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

Article abstract of DOI:10.3390/ijms22031194

Protein kinases regulate protein phosphorylation, which are involved in fundamental cellular processes such as inflammatory response. In this study, we discovered a novel multi-protein kinase inhibitor, KMU-1170, a derivative of indolin-2-one, and investigated the mechanisms of its inflammation-inhibiting signaling in both THP-1 cells and human osteoarthritic fibroblast-like synoviocytes (FLS). We demonstrated that in THP-1 cells, KMU-1170 inhibited lipopolysaccharide (LPS)-induced upregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and, furthermore, suppressed LPS-induced phosphorylation of transforming growth factor- β-activated kinase 1, JNK, ERK, inhibitor of NF-κB kinase α/β (IKKα/α), and NF-κB p65 as well as nuclear translocation of NF-κB p65. Moreover, KMU-1170 suppressed LPS-induced upregulation of proinflammatory cytokines such as IL-1β, TNF-α, and IL-6, and, notably, inhibited LPS-induced upregulation of the NLRP3 inflammasome in THP-1 cells. Importantly, KMU-1170 attenuated LPS-mediated inflammatory responses in human osteoarthritic FLS, such as the upregulation of IL-1β, TNF-α, IL-6, iNOS, and COX-2 and the phosphorylation of IKKα/β and NF-κB p65. Collectively, these results suggest that KMU-1170 inhibits inflammatory signal transduction and could be developed as a potential anti-inflammatory agent.

Full text of DOI:10.3390/ijms22031194

International Journal of
Molecular Sciences
Article
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
Hye Suk Baek 1,†, Victor Sukbong Hong 2,†, Sang Hyon Kim 3,4, Jinho Lee 2,* and Shin Kim 1,4,
*
1
Department of Immunology, School of Medicine, Keimyung University, Daegu 42601, Korea;
thortiw@naver.com
Department of Chemistry, Keimyung University, Daegu 42601, Korea; victorh@kmu.ac.kr
Division of Rheumatology, Department of Internal Medicine, School of Medicine, Keimyung University,
Daegu 42601, Korea; mdkim9111@dsmc.or.kr
Institute of Medical Science, Keimyung University, Daegu 42601, Korea
Correspondence: jinho@kmu.ac.kr (J.L.); god98005@dsmc.or.kr (S.K.);
Tel.: +82-53-580-5183 (J.L.); +82-53-258-7359 (S.K.);
2
3
4
*
Fax: +82-050-4154-2213 (J.L.); +82-53-258-7355 (S.K.)
†
These authors contributed equally.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Protein kinases regulate protein phosphorylation, which are involved in fundamental
cellular processes such as inflammatory response. In this study, we discovered a novel multi-protein
kinase inhibitor, KMU-1170, a derivative of indolin-2-one, and investigated the mechanisms of
its inflammation-inhibiting signaling in both THP-1 cells and human osteoarthritic fibroblast-like
synoviocytes (FLS). We demonstrated that in THP-1 cells, KMU-1170 inhibited lipopolysaccha-
ride (LPS)-induced upregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2
(COX-2), and, furthermore, suppressed LPS-induced phosphorylation of transforming growth factor-
Citation: 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/ijms
22031194
β
-activated kinase 1, JNK, ERK, inhibitor of NF-
nuclear translocation of NF- B p65. Moreover, KMU-1170 suppressed LPS-induced upregulation
of proinflammatory cytokines such as IL-1 , TNF- , and IL-6, and, notably, inhibited LPS-induced
κB kinase α/β (IKKα/β), and NF-κB p65 as well as
κ
β
α
upregulation of the NLRP3 inflammasome in THP-1 cells. Importantly, KMU-1170 attenuated
LPS-mediated inflammatory responses in human osteoarthritic FLS, such as the upregulation of
IL-1β, TNF-α, IL-6, iNOS, and COX-2 and the phosphorylation of IKKα/β and NF-κB p65. Collec-
tively, these results suggest that KMU-1170 inhibits inflammatory signal transduction and could be
developed as a potential anti-inflammatory agent.
Received: 7 January 2021
Accepted: 22 January 2021
Published: 26 January 2021
Keywords: KMU-1170; protein kinase; lipopolysaccharide; inflammation; NF-κB; NLRP3 inflamma-
some; osteoarthritis; fibroblast-like synoviocyte
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iations.
1. Introduction
Inflammation is a crucial and fundamental immune system response in the body that
protects the host from harmful stimuli and maintains tissue homeostasis [ ]. Although an
1,2
Copyright:
© 2021 by the authors.
effective inflammatory response allows survival in the case of infection or injury, excessive
or sustained inflammation can lead to diverse pathological conditions, such as asthma,
cancer, chronic pain, gout, mental health disorders, nervous breakdown, psoriasis, rheuma-
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This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
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4.0/).
toid arthritis, and vasculitis [3]. Moreover, it is equally important that the inflammatory
response to a noxious stimulus is terminated when the stimulus is removed [
3]. There-
fore, the balance between an effective inflammatory response and the anti-inflammatory
response is one of the most important conditions for maintaining a healthy state.
Int. J. Mol. Sci. 2021, 22, 1194. https://doi.org/10.3390/ijms22031194
https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, 1194
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Toll-like receptors (TLRs) constitute a family of critical receptors in the innate immune
system that recognize various pathogens and damaged tissues [4]. TLRs are divided
into several types according to their specific characteristics, such as structure, subcellular
localization, and recognized molecular properties. Lipopolysaccharide (LPS), a bacterial
endotoxin that is a component of the outer wall of gram-negative bacteria, is recognized
by TLR4 [
through the transcription factor NF-
immune signaling molecules called cytokines [
interleukin (IL)-1 , IL-6, and tumor necrosis factor-
5
]. When LPS binds to TLR4, inflammation-related gene expression is regulated
B [ ]. Inflammation is controlled by water-soluble
], and inflammatory cytokines such as
(TNF- ) are induced by LPS [ ].
κ
6
7
β
α
α
8
These cytokines are involved in various inflammatory diseases and inhibiting them can
potently help treat inflammatory diseases.
Inflammasome, a complex of inflammatory molecules, is an innate immune response
to pathogenic infections and plays an important role in the formation of proinflammatory
cytokine [9]. Among them, the NOD-, LRR- and pyrin domain-containing protein 3
(NLRP3) inflammasome function has been studied a lot and it is composed of essential
regulatory proteins NLRP3, the adaptor molecule apoptosis-associated speck-like protein
containing a CARD (ASC), procaspase-1, and NIMA-related kinase 7 [10]. After the
transcription and translation of NLRP3 and IL-1β by NF-κB, active caspase-1 can process
pro-IL-1 into its mature secreted form [11]. Therefore, it is very important to effectively
β
regulate NLRP3 inflammasome-related signaling in inflammatory diseases.
Protein kinase is responsible for phosphorylation of protein, which plays a critical role
in intracellular signaling of inflammation [12]. Protein kinases are very important drug
targets in drug development for the treatment of inflammatory diseases [13]. For example,
tofacitinib, a potent Janus-associated kinase (JAK) 1 and JAK3 antagonist developed by
Pfizer, reduced signs and symptoms of rheumatoid arthritis (RA) and improved physical
function of patients with RA [14]. Moreover, semapimod, a p38 kinase inhibitor improved
clinical outcome in patient with Crohn’s disease [15]. A spleen tyrosine kinase inhibitor
BAY 61-3606 was approved by the US Food and Drug Administration (FDA) of indication
for patients with allergic asthma [16]. Additionally, various Bruton’s tyrosine kinase (BTK)
inhibitors are in clinical trials [17]. However, promising protein kinase inhibitors still need
to be developed.
In this study, we developed a novel multi-protein kinase inhibitor, KMU-1170, a
derivative of indolin-2-one, and evaluated the anti-inflammatory effect of KMU-1170 in the
TLR4-NF-κB-NLRP3 signaling pathway by using the human monocyte cell line THP-1 and
primary human osteoarthritic fibroblast-like synoviocytes (FLS).
2. Results
2.1. Effect of KMU-1170 on the Activity of Various Protein Kinases
KMU-1170 was synthesized from 5-bromoindoline-2,3-dione as follows (Figure 1A):
Compound
of compound
1
was obtained through reduction and borylation, and Suzuki coupling reaction
with a pyrazine derivative, compound , yielded compound . Lastly,
1
2
3
Knoevenagel condensation with 1H-imidazole-4-carbaldehyde generated KMU-1170. The
kinase assay results showed that KMU-1170 inhibited numerous kinases associated with
inflammatory reactions, with particularly strong inhibition for mitogen-activated protein
kinase 1 (MAPK1), Lck, TYK2, JAK3, and Txk (Table 1).

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Figure 1. Discovery of KMU-1170, and the effect of KMU-1170 on LPS-induced iNOS and COX-2 in THP-1 cells. (A) Synthetic
scheme for the synthesis of KMU-1170. Reagents and experimental conditions: (a) (i) NH2NH2, NaOH, EtOH; (ii) bis
(pincholato)diboron, PdCl2(dppf), KOAc, 1,4-dioxane: EtOH(1:1.5), microwave; (b) K2CO3, cyclopropylamine, DMF; (c)
PdCl2(PPh3)2, 2M K2CO3, 1,4-dioxane: EtOH (1:1.5), microwave; (d) piperidine, 1H-imidazole-4-carbaldehyde, EtOH,
microwave. (
macrophages for 24 h using PMA (100 nM), then the cells were treated with different doses of KMU-1170 (0.01, 0.1, 0.5,
1, 5, and 10 M) for 24 h. ( ) Cells were treated with LPS (1 g/mL) for 6 h after pretreatment with different doses of
KMU-470 (0.1, 0.5, and 1 M) for 1 h. Total RNA was extracted, and used to determine the mRNA expression levels of iNOS,
COX-1, and COX-2 ( , respectively). Whole cell lysates were isolated and used to measure the protein expression levels
-actin by Western blot analysis ( ). Image-J software was used to analyze the relative optical density of the
). Whole cell lysates were isolated and used to measure the protein expression levels of COX-1, COX-2, and
). Image-J software was used to analyze the relative optical density of the COX-1 and
Cox-2 band (G). † p < 0.05, * p < 0.01, and # p < 0.001 compared to LPS alone.
B) Cytotoxic effect of KMU-1170 in THP-1 cells was performed by XTT assay. Cell were differentiated into
µ
C
–
I
µ
µ
C,H,I
of iNOS and
iNOS band (
β
D
E
β-actin by Western blot analysis (F

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Table 1. Kinase activity of 15 protein kinases upon treatment with 1 µM KMU-1170.
KMU-1170 1 µM
Kinase
Activity in % *
MAPK1 (h)
Yes (h)
Blk (h)
Fgr (h)
Lyn (h)
2
23
20
29
13
7
Lck (h)
TYK2 (h)
JAK3 (h)
Fyn (h)
Itk (h)
Syk (h)
JAK2 (h)
JNK1α1 (h)
Hck (h)
Pyk2 (h)
cSRC (h)
Bmx (h)
6
3
26
45
55
41
67
30
67
36
26
−7
41
108
40
108
Txk (h)
Tec (h) activated
SAPK2a (h)
BTK (h)
ZAP-70 (h)
* Values were obtained from the KinaseProfilerTM project of Eurofins.
2.2. KMU-1170 Reduces LPS-Stimulated Upregulation of Inducible Nitric Oxide Synthase (iNOS)
and Cyclooxygenase-2 (COX-2) in THP-1 Cells
To investigate the toxicity of KMU-1170 in THP-1 cells, 2,3-bis-(2-methoxy-4-nitro-5-
sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay was used. The result revealed
that KMU-1170 exhibited no toxicity up a concentration of 1
µM (Figure 1B). Notably,
pretreatment of cells with 1 M KMU-1170 inhibited LPS-induced upregulation of iNOS
µ
mRNA and protein (Figure 1C–E), and the pretreatment also inhibited LPS-induced upreg-
ulation of COX-2 mRNA and protein but not COX-1 mRNA/protein (Figure 1F–I).
2.3. KMU-1170 Inhibits LPS-Induced Proinflammatory Cytokine Production in THP-1 Cells
To evaluate the anti-inflammatory mechanism of action of KMU-1170, we examined
the regulation of the levels of proinflammatory cytokines such as IL-1
β, TNF-
α, and IL-6.
Pretreatment of cells with 1
µM KMU-1170 inhibited LPS-induced upregulation of IL-1β,
TNF-α, and IL-6 (Figure 2).

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Figure 2. Inhibitory effect of KMU-1170 on LPS-induced upregulation of proinflammatory cytokines in THP-1 cells. Cells
were differentiated into macrophages for 24 h using PMA (100 nM). And then the cells were treated with LPS (1 g/mL) for
6 h after pretreatment with different doses of KMU-1170 (0.1, 0.5, and 1 M) for 1 h. ( ) Whole cell lysates were isolated
and used to measure the protein expression levels of pro-IL-1 , TNF- , IL-6, and -actin by Western blot analysis. (
Image-J software was used to analyze the relative optical density of the pro-IL-1 , TNF- , and IL-6 band, respectively. (
Total RNA was extracted and used to determine the mRNA expression levels of IL-1 , TNF-
Image-J software was used to analyze the relative optical density of the IL-1 , TNF- , and IL-6 band, respectively. (
, TNF-
µ
µ
A
β
α
β
B
C
D
G
)
)
)
)
β
α
β
α
, and IL-6 using RT-PCR. (
β
α
E–
Total RNA was extracted and used to determine the mRNA expression levels of IL-1
β
α
, and IL-6 using real time PCR.
† p < 0.05, * p < 0.01, and # p < 0.001 compared to LPS alone.
2.4. KMU-1170 Suppresses LPS-Induced Phosphorylation of TAK1 and MAPKs in THP-1 Cells
Next, we investigated whether KMU-1170 affects TLR4 and MyD88 expression. Pre-
treatment of THP-1 cells with 1 µM KMU-1170 did not attenuate LPS-induced upregu-
lation of TLR4 and MyD88 (Figure 3A,B). To identify the potential molecular targets of
KMU-1170 in inflammation, we examined the phosphorylation of transforming growth
factor-β-activated kinase 1 (TAK1), which regulates MAPKs and the function of NF-κB.
Pretreatment of KMU-1170 inhibited LPS-induced phosphorylation of TAK1 in THP-1 cells
(Figure 3C,D). Among MAPKs, only ERK and JNK were phosphorylated in response to
LPS, and pretreatment with 1
µM KMU-1170 suppressed LPS-induced phosphorylation of
ERK and JNK in THP-1 cells (Figure 3E,F).

