ABSTRACT
Toll-like receptor 4 (TLR4) recognizes lipopolysaccharide (LPS) and triggers the activation of myeloid differention factor 88 (MyD88) and the Toll/interleukin-1 receptor domain-containing adapter, inducing interferon-ß (TRIF)-dependent major downstream signaling pathways. To evaluate the therapeutic potential of 1-[5-methoxy-2-(2-nitrovinyl)phenyl]pyrrolidine (MNP), previously synthesized in our laboratory, its effect on signal transduction via the TLR signaling pathways was examined. Here, we investigated whether MNP modulates the TLR4 signaling pathways and which anti-inflammatory target in TLR4 signaling is regulated by MNP. MNP inhibited the activation of nuclear factor-κB (NF-κB) induced by LPS (TLR4 agonist), and it also inhibited the expression of cyclooxygenase-2 and inducible nitric oxide synthase. MNP inhibited LPS-induced NF-κB activation by targeting TLR4 dimerization in addition to IKKß. These results suggest that MNP can modulate the TLR4 signaling pathway at the receptor level to decrease inflammatory gene expression.
Subject(s)
Nitro Compounds/pharmacology , Protein Multimerization/drug effects , Pyrrolidines/pharmacology , Toll-Like Receptor 4/metabolism , Animals , Anti-Inflammatory Agents/pharmacology , Cells, Cultured , Cyclooxygenase 2/biosynthesis , Dose-Response Relationship, Drug , I-kappa B Kinase/antagonists & inhibitors , Lipopolysaccharides , Mice , NF-kappa B/biosynthesis , Nitric Oxide Synthase Type II/biosynthesis , Signal Transduction/drug effectsABSTRACT
Toll-like receptors (TLRs) play significant roles in recognizing the pathogen-associated molecular patterns that induce innate immunity, and subsequently, acquired immunity. In general, TLRs have two downstream signaling pathways, the myeloid differential factor 88 (MyD88)-dependent and toll-interleukin-1 receptor domain-containing adapter-inducing interferon-ß (TRIF)-dependent pathways, which lead to the activation of nuclear factor-kappa B (NF-κB) and interferon regulatory factor 3 (IRF3). 1-[5-methoxy-2-(2-nitrovinyl)phenyl]pyrrolidine (MNP) has been previously synthesized in our laboratory. To evaluate the therapeutic potential of MNP, its effect on signal transduction via the TLR signaling pathways was examined. MNP was shown to inhibit the activation of NF-κB and IRF3 induced by TLR agonists, as well as to inhibit the expression of cyclooxygenase-2, inducible nitric oxide synthase, and interferon inducible protein-10. MNP also inhibited the activation of NF-κB and IRF3 induced by the overexpression of downstream signaling components of the MyD88- or TRIF-dependent signaling pathways. These results suggest that MNP can modulate MyD88- and TRIF-dependent signaling pathways of TLRs, leading to decreased inflammatory gene expression.
Subject(s)
Nitro Compounds/pharmacology , Pyrrolidines/pharmacology , Toll-Like Receptors/agonists , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Chemokine CXCL10/metabolism , Cyclooxygenase 2/metabolism , HEK293 Cells , Humans , Immunity, Innate , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Mice , Myeloid Differentiation Factor 88/metabolism , NF-kappa B/metabolism , Nitric Oxide Synthase Type II/metabolism , Nitro Compounds/chemistry , Pyrrolidines/chemistry , RAW 264.7 Cells , Signal Transduction/drug effectsABSTRACT
Toll-like receptor 4 (TLR4) recognizes LPS and triggers the activation of the myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain-containing adapter, inducing interferon-ß (TRIF)-dependent major downstream signaling pathways. Previously, we presented biochemical evidence that 1-[4-Fluoro-2-(2-nitrovinyl)phenyl]pyrrolidine (FPP), which was synthesized in our laboratory, inhibits NF-κB activation induced by LPS. Here, we investigated whether FPP modulates the TLR4 downstream signaling pathways and what anti-inflammatory target in TLR4 signaling is regulated by FPP. FPP inhibited LPS-induced NF-κB activation by targeting TLR4 dimerization. These results suggest that FPP can modulate the TLR4 signaling pathway at the receptor level to decrease inflammatory gene expression.
