Pyrimidine Triazole Thioether Derivatives as Toll‐Like Receptor 5 (TLR5)/Flagellin Complex Inhibitors

Abstract

Protein-protein interactions have been regarded as "undruggable" despite their importance in many biological processes. The complex formed between host toll-like receptor 5 (TLR5) and flagellin, a globular protein that is the main component of a bacterial flagellum, plays a vital role in a number of pathogen defenses, immunological diseases and cancers. Through high-throughput screening, we identified two hits with a common pharmacophore, which were used to successfully develop a series of small-molecule probes as novel inhibitors of flagellin binding to TLR5. In a multitude of assays, 4-((4-benzyl-5-(pyridin4yl)-4H-1,2,4-triazol-3-yl)thio)pyrido[3',2':4,5]thieno[3,2-d]pyrimidine (TH1020) was identified as a potent antagonist of TLR5 signaling with promising activity (IC50 =0.85±0.12 μm) and specificity. Furthermore, TH1020 was shown to repress the expression of downstream TNF-α signaling pathways mediated by the TLR5/flagellin complex formation. Based on molecular docking simulation, TH1020 is suggested to compete with flagellin and disrupt its association with TLR5. TH1020 provides a much-needed molecular probe for studying this important protein-protein interaction and a lead compound for identifying novel therapeutics targeting TLR5.

HMGB1 Activates Proinflammatory Signaling via TLR5 Leading to Allodynia

“…Control experiments confirmed that TH1020 also inhibited flagellin-induced TLR5 activation in HEK-hTLR5 cells as reported previously (Yan et al, 2016) with no apparent cytotoxicity (Figure S2G). Furthermore, NF-κB activation was inhibited by treating HMGB1 stimulated HEK-hTLR5 cells with triptolide, a small molecule inhibitor of NF-κB (Qiu et al, 1999) (Figure 2E).…”

“…First, an anti-TLR5 NAb was used to block direct activation by flagellin or HMGB1, followed by a small molecule inhibitor of MyD88 (Davis et al, 2006) and a peptide inhibitor of the TIRAP pathway (Brown and McIntyre, 2011) to target the downstream adaptor proteins (Figure 2). Finally, two more inhibitors, triptolide (NF-κB inhibitor) (Qiu et al, 1999) and TH1020 (TLR5 inhibitor) (Yan et al, 2016) were applied to decipher how the HMGB1-TLR5 signaling activates the NF-κB-driven proinflammatory pathway.…”

“…Upon TLR5 activation by HMGB1 the NF-κB p65 subunit translocates from the cytosol to the nucleus resulting in the transcription and release of proinflammatory cytokines and nitric oxide (NO) in the extracellular milieu of engineered and primary peripheral blood mononuclear cells (PBMCs). In PBMCs, both HMGB1 and flagellin (the classical TLR5 agonist) induced NO production and the effects were significantly suppressed by the co-application of TH1020, a TLR5-specific inhibitor (Yan et al, 2016), thereby further supporting a role for HMGB1 as a TLR5 agonist. Second, we characterized this interaction biophysically using fluorescence polarization and solution NMR spectroscopy, which revealed that the C-terminal acidic tail region of HMGB1 is critical for binding to the extra cellular domain of TLR5 in vitro .…”

Toll-like receptor 5-mediated IL-17C expression in intestinal epithelial cells enhances epithelial host defense against F4+ ETEC infection

“…As the upregulation of TLR 5 and -8 correlated with the increase in IL-17C production in both the porcine intestinal tissues and IPEC-J2 cells upon F4 + ETEC stimulation, in a next effort we wanted to determine which TLR controls IL-17C expression in IECs upon ETEC infection. Given the fact that TLR5 engagement elicits the expression of IL-17C in human IECs [23], we further elucidated IL-17C expression in the presence of the TLR5 signaling inhibitor TH1020 [22] and oligodeoxyribonucleotides (ODN) 2088, which inhibit TLR7/8/9 signaling [20, 21]. As TH1020 and ODN 2088 were dissolved in DMSO and TE buffer, respectively, the effect of these two solvents on IL-17C production by IPEC-J2 monolayers was also investigated.…”

“…Two hours before bacterial inoculation, IPEC-J2 monolayers were incubated with 0.5 μM oligodeoxyribonucleotide (ODN 2088, Miltenyi Biotec, Bergisch Gladbach, Germany; blocks TLR7/8/9 signaling as described previously [20, 21]), TH1020 (SML1741, Sigma-Aldrich, inhibits TLR5 signaling as described previously [22]), 20 or 40 ng/mL IL-17C (MyBioSource, San Diego, CA, USA) and then inoculated for 24 h with the different bacterial strains as described above. As flagellin has been reported to induce IL-17C production in several epithelial cells [7, 23, 24], IPEC-J2 monolayers were also stimulated with flagellin (100 ng/mL, FLA-ST Ultrapure, Invivogen, San Diego, CA, USA) in the presence or absence of TH1020.…”