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Figure 3. Effect of KMU-1170 on LPS-TLR4 signaling pathway-related proteins in THP-1 cells. (
A
,
C
) Cells were differentiated
g/mL) for 6 h after pretreatment
M) for 1 h. Whole cell lysates were isolated and used to measure the
-actin by Western blot analysis ( ). ( ) Image-J software was used to analyze
the relative optical density of the TLR4 and MyD88 band. Whole cell lysates were isolated and used to measure the protein
expression levels of p-TAK1, TAK1, and -actin by Western blot analysis ( ). ( ) Image-J software was used to analyze the
relative optical density of the p-TAK1 band. ( ) Cells were differentiated into macrophages for 24 h using PMA (100 nM),
then the cells were treated with or without 1 M KMU-1170 for 24 h and stimulated with LPS (1 g/mL) at the indicated
times. Whole cell lysates were isolated and used to measure the protein expression levels of p-ERK, ERK, p-JNK, JNK,
p-p38, p38, and -actin by Western blot analysis. ( ) Image-J software was used to analyze the relative optical density of the
p-ERK, p-JNK, and p-p38 band. # p < 0.001 compared to LPS alone.
into macrophages for 24 h using PMA (100 nM). And then the cells were treated with LPS (1
with different doses of KMU-1170 (0.1, 0.5, and 1
protein expression levels of TLR, MyD88, and
µ
µ
β
A
B
β
C
D
E
µ
µ
β
F
2.5. KMU-1170 Inhibits LPS-Induced Activation of NF-κB in THP-1 Cells
LPS induced the phosphorylation of inhibitor of NF- B kinase
B p65 in THP-1 cells, but pretreatment with KMU-1170 inhibited their LPS-induced
phosphorylation (Figure 4A,B). We also investigated whether KMU-1170 affects the nuclear
translocation of NF- B p65 by examining cells using fluorescence microscopy, which
κ
α/β (IKKα/β) and
NF-κ
κ

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revealed that KMU-1170 pretreatment suppressed LPS-induced nuclear translocation of
NF-κB p65 in THP-1 cells (Figure 4C).
Figure 4. Inhibitory effect of KMU-1170 on LPS-induced phosphorylation of IKK
of NF- B in THP-1 cells. Cells were differentiated into macrophages for 24 h using PMA (100 nM). And then the cells were
treated with LPS (1 g/mL) for 6 h after pretreatment with the indicated concentrations of KMU-1170 for 1 h. ( ) Whole cell
lysates were isolated and used to measure the protein expression levels of p-IKK , IKK , p-NF- B p65, NF- B p65,
α/β and NF-κB, and nuclear translocation
κ
µ
A
α
/
β
α
/
β
κ
κ
and
and p-NF-
blue) and captured at
# p < 0.001 compared to LPS alone.
β
-actin by Western blot analysis. (
B
) Image-J software was used to analyze the relative optical density of the p-IKK
α
/
β
0
κ
B p65 band. (
C
) Cells were stained with antibodies to the NF-
κ
B p65 (green) and 4 ,6-diamidino-2-phenylindole
(
×
200 using fluorescence microscope. Arrows indicate 400 times magnification. * p < 0.01 and
2.6. KMU-1170 Attenuates LPS-Induced Activation of NLRP3 in THP-1 Cells
We next investigated whether KMU-1170 affects NLRP3 inflammasome signaling.
Pretreatment of THP-1 cells with 1 M KMU-1170 inhibited LPS-induced cytosolic release of
pro-IL-1 and IL-1 (Figure 5A). Moreover, pretreatment with 1
the upregulation of NLRP3, ASC, pro-caspase-1, and pro-IL-1
µ
β
β
µ
M KMU-1170 attenuated
induced by LPS and by
β
co-treatment with LPS and ATP (Figure 5A). Our quantified results showed that KMU-1170
significantly inhibited NLRP3 inflammasome activity (Figure 5B).

