TAK-242 (Resatorvid), a Small-Molecule Inhibitor of Toll-Like Receptor (TLR) 4 Signaling, Binds Selectively to TLR4 and Interferes with Interactions between TLR4 and Its Adaptor Molecules

Abstract

TAK-242 (resatorvid), a small-molecule-specific inhibitor of Toll-like receptor (TLR) 4 signaling, inhibits the production of lipopolysaccharide-induced inflammatory mediators by binding to the intracellular domain of TLR4. Cys747 in TLR4 has been identified previously as the binding site of TAK-242. However, the mechanism by which TAK-242 inhibits TLR4 signaling after binding to TLR4 remains unknown. The present study demonstrated, using coimmunoprecipitation, that TAK-242 interferes with protein-protein interactions between TLR4 and its adaptor molecules. Among 10 different human TLRs, TAK-242 selectively bound to TLR4. The time course of the inhibitory effect of TAK-242 on inflammatory mediator production corresponded to that of the binding of TAK-242 to TLR4. TAK-242 inhibited the association of TLR4 with Toll/interleukin-1 receptor domain-containing adaptor protein (TIRAP) or Toll/interleukin-1 receptor domain-containing adaptor protein inducing interferon-␤-related adaptor molecule (TRAM) in human embryonic kidney (HEK) 293 cells overexpressing TLR4, MD-2, and TIRAP or TRAM, respectively. TAK-242 inhibited the TIRAPmediated activation of nuclear factor B (NF-B) and the TRAM-mediated activation of NF-B and interferon-sensitive response element in HEK293 cells stably expressing TLR4, MD-2, and CD14. The activation of endogenous interleukin-1 receptor-associated kinase in RAW264.7 cells was also inhibited by TAK-242 treatment. These findings suggest that TAK-242 binds selectively to TLR4 and subsequently disrupts the interaction of TLR4 with adaptor molecules, thereby inhibiting TLR4 signal transduction and its downstream signaling events. This work proposes a novel paradigm of a small molecule capable of disrupting protein-protein interactions.

OPN‐a induces muscle inflammation by increasing recruitment and activation of pro‐inflammatory macrophages

“…In murine RAW264.7 macrophages, TAK‐242 inhibits TLR4 signalling by binding to the TIR domain (Cys747) of TLR4 to inhibit downstream signalling activation (Matsunaga et al . 2011). Our in vitro and in vivo profiling data suggest that OPN upregulates TNC expression.…”

Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease

“…We inhibited TLR4 signaling with TAK-242 (resatorvid), a specific small-molecule inhibitor of TLR4 signaling that selectively binds to Cys747 on the intracellular domain of TLR4 and interrupts interactions between TLR4 and its adaptor molecules. 34 TAK-242 inhibited heme-induced P-selectin and VWF expression on the surface of HUVECs (Figure 4A), indicating that heme induces the release of P-selectin and VWF via TLR4 signaling. …”

Platelets promote tumour metastasis via interaction between TLR4 and tumour cell-released high-mobility group box1 protein

“…Next, we determined the effect of TLR4 blockage on tumour metastasis. TAK-242, a cyclohexene derivative, is a novel smallmolecule compound that selectively inhibits TLR4 signalling 27 and has been shown effective in protecting mice against LPS-induced lethality 28 . Mice were administered with TAK-242, followed by tumour cell injection 1 h later.…”