Anti-inflammatory activity of small-molecule antagonists of Toll-like receptor 2 (TLR2) in mice

Toll-like receptor 2 (TLR2) is currently investigated as a potential therapeutic target in diseases with underlying inflammation like sepsis and arthritis. We reported the discovery, by virtual screening and biological testing, of eight TLR2 antagonists (AT1-AT8) which showed TLR2-inhibitory activity in human cells (Murgueitio et al., 2014). In this study, we have deepened in the mechanism of action and selectivity (TLR2/1 or TLR2/6) of those compounds in mouse primary cells and in vivo. The antagonists reduced, in a dose-dependent way the TNF alpha production (e.g. AT5 IC50 7.4 mu M) and also reduced the nitric oxide (NO) formation in mouse bone marrow derived macrophages (BMDM). Treatment of BMDM with the antagonists showed that downstream of TLR2, MAPKs phosphorylation and IkB alpha degradation was reduced. Notably, in a mouse model of tri-acylated lipopeptide (Pam3CSK4)-induced inflammation, AT5 attenuated the TNF alpha and IL-6 inflammatory response. Further, the effect of AT5 in the stimulation of BMDM by the endogenous alarmin HMGB1 was investigated. Our results indicate that AT4-AT7 and, particularly AT5 appear as good starting points for the development of inhibitors targeting TLR2 in inflammatory disorders.