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Figure 5. Inhibitory effect of KMU-1170 on LPS-induced activation of NLRP3 inflammasome and anti-inflammatory
potentiality of KMU-1170 in THP-1 cells. ( ) Cells were differentiated into macrophages for 24 h using PMA (100 nM). And
then the cells were treated with LPS (1 g/mL) and/or ATP (1 mM) for 6 h after pretreatment with 1 M KMU-1170 for 1
h. Cell lysate (Lysate) and media supernatant (Sup) were isolated and used to measure the protein expression levels of
pro-IL-1 and IL-1 for the Sup as well as NLRP3, ASC, pro-caspase-1, pro-IL-1 , and -actin for the Lysate by Western
blot analysis. ( ) Image-J software was used to analyze the relative optical density of the NLRP3, ASC, pro-caspase-1, and
pro-IL-1 band in the Lysate. ( ) Cells were differentiated into macrophages for 24 h using PMA (100 nM). And then
the cells were treated with LPS (1 g/mL) for 6 h after pretreatment with KMU-1170 (1 M), celecoxib (25 M), ibrutinib
(5 M), luxolitinib (50 M), and metotrexate (1 M) for 1 h. Whole cell lysates were isolated and used to measure the protein
expression levels of pro-IL-1 and -actin by Western blot analysis. ( ) Image-J software was used to analyze the relative
A
µ
µ
β
β
β
β
B
β
C
µ
µ
µ
µ
µ
µ
β
β
D
optical density of the pro-IL-1β band. † p < 0.05, * p < 0.01, and # p < 0.001 compared to LPS alone.
2.7. KMU-1170 Inhibits LPS-Induced Inflammation in THP-1 Cells
Considering the action of KMU-1170 as an anti-inflammatory agent, we analyzed
the potential superiority of the anti-inflammatory efficacy of KMU-1170 by comparing it
with various other anti-inflammatory drugs. At 1 M, KMU-1170 significantly suppressed
LPS-induced increase of IL-1 protein in THP-1 cells, whereas this effect was not observed
µ
β
even at higher concentrations of other tested drugs, such as celecoxib (COX-2 inhibitor),
ibrutinib (BTK inhibitor), ruxolitinib (JAK inhibitor) and methotrexate (Anti-inflammatory
drug of RA) (Figure 5C,D). Only 1 µM methotrexate inhibited LPS-induced upregulation
of IL-1β to a similar degree as KMU-1170 did (Figure 5C,D).
2.8. KMU-1170 Suppresses LPS-Induced Inflammation in Osteoarthritic FLS
Lastly, we investigated whether KMU-1170 inhibits inflammatory signal transduction
in osteoarthritis (OA) using human osteoarthritic FLS. We obtained human osteoarthritic
FLS from the synovial tissues of patients with knee OA. Pretreatment of FLS with KMU-

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1170 inhibited LPS-induced upregulation of IL-1
β, TNF-
α
, and IL-6 mRNA and protein
levels (Figure 6A–D) and iNOS and COX-2 protein levels (Figure 6E). Furthermore, the
KMU-1170 pretreatment inhibited LPS-induced phosphorylation of IKK
α/β and NF-κB
p65 in FLS (Figure 6F).
Figure 6. Inhibitory effect of KMU-1170 on LPS-mediated inflammatory response in primary human osteoarthritic FLS.
Cells were treated with LPS (1 g/mL) for 6 h after pretreatment with different doses of KMU-1170 (0.1, 0.5, and 1 M) for
1 h. ( ) Total RNA was extracted and used to determine the mRNA expression levels of IL-1 , TNF- , and IL-6 using
real time PCR. ( ) Whole cell lysates were isolated and used to measure the protein expression levels of pro-IL-1 , TNF-
IL-6, and -actin by Western blot analysis. ( ) Whole cell lysates were isolated and used to measure the protein expression
levels of iNOS, COX-2, and -actin by Western blot analysis. ( ) Whole cell lysates were isolated and used to measure
-actin by Western blot analysis. * p < 0.01 and # p < 0.001
µ
µ
A
–C
β
α
D
β
α,
β
E
β
F
the protein expression levels of p-IKK
compared to LPS alone.
α/β, p-NF-κB p65, and β
3. Discussion
Protein kinases are enzymes that phosphorylates protein molecules by attaching a
phosphoryl group to serine, threonine, or tyrosine residue, and binding with ATP is es-
sential for kinase activity [18]. Accumulating evidence has recently indicated that protein
kinases are implicated in various human disease including cancer and inflammatory dis-
eases [12,19], resulting in development of the FDA-approved protein kinase inhibitors [20].
In this study, we discovered a novel multi-target protein kinase inhibitor KMU-1170 and
identified its anti-inflammatory mechanisms in both THP-1 cells and primary human
osteoarthritic FLS.
KMU-1170, indolin-2-one derivative, was found during the screening of the in-house
protein kinase inhibitor library for anti-inflammatory activities. Indolin-2-one is regarded
as a privileged scaffold for the discovery of kinase inhibitors [21]. Antitumor drugs such as
sunitinib and nintedanib are representative kinase inhibitors that were developed using
the indolin-2-one scaffold [21]. A series of indolin-2-one analogues were synthesized based
on a de novo design for the inhibitor of mitogen activated protein kinase 1 (MAPK1).
Indolin-2-one was designed to bind at the ATP binding pocket of MAPK1 using two
H-bond interactions. Two substituents were attached at the 3- and 5-postion of indolin-
2-one to increase binding affinity: pyrazine at the 5-positon for the interaction with the
ε
-amino group of Lys54, imidazole at the 3-position for the H-bond interaction with the
backbone carbonyl group of Met108 of MAPK1 (PDB code: 3W55). The introduction of
cyclopropylamine at the 5-position of pyrazine yielded KMU-1170. The novel compound

