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-β-related adaptor molecule (TRAM) in human embryonic kidney (HEK) 293 cells overexpressing TLR4, MD-2, and TIRAP or TRAM, respectively. TAK-242 inhibited the TIRAP-mediated activation of nuclear factor κB (NF-κB) and the TRAM-mediated activation of NF-κB 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.
Footnotes
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.110.068064.
↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- TLR
- Toll-like receptor
- LPS
- lipopolysaccharide
- IL
- interleukin
- IFN
- interferon
- TNF
- tumor necrosis factor
- TIR
- Toll/interleukin-1 receptor
- MyD88
- myeloid differentiation primary response gene 88
- TIRAP
- Toll/interleukin-1 receptor domain-containing adaptor protein
- TRIF
- Toll/interleukin-1 receptor domain-containing adaptor protein inducing interferon-β
- TRAM
- Toll/interleukin-1 receptor domain-containing adaptor protein inducing interferon-β-related adaptor molecule
- IRAK
- interleukin-1 receptor-associated kinase
- HEK
- human embryonic kidney
- TAK-242 (resatorvid)
- ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate
- NF-κB
- nuclear factor-κB
- ISRE
- interferon-sensitive response element
- Ab
- antibody
- mAb
- monoclonal antibody
- PAGE
- polyacrylamide gel electrophoresis
- HA
- hemagglutinin
- FCS
- fetal calf serum
- DMEM
- Dulbecco's modified Eagle's medium
- TK
- thymidine kinase
- BAY 11-7082
- 3-[(4-methylphenyl)sulfonyl]-(2E)-propenenitrile
- SB203580
- 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole
- U0126
- 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene
- SP600125
- 1,9-pyrazoloanthrone.
- Received August 9, 2010.
- Accepted September 29, 2010.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics