Identification and validation of a novel dual small-molecule TLR2/8 antagonist

• Published in: Biochemical pharmacology 2020-07, Vol.177, p.113957, Article 113957
• Main Authors: Grabowski, Maria, Bermudez, Marcel, Rudolf, Thomas, Šribar, Dora, Varga, Péter, Murgueitio, Manuela S., Wolber, Gerhard, Rademann, Jörg, Weindl, Günther
• Format: Article
• Language: English
• Subjects: Benzothiazoles - chemistry; Benzothiazoles - pharmacology; Cell Survival - drug effects; Computer modeling; Dose-Response Relationship, Drug; Drug discovery; Dual antagonist; HEK293 Cells; Humans; Inflammation; Innate immunity; Interleukin-8 - metabolism; Macrophages - cytology; Macrophages - drug effects; Macrophages - metabolism; Molecular Structure; Non-competitive antagonism; Protein Multimerization - drug effects; Signal Transduction - drug effects; Small Molecule Libraries - chemistry; Small Molecule Libraries - pharmacology; THP-1 Cells; Toll-like receptor (TLR); Toll-Like Receptor 2 - antagonists & inhibitors; Toll-Like Receptor 2 - chemistry; Toll-Like Receptor 2 - metabolism; Toll-Like Receptor 8 - antagonists & inhibitors; Toll-Like Receptor 8 - chemistry; Toll-Like Receptor 8 - metabolism; Virtual screening
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/j.bcp.2020.113957

[Display omitted] •Endosomal- and surface-located TLRs can be targeted by a small-molecule antagonist.•Modeling-guided synthesis approach identified compound 24 as dual TLR2/8 antagonist.•Compound 24 potently and selectively reduced TLR2- and TLR8-mediated signaling.•Compound 24 showed characteristics of non-competitive antagonism at TLR2.•Computational docking studies predicted a plausible binding mode at TLR2 and TLR8. Toll-like receptor 2 (TLR2) and TLR8 are involved in the recognition of bacterial and viral components and are linked not only to protective antimicrobial immunity but also to inflammatory diseases. Recently, increasing attention has been paid to the receptor crosstalk between TLR2 and TLR8 to fine-tune innate immune responses. In this study, we report a novel dual TLR2/TLR8 antagonist, compound 24 that was developed by a modeling-guided synthesis approach. The modulator was optimized from the previously reported 1,3-benzothiazole derivative, compound 8. Compound 24 was pharmacologically characterized for the ability to inhibit TLR2- and TLR8-mediated responses in TLR-overexpressing reporter cells and THP-1 macrophages. The modulator showed high efficacy with IC50 values in the low micromolar range for both TLRs, selectivity towards other TLRs and low cytotoxicity. At TLR2, a slight predominance for the TLR2/1 heterodimer was found in reporter cells selectively expressing TLR2/1 or TLR2/6 heterodimers. Concentration ratio analysis in the presence of Pam3CSK4 or Pam2CSK4 indicated non-competitive antagonist behavior at hTLR2. In computational docking studies, a plausible alternative binding mode of compound 24 was predicted for both TLR2 and TLR8. Our results provide evidence that it is feasible to simultaneously and selectively target endosomal- and surface-located TLRs. We identified a small-molecule dual TLR2/8 antagonist that may serve as a valuable pharmacological tool to decipher the role of TLR2/8 co-signaling in inflammation.