Int. J. Mol. Sci. 2021, 22, 1194
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was found to strongly inhibit the activities of inflammation-associated protein kinases
(Table 1).
A critical mediator of inflammatory responses is iNOS, which belongs to a family of
catalytic enzymes is necessary for nitric oxide (NO) production [22]. Because excessive NO
produced by iNOS is involved in various pathophysiological conditions [23], iNOS activ-
ity/level must be controlled. We found that KMU-1170 inhibited LPS-induced upregulation
of iNOS in THP-1 cells (Figure 1C,D). COX, which is composed of two isozymes, COX-1
and COX-2, is associated with diverse human diseases, such as inflammatory diseases,
neoplastic diseases, and Alzheimer’s disease [24]. Selective inhibition of COX-2 represents
a breakthrough therapeutic strategy that has been found to control inflammation and pain
while producing few side effects such as gastrointestinal irritation [24]. Therefore, we
investigated the inhibitory effect on COX-2 in both THP-1 cells and human osteoarthritic
FLS, and found that KMU-1170 selectively suppressed LPS-induced upregulation of COX-2
in these cells (Figure 1F,G and Figure 6E).
TAK1 is associated with various cellular processes, including cell death, differentiation,
immunity, inflammation, and survival [25
–
27]. Notably, TAK1 acts upstream of NF-
κB
and MAPKs in LPS-TLR4 signaling-mediated inflammation process [28
,29]. Additionally,
phosphorylation of TAK1 at Ser412 is critical for LPS-TLR4-TAK1 signaling-mediated pro-
duction of proinflammatory cytokines [30]. MAPK pathways are widely recognized to play
critical roles in TLR-mediated inflammatory responses, such as the production of proin-
flammatory cytokines stimulated by LPS [31]. Here, we found that KMU-1170 suppressed
LPS-induced phosphorylation of TAK1, ERK, and JNK in THP-1 cells (Figure 3C–F).
The transcription factor NF-
regulates key factors, such as cytokines and the inflammasome, in an inflammatory re-
sponse [31]. NF- B protein is sequestered in the cytoplasm through its binding to inhibitor
of NF- B (I B) [31]. Moreover, in the canonical NF- B pathway, phosphorylation of I B by
IKK results in I B degradation through ubiquitination, following which the NF- B complex
κB is a major mediator in the immune system that
κ
κ
κ
κ
κ
κ
κ
enters the nucleus and acts as a transcription factor to enable the expression of specific
genes involved in the inflammatory response [31]. Notably, upon LPS binding, the TLR-
MyD88 signal activates the transcription factor NF-
of proinflammatory cytokines such as IL-1 , TNF- , and IL-6 [32
that KMU-1170 exerts inhibitory effects on LPS-induced phosphorylation of IKK
NF- B p65, nuclear translocation of NF- B p65, and upregulation of IL-1 , TNF- , and IL-6
κ
B, which then leads to the induction
33]. Our results showed
and
β
α
,
α/β
κ
κ
β
α
(Figure 2, Figure 3C,D and Figure 4). However, KMU-1170 did not inhibit LPS-mediated up-
regulation of TLR4 and MyD88 in THP-1 cells (Figure 3A). Therefore, these results suggest
that KMU-1170 inhibits inflammatory signal transduction downstream of TLR4/MyD88 in
the LPS-mediated inflammatory response of THP-1 cells.
The inflammasome is a multiprotein complex that is considered to function in the
recognition of pathogen-associated molecular patterns and danger-associated molecular
patterns in the inflammatory responses of the innate immune system [34]. In the two-signal
model, the NLRP3 inflammasome, which consists of NLRP3, ASC, and pro-caspase-1, is
regulated by a priming signal and an activation signal [10]. The first signal, the priming
signal, is induced by endogenous cytokines or microbial molecules such as LPS, and this
leads to the upregulation of NLRP3 and pro-IL-1β through NF-κB activation. Next, the
second signal, the activation signal, is initiated by a plethora of stimuli, such as ATP, and
activates the NLRP3 inflammasome [10]. In this study, we investigated whether KMU-1170
can inhibit the two signals in THP-1 cells, which revealed that KMU-1170 attenuated
LPS-induced upregulation of NLRP3 and pro-IL-1β as well as ATP-mediated production
of IL-1β (Figure 5A,B).
Recently, various signaling pathways have been identified as therapeutic targets
for inflammation, and numerous anti-inflammatory agents are currently under devel-
opment [35]. Here, we sought to compare the anti-inflammatory efficacy of KMU-1170
with that of other targeted anti-inflammatory drugs. KMU-1170 exhibited superior in-
hibitory efficiency in inflammatory signaling pathway relative to other drugs, including a

