Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain.

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TitleInhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain.
Publication TypeJournal Article
Year of Publication2015
AuthorsMistry, P, Laird, MHW, Schwarz, RS, Greene, S, Dyson, T, Snyder, GA, Xiao, TSam, Chauhan, J, Fletcher, S, Toshchakov, VY, Mackerell, AD, Vogel, SN
JournalProc Natl Acad Sci U S A
Date Published2015 Apr 28
KeywordsAnimals, Anti-Inflammatory Agents, Antioxidants, Benzaldehydes, Drug Design, Drug Evaluation, Preclinical, HEK293 Cells, Humans, Inflammation, Interleukin-8, Mice, Protein Structure, Tertiary, RNA, Messenger, Signal Transduction, Toll-Like Receptor 1, Toll-Like Receptor 2, Toll-Like Receptor 6

Toll-like receptor (TLR) signaling is initiated by dimerization of intracellular Toll/IL-1 receptor resistance (TIR) domains. For all TLRs except TLR3, recruitment of the adapter, myeloid differentiation primary response gene 88 (MyD88), to TLR TIR domains results in downstream signaling culminating in proinflammatory cytokine production. Therefore, blocking TLR TIR dimerization may ameliorate TLR2-mediated hyperinflammatory states. The BB loop within the TLR TIR domain is critical for mediating certain protein-protein interactions. Examination of the human TLR2 TIR domain crystal structure revealed a pocket adjacent to the highly conserved P681 and G682 BB loop residues. Using computer-aided drug design (CADD), we sought to identify a small molecule inhibitor(s) that would fit within this pocket and potentially disrupt TLR2 signaling. In silico screening identified 149 compounds and 20 US Food and Drug Administration-approved drugs based on their predicted ability to bind in the BB loop pocket. These compounds were screened in HEK293T-TLR2 transfectants for the ability to inhibit TLR2-mediated IL-8 mRNA. C16H15NO4 (C29) was identified as a potential TLR2 inhibitor. C29, and its derivative, ortho-vanillin (o-vanillin), inhibited TLR2/1 and TLR2/6 signaling induced by synthetic and bacterial TLR2 agonists in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages. C29 failed to inhibit signaling induced by other TLR agonists and TNF-α. Mutagenesis of BB loop pocket residues revealed an indispensable role for TLR2/1, but not TLR2/6, signaling, suggesting divergent roles. Mice treated with o-vanillin exhibited reduced TLR2-induced inflammation. Our data provide proof of principle that targeting the BB loop pocket is an effective approach for identification of TLR2 signaling inhibitors.

Alternate JournalProc. Natl. Acad. Sci. U.S.A.
PubMed ID25870276
PubMed Central IDPMC4418912
Grant ListAI018797 / AI / NIAID NIH HHS / United States
T32ES007263 / ES / NIEHS NIH HHS / United States
R37 AI018797 / AI / NIAID NIH HHS / United States
T32 ES007263 / ES / NIEHS NIH HHS / United States
R01 AI018797 / AI / NIAID NIH HHS / United States
R56 AI018797 / AI / NIAID NIH HHS / United States