediated NF-kb activation. Because TLRsbind to adaptor protein MyD88 (Myeloid differentiationprimary response 88) we used co-immunoprecipitationto test whether DMPO can prevent TLR2-MyD88 bindingand found no effect. Taking together our results show thatDMPO blocks TLR2 signaling without preventing completelythe coupling of adaptor protein to its TIR domain.This can be due to DMPO disrupting proper coupling ofTLRs with MyD88 by direct binding to BB-loop region responsiblefor signal transduction. These data encouragesthe use of DMPO derivatives as mechanism-basedTLR inhibitors. Supported by PIP916-PICT3369 to DCRand SEGM.