Subject(s)
Lipopolysaccharides/pharmacology , Protein Multimerization/drug effects , Pyrrolidines/pharmacology , Toll-Like Receptor 4/metabolism , Vinyl Compounds/pharmacology , Animals , Cells, Cultured , HEK293 Cells , Humans , Mice , Molecular Structure , NF-kappa B/metabolism , Protein Binding/drug effects , Toll-Like Receptor 4/chemistryABSTRACT
When various pathogens invade a host, toll-like receptors (TLRs) play a significant role in recognizing the pathogen-associated molecular patterns carried by the pathogens to induce innate immune reaction, followed by acquired immunity reaction. TLRs have two downstream signaling pathways, the myeloid differential factor 88 (MyD88)-dependent and toll-interleukin-1 receptor domain-containing adapter inducing interferon-ß (TRIF)-dependent pathways. To evaluate the therapeutic potential of 1-[4-fluoro-2-(2-nitrovinyl)phenyl]pyrrolidine (FPP), previously synthesized in our laboratory, its effect on signal transduction via the TLR signaling pathways was examined. FPP inhibited the activation of nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3) induced by TLR agonists, as well as inhibited the expression of cyclooxygenase-2, inducible nitric oxide synthase, and interferon inducible protein-10. FPP also inhibited the activation of NF-κB and IRF3 when induced by the overexpression of downstream signaling components of the TLRs. As a result, FPP has potential to become a new therapeutic drug for many inflammatory diseases.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Inflammation/drug therapy , Pyrrolidines/therapeutic use , Vinyl Compounds/therapeutic use , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Cell Line , Chemokine CXCL10/genetics , Chemokine CXCL10/metabolism , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Down-Regulation/drug effects , Humans , Interferon Regulatory Factor-3/metabolism , Mice , NF-kappa B/metabolism , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Pyrrolidines/chemistry , Signal Transduction/drug effects , Toll-Like Receptors/agonists , Vinyl Compounds/chemistryABSTRACT
Toll-like receptors (TLRs) play an important role in the recognition of microbial pathogens and induce innate immune responses. The recognition of microbial components by TLRs triggers the activation of myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain-containing adapter inducing interferon-ß (TRIF)-dependent downstream signaling pathways. Previously, we synthesized (E)-1-(2-(2-nitrovinyl)phenyl)pyrrolidine (NVPP), which contains a nitrovinyl-phenyl and pyrrolidine. To evaluate the therapeutic potential of NVPP, its effect on signal transduction via the TRIF-dependent pathway of TLRs induced by lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid (poly[I:C]) was examined. NVPP inhibited LPS or poly[I:C]-induced activation of nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3), and the phosphorylation of IRF3, as well as inhibiting the activation of interferon-inducible genes such as interferon inducible protein-10 (IP-10). These results suggest that NVPP can modulate TRIF-dependent signaling pathways of TLRs, potentially resulting in effective therapeutics for chronic inflammatory diseases.
Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , Anti-Inflammatory Agents/pharmacology , Pyrrolidines/pharmacology , Signal Transduction/drug effects , Styrenes/pharmacology , Toll-Like Receptors/metabolism , Animals , Cell Line , Mice , Myeloid Differentiation Factor 88/metabolismABSTRACT
Toll-like receptors (TLRs) recognize many pathogen-associated molecular patterns and induce innate immunity. TLR signaling pathways induce the activation of various transcription factors, such as nuclear factor-κB (NF-κB), leading to the induction of pro-inflammatory gene products, such as inducible nitric oxide synthase (iNOS). Here, we investigated the effect of an (E)-1-(2-(2-nitrovinyl)phenyl)pyrrolidine (NVPP), previously synthesized in our laboratory, on inflammation by modulating NF-κB activation and iNOS expression induced by TLR agonists in murine macrophages. NVPP suppressed NF-κB activation and iNOS expression induced by lipopolysaccharide (TLR4 agonist), polyriboinosinic polyribocytidylic acid (TLR3 agonist), and macrophage-activating lipopeptide 2kDa (TLR2 and TLR6 agonist). All the results suggest that NVPP is suitable for development as a new anti-inflammatory drug.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Monocytes/drug effects , Nitric Oxide Synthase Type II/antagonists & inhibitors , Pyrrolidines/pharmacology , Styrenes/pharmacology , Toll-Like Receptors/antagonists & inhibitors , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Cell Line , Gene Expression Regulation/drug effects , Lipopeptides/pharmacology , Lipopolysaccharides/pharmacology , Mice , Monocytes/immunology , NF-kappa B/metabolism , Nitric Oxide/metabolism , Poly I-C/pharmacology , Pyrrolidines/chemical synthesis , Styrenes/chemical synthesis , Toll-Like Receptors/agonists , Transcriptional Activation/drug effectsABSTRACT
Toll-like receptors (TLRs) are pattern recognition receptors that recognize molecular structures derived from microbes and initiate innate immunity. TLRs have two downstream signaling pathways, the MyD88- and TRIF-dependent pathways. Dysregulated activation of TLRs is closely linked to increased risk of many chronic diseases. Previously, we synthesized fumaryl pyrrolidinone, (E)-isopropyl 4-oxo-4-(2-oxopyrrolidin-1- yl)-2-butenoate (IPOP), which contains a fumaric acid isopropyl ester and pyrrolidinone, and demonstrated that it inhibits the activation of nuclear factor kappa B by inhibiting the MyD88-dependent pathway of TLRs. However, the effect of IPOP on the TRIF-dependent pathway remains unknown. Here, we report the effect of IPOP on signal transduction via the TRIF-dependent pathway of TLRs. IPOP inhibited lipopolysaccharide- or polyinosinic-polycytidylic acid-induced interferon regulatory factor 3 activation, as well as interferon- inducible genes such as interferon inducible protein-10. These results suggest that IPOP can modulate the TRIF-dependent signaling pathway of TLRs, leading to decreased inflammatory gene expression.
Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , Butyrates/pharmacology , Fumarates/pharmacology , Pyrrolidinones/pharmacology , Signal Transduction/drug effects , Toll-Like Receptors/metabolism , Animals , Butyrates/chemistry , Fumarates/chemistry , Humans , Interferon Regulatory Factor-3/metabolism , Lipopolysaccharides/pharmacology , Luciferases/metabolism , Mice , NF-kappa B/metabolism , Poly I-C/pharmacology , Pyrrolidinones/chemistryABSTRACT
Toll-like receptors (TLRs) recognize molecular structures derived from microbes and initiate innate immunity. The stimulation of TLR4 by lipopolysaccharide (LPS) triggers the activation of the myeloid differential factor 88 (MyD88)-dependent and toll-interleukin-1 receptor domain-containing adapter inducing interferon-ß (TRIF)-dependent major downstream signaling pathways. Previously, we synthesized a fumaryl oxazolidinone derivative, 4-oxo-4-(2-oxo-oxazolidin-3-yl)-but-2-enoic acid ethyl ester (OSL07) and demonstrated that it inhibits activation of nuclear factor kappa B (NF-κB) by inhibiting the MyD88-dependent pathway of TLRs. TLR4 and the downstream signaling components are good therapeutic targets for many chronic inflammatory diseases. Here, it is investigated whether OSL07 modulates TLR4 downstream signaling pathways and what anti-inflammatory target in TLR4 signaling is regulated by OSL07. OSL07 inhibited LPS-induced NF-κB and interferon regulatory factor 3 activation by targeting TLR4 dimerization. These results suggest that OSL07 can modulate TLR4 signaling pathway leading to decreased inflammatory gene expression.