Int. J. Mol. Sci. 2021, 22, 1194
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COX-2 inhibitor (celecoxib), a BTK inhibitor (ibrutinib), a JAK inhibitor (ruxolitinib), and
methotrexate (Figure 5C,D); however, because KMU-1170 exerts a multi-kinase inhibitory
effect (Table 1), additional studies will be required before the application of this inhibitor to
a multicellular system.
Lastly, we investigated whether KMU-1170 suppresses LPS-mediated inflammatory
responses in human osteoarthritic FLS. Osteoarthritic FLS play a pivotal role in the pro-
cesses of OA characterized by continuous synovial inflammation and progressive cartilage
degradation [36]. Moreover, macrophage-mediated inflammation play an important role in
pathophysiology of OA [37,38]. As shown in Figure 6, KMU-1170 inhibited LPS-induced in-
flammatory responses in human osteoarthritic FLS, suggesting that KMU-1170 can alleviate
OA exacerbation.
In order to develop the KMU-1170 as a promising anti-inflammatory agent, it is neces-
sary to improve therapeutic window of concentration, reduce toxicity, improve the efficacy
on various cell model of inflammatory disease, and conduct in vivo study. Nevertheless,
we expected that KMU-1170, which has the inhibitory effect on LPS-mediated inflammatory
pathway, will be a candidate agent for the treatment of inflammatory diseases.
In conclusion, this study demonstrated that a novel multi-protein kinase inhibitor,
KMU-1170, suppresses inflammatory signal transduction by controlling the p-TAK1/p-
IKKα/β/p-NF-κB/proinflammatory cytokine signaling pathway and the NLRP3 inflam-
masome (Figure 7). Our findings suggest that KMU-1170 is a highly favorable compound
for development as a potent anti-inflammatory drug.
Figure 7. Mechanistic scheme summarizing the anti-inflammatory effect of KMU-1170. LPS, lipopolysaccharide; TLR4,
toll-like receptor 4; MyD88, myeloid differentiation factor 88; TAK1, transforming growth factor- -activated kinase 1; IKK,
inhibitor NF- B kinase; MAPKs, mitogen-activated protein kinases; NF- B, nuclear factor- B; iNOS, inducible nitric oxide
synthase; COX-2, cyclooxygenase-2; TNF- , tumor necrosis factor- ; IL-1 , interleukin-1 ; NLRP3, NOD-like receptor
β
κ
κ
κ
α
α
β
β
family, pyrin domain containing 3; ASC, apoptosis-associated speck-like protein containing a CARD.
4. Materials and Methods
4.1. Synthesis of KMU-1170
4.1.1. General Information
All reactions were performed using commercially available reagents and solvents
without further purification, under proper conditions as stated by the manufacturers. A
CEM Discover BenchMate was used for microwave-assisted reactions. Reaction completion

Int. J. Mol. Sci. 2021, 22, 1194
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was monitored on E. Merck silica gel F254 TLC plates. Synthesized compounds were puri-
fied by means of flash column chromatography using Merck Silica Gel 60 (230–400 mesh).
Synthesized compounds were characterized by performing 1H-NMR on a JEOL ECA 500
(1H: 500 MHz) spectrometer and chemical shift
δ values were measured in ppm with TMS
as the reference standard. Mass spectra were obtained using a Waters ACQUITY UPLC,
Micromass Quattro microTM API.
4.1.2. 5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)indolin-2-one (1)
A microwave vessel was filled with 5-bromoindoline-2,3-dione (0.50 g, 2.2 mmol),
hydrazine hydrate (0.14 g,◦4.4 mmol), and ethanol (EtOH, 2 mL). The mixture was ir-
radiated for 10 min at 100 C by applying 100 W. After addition of sodium hydroxide
(0.18 g, 4.4 mmol), the mixture was irradiated for 10 min at 80 ◦C by applying 100 W. The
mixture was acidified using 2 M hydrochloric acid (HCl), and the precipitate formed by
the addition of cold water was collected through filtration and washed with 2 M HCl
and then with water. Drying under vacuum generated 0.42 g of 5-bromoindolin-2-one (at
89% yield), which was used for the next step without further purification. A microwave
vessel was filled with 5-bromoindolin-2-one (0.40 g, 1.9 mmol) and 1,4-dioxane (2.0 mL),
and after adding bis(pinacolato)diboron (0.72 g, 3.8 mmol) and potassium acetate (KOAc;
0.56 g, 5.7 mmol) to the mixture, the mixture was purged with N2 gas for 5 min. To the
reaction mixture was added 1,10-bis(diphenylphosphino)ferrocene]dichloropalladium(II)
(PdCl2(dppf)) (0.069 g, 0.094 mmol) in N2 gas, and the mixture was irradiated for 10 min
◦
at 110 C by applying 100 W. After solvent removal in vacuo, the residue was purified
by means of silica gel chromatography using 1:1 hexane (HEX)/ethyl acetate (EA), which
yielded 0.41 g (82%) of the title compound.
4.1.3. 6-Chloro-N-cyclopropylpyrazin-2-amine (2)
Cyclopropylamine (5.1 mmol, 0.35 mL) and potassium carbonate (K2CO3; 6.7 mmol,
0.93 g) were added to 5.0 mL of N,N-dimethylformamide, and the mixture was stirred for
30 min at room temperature. After addition of 2,6-dichloropyrazine (3.4 mmol, 0.50 g),
the reaction mixture was stirred overnight at room temperature, and then the solvent was
removed in vacuo. The residue was next treated with dichloromethane (DCM) and filtered
with the aid of celite, and the filtrate was collected and the solvent was removed in vacuo.
The final residue was purified through silica gel chromatography by using 5:1 DCM/EA,
which yielded 0.21 g (36%) of the title compound.
4.1.4. 5-(6-(Cyclopropylamino)pyrazin-2-yl)indolin-2-one (3)
A microwave vessel was filled with compound
1 (0.45 g, 1.8 mmol), compound 2
(0.20 g, 1.2 mmol), 1,4-dioxane (2.0 mL), EtOH (0.40 mL), and 2 M aqueous potassium
carbonate (1.8 mL, 2.54 mmol). After adding tetrakis (triphenylphosphine) palladium(0)
(PdCl2(PPh3)2) (0.041 g, 0.059 mmol) in an N2 atmosphere, the mixture was irradiated for
10 min at 110 ◦C by applying 100 W. The solvent was removed in vacuo, and the residue
was treated with DCM and then filtered with the aid of celite. The filtrate was collected
and the solvent was removed in vacuo. The residue was purified by performing silica gel
chromatography with 5:1 DCM/EA, which yielded 0.19 g (41%) of the title compound.
4.1.5. (Z)-3-((1H-Imidazol-5-yl)methylene)-5-(6-(cyclopropylamino)pyrazin-2-yl)
indolin-2-one (KMU-1170)
A microwave vessel was filled with compound 3 (0.15 g, 0.056 mmol), piperidine
(6.0 µL, 0.06 mmol), 1H-imidazole-4-carbaldehyde (0.081 g, 0.85 mmol), and EtOH (2.0 mL),
and the mixture was irradiated for 10 min at 80 ◦C by applying 100 W. The solvent was
removed in vacuo and the residue was purified by performing silica gel chromatography
1
with 5:95 methanol/DCM, which yielded 0.042 g (22%) of the title compound. H-NMR
(500 MHz, DMSO-d6) δ values: 11.27–11.14 (br, 1H), 8.41 (s, 1H), 8.38 (s, 1H), 8.06 (s, 1H),
7.99 (s, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.95 (s, 1H), 7.69 (s, 1H), 7.29 (s, 1H), 7.01 (d, J = 8.0 Hz,
1H), 2.71 (s, 1H), 0.79 (m, 2H), 0.57–0.45 (m, 2H). ESI MS: m/z = 345 [M + H]+.

