Inhibition of TLR2 signaling by small molecule inhibitors targeting a novel putative pocket within the TLR2 TIR domain (INM9P.448)

Abstract

Overexuberant TLR2 signaling has been implicated in numerous diseases. Visual analysis of the TLR2 TIR domain crystal structure suggested the presence of a “pocket” adjacent to the highly conserved and functionally important proline and glycine residues of the BB loop. Given the importance of the BB loop in mediating TLR signal transduction, we hypothesized that Computer-Aided Drug Design (CADD) could be used to identify a small molecule inhibitor(s) that would fit within this “pocket” and blunt TLR2 signaling. Using CADD, ~150 small compounds were identified based on their predicted ability to bind in this pocket. Based on inhibition of IL-8 mRNA induced by TLR2 agonists, compound “C29” was found to inhibit TLR2/1 and TLR2/6 signaling in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages. C29 blocks heat-killed and live bacterial TLR2 agonist-induced proinflammatory cytokine mRNA. C29 prevents the early activation of TLR2-mediated signaling, including NF-κB and MAPKs, as well as MyD88 recruitment to TLR2 in THP-1 cells. C29 is cleavable and the byproduct, o-vanillin, reproduces comparable TLR2 inhibitory activity. O-vanillin covalently interacts with recombinant human TLR2 TIR domain dose-dependently. Mutagenesis of “pocket” residues revealed an indispensable role for TLR2/1, but not TLR2/6, signaling, suggesting divergent roles. Collectively, these results provide proof-of-principle for using CADD to identify inhibitors of TLR2 signaling.