Discovery of TLR2 antagonists as anti-inflammatory drugs

The contribution of Toll-like receptors (TLRs) to several inflammatory chronic diseases and to the pathogenesis of sepsis is well recognized. For this reason, modulation of the response to TLR ligands is a promising therapeutic tool in a broad spectrum of inflammatory diseases. In the proposed study we set up to discover new antagonists for TLR2. The recent resolution of the TLR2 crystal structure in complex with its co-receptors (TLR1/6) and with its designated lipopeptide ligands (Pam3CSK4/Pam2CSK4) has opened the door to directed ligand-search studies. Based on the chemical interactions of the lipopeptide binding site in TLR2/1 and TLR2/6, a 3D pharmacophore model will be developed. In silico virtual compound library screening of molecules with the required physico- chemical characteristics will significantly reduce the number of candidate molecules to be biologically tested. In vitro testing of the pre-selected molecules for inhibition of the TLR2 response to Pam3CSK4/Pam2CSK4 will provide experimental validation of the in silico predictions and lead to the refinement of the predictive model. A final set of antagonist-hits with anti-inflammatory activity on TLR2 stimulation will be selected and characterised in depth e.g: effect on the inflammatory cytokine response, IC50 determination, selectivity and binding affinity for TLR1 and TLR6, cytotoxicity and mechanism of action. In the future, the selected antagonist drug candidates will be used in preclinical mouse models for diseases with TLR2-mediated uncontrolled cytokine production such as group B streptococcus sepsis and atherosclerosis. The directed search for TLR2 antagonists here proposed has been successfully used for other receptors and enzymes however it is novel in the TLR receptor field. Overall, we believe that this study will set the ground for future discovery of novel drugs with anti-inflammatory activity.

The immune system and in particular the innate-immune receptors TLRs (Toll-like receptors) play a fundamental role in several inflammatory chronic diseases and in the pathogenesis of sepsis. Thus, modulation of the response to TLR ligands is a promising therapeutic tool in a broad spectrum of inflammatory diseases. In general, the ten described human TLRs recognize bacterial and viral-derived components. TLR2 binds di- and tri-acylated lipoproteins from Gram positive bacteria. In the proposed study, we did set up to discover new small-molecules that might bind TLR2 and modify its activity. This enterprise was facilitated by the available crystal structure of TLR2 in complex with its co-receptors (TLR1 and TLR6) and with two synthetic lipopeptide ligands (Pam3CSK4 and Pam2CSK4). Based on the chemical interactions of the lipopeptide binding site in TLR2-1 and TLR2-6, a 3D pharmacophore model was generated. In silico virtual screening of a library of compounds with the required physico-chemical characteristics reduced the number of compounds which could potentially bind TLR2 and that had to be experimentally tested. From those, two sets of compounds were selected: on one hand compounds that inhibited the activity of the TLR2-ligands Pam3CSK4/Pam2CSK4, and on the other hand compounds that increased their stimulatory activity. The data obtained within this project indicate that future drugs for inflammatory diseases with a TLR2 component might be developed from the first set of compounds. Besides, chemical modifications to improve the activity of the second group of compounds might result in vaccine adjuvants in dendritic-cell cancer vaccination.