KMU-1170, a Novel Multi-Protein Kinase Inhibitor, Suppresses Inflammatory Signal Transduction in THP-1 Cells and Human Osteoarthritic Fibroblast-Like Synoviocytes by Suppressing Activation of NF-κB and NLRP3 Inflammasome Signaling Pathway
Abstract
:1. Introduction
2. Results
2.1. Effect of KMU-1170 on the Activity of Various Protein Kinases
2.2. KMU-1170 Reduces LPS-Stimulated Upregulation of Inducible Nitric Oxide Synthase (iNOS) and Cyclooxygenase-2 (COX-2) in THP-1 Cells
2.3. KMU-1170 Inhibits LPS-Induced Proinflammatory Cytokine Production in THP-1 Cells
2.4. KMU-1170 Suppresses LPS-Induced Phosphorylation of TAK1 and MAPKs in THP-1 Cells
2.5. KMU-1170 Inhibits LPS-Induced Activation of NF-κB in THP-1 Cells
2.6. KMU-1170 Attenuates LPS-Induced Activation of NLRP3 in THP-1 Cells
2.7. KMU-1170 Inhibits LPS-Induced Inflammation in THP-1 Cells
2.8. KMU-1170 Suppresses LPS-Induced Inflammation in Osteoarthritic FLS
3. Discussion
4. Materials and Methods
4.1. Synthesis of KMU-1170
4.1.1. General Information
4.1.2. 5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)indolin-2-one (1)
4.1.3. 6-Chloro-N-cyclopropylpyrazin-2-amine (2)
4.1.4. 5-(6-(Cyclopropylamino)pyrazin-2-yl)indolin-2-one (3)
4.1.5. (Z)-3-((1H-Imidazol-5-yl)methylene)-5-(6-(cyclopropylamino)pyrazin-2-yl)indolin-2-one (KMU-1170)
4.2. Reagents
4.3. Cell Line and Culture
4.4. Isolation and Culture of Primary Human Osteoarthritic FLS
4.5. Kinase-Profiling Analysis
4.6. Cell Viability Assay
4.7. Western Blotting Analysis
4.8. RNA Isolation and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Real Time Quantitative PCR (qPCR)
4.9. Immunofluorescence Staining
4.10. Statistical Analysis
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
FLS | fibroblast-like synoviocyte |
LPS | lipopolysaccharide |
iNOS | inducible nitric oxide synthase |
COX | cyclooxygenase |
TLR | toll-like receptor |
IL | interleukin |
TNF | tumor necrosis factor |
NLRP3 | the NOD-, LRR- and pyrin domain-containing protein 3 |
ASC | the adaptor molecule apoptosis-associated speck-like protein containing a CARD |
JAK | Janus-associated kinase |
FDA | the US Food and Drug Administration |
BTK | Bruton’s tyrosine kinase |
MAPK | mitogen-activated protein kinase |
XTT | 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide |
TAK1 | transforming growth factor-β-activated kinase 1 |
IKK | inhibitor of NF-κB kinase |
OA | osteoarthritis |
HRP | horseradish peroxidase |
FBS | fetal bovine serum |
IRB | the Institutional Review Board |
DMEM | Dulbecco’s modified Eagle medium |
OD | optical density |
TBS | Tris-buffered saline |
RT-PCR | reverse transcription-polymerase chain reaction |
qPCR | quantitative PCR |
Ct | the threshold cycle number |
SPSS | the Statistical Package for Social Science |
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KMU-1170 1 μM | |
---|---|
Kinase | Activity in % * |
MAPK1 (h) | 2 |
Yes (h) | 23 |
Blk (h) | 20 |
Fgr (h) | 29 |
Lyn (h) | 13 |
Lck (h) | 7 |
TYK2 (h) | 6 |
JAK3 (h) | 3 |
Fyn (h) | 26 |
Itk (h) | 45 |
Syk (h) | 55 |
JAK2 (h) | 41 |
JNK1α1 (h) | 67 |
Hck (h) | 30 |
Pyk2 (h) | 67 |
cSRC (h) | 36 |
Bmx (h) | 26 |
Txk (h) | −7 |
Tec (h) activated | 41 |
SAPK2a (h) | 108 |
BTK (h) | 40 |
ZAP-70 (h) | 108 |
Primers | Sequences (5′ → 3′) | |
---|---|---|
iNOS | Forward | CTG TCT GGT TCC TAC GTC ACC |
Reverse | CCC ACG TTA CAT GGG AGG ATA | |
COX-1 | Forward | ACC TTG AAG GAG TCA GGC ATG AG |
Reverse | TGT TCG GTG TCC AGT TCC AAT A | |
COX-2 | Forward | ATC ACA GGC TTC CAT TGA CC |
Reverse | TAT CAT CTA GTC CGG AGG GG | |
IL-1β | Forward | CCT TGG GCC TCA AGG AAA A |
Reverse | CTC CAG CTG TAG AGT GGG CTT A | |
TNF-α | Forward | GGA GAA GGG TGA CCG ACT CA |
Reverse | CTG CCC AGA CTC GGC AA | |
IL-6 | Forward | ATG GCA CAG TAT CTG GAG GAG |
Reverse | TAA GCT GGA CTC ACT CTC GGA | |
β-actin | Forward | AAT CTG GCA CCA CAC CTT CTA |
Reverse | ATA GCA CAG CCT GGA TAG CAA |
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Baek, H.S.; Hong, V.S.; Kim, S.H.; Lee, J.; Kim, S. KMU-1170, a Novel Multi-Protein Kinase Inhibitor, Suppresses Inflammatory Signal Transduction in THP-1 Cells and Human Osteoarthritic Fibroblast-Like Synoviocytes by Suppressing Activation of NF-κB and NLRP3 Inflammasome Signaling Pathway. Int. J. Mol. Sci. 2021, 22, 1194. https://doi.org/10.3390/ijms22031194
Baek HS, Hong VS, Kim SH, Lee J, Kim S. KMU-1170, a Novel Multi-Protein Kinase Inhibitor, Suppresses Inflammatory Signal Transduction in THP-1 Cells and Human Osteoarthritic Fibroblast-Like Synoviocytes by Suppressing Activation of NF-κB and NLRP3 Inflammasome Signaling Pathway. International Journal of Molecular Sciences. 2021; 22(3):1194. https://doi.org/10.3390/ijms22031194
Chicago/Turabian StyleBaek, Hye Suk, Victor Sukbong Hong, Sang Hyon Kim, Jinho Lee, and Shin Kim. 2021. "KMU-1170, a Novel Multi-Protein Kinase Inhibitor, Suppresses Inflammatory Signal Transduction in THP-1 Cells and Human Osteoarthritic Fibroblast-Like Synoviocytes by Suppressing Activation of NF-κB and NLRP3 Inflammasome Signaling Pathway" International Journal of Molecular Sciences 22, no. 3: 1194. https://doi.org/10.3390/ijms22031194