Int. J. Mol. Sci. 2021, 22, 1194
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4.2. Reagents
LPS (Escherichia coli serotype) was purchased from Sigma-Aldrich (St. Louis, MO,
USA). Celecoxib, ibrutinib, methotrexate, and ruxolitinib were purchased from Selleck
Chemicals (Houston, TX, USA). Primary antibodies against iNOS, TLR4, MyD88, p-TAK1
(Ser412), p-ERK, ERK, p-JNK, p-IKK
and NLRP3 were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-IL-
antibody was purchased from Novus Biologicals (Centennial, CO, USA). Antibodies
α/β, IKKα/β, p-NF-κB p65, NF-κB, ASC, caspase-1,
1β
against IL-6, COX-1, COX-2, TAK1, p-p38, and p38 were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA, USA) and Sigma-Aldrich (St. Louis, MO, USA), respec-
tively. Anti-TNF-α antibody was purchased from Abcam (Cambridge, MA, USA). Anti-
β-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-mouse
IgG-horseradish peroxidase (HRP) and anti-mouse IgG-HRP antibodies were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
4.3. Cell Line and Culture
The human monocytic cell line THP-1 cells (Korean Cell Line Bank, Seoul, Korea) were
cultured in RPMI1640 medium (Welgene Inc., Gyeongsan, Korea), 10% fetal bovine serum
(FBS; Welgene Inc., Gyeongsan, Korea), and 1% antibio◦tic-antimycotic solution (Welgene
Inc., Gyeongsan, Korea). The cells were cultured at 37.5 C, 5% CO2 in fully humidified air.
4.4. Isolation and Culture of Primary Human Osteoarthritic FLS
Isolation of human osteoarthritic FLS was performed by enzymatic digestion of syn-
ovial tissues from osteoarthritic patients who underwent a synovectomy at Keimyung
University Dongsan Medical Center. The enrolled patients with OA fulfilled the Amer-
ican College of Rheumatology criteria and agreed to give their informed consent. The
experimental study was approved by the Institutional Review Board (IRB) of Keimyung
University Dongsan Medical Center on November 20th, 2020 [IRB No. 2020-11-031]). After
crushing the osteoarthritic tissues into 2–3-mm pieces, the minced tissues were treated
with 0.5 mg/mL type II collagenases (Thermo Fisher Scientific, Waltham, MA, USA) in
Dulbecco’s modified Eagle medium (DMEM; Welgene Inc., Gyeongsan, Korea) under 5%
CO2 at 37.5 ◦C for 2 h. The separated cells were centrifuged (3000 rpm, 5 min) and then
resuspended in DMEM), 10% fetal bovine serum (FBS; Welgene Inc., Gyeongsan, Korea),
and 1% antibiotic-antimycotic solution (Welgene Inc., Gyeongsan, Korea), and agitated
in a ◦75-cm2 flask. One day later, the adherent cells were incubated under 5% CO2 at
37.5 C in DMEM), 10% fetal bovine serum (FBS; Welgene Inc., Gyeongsan, Korea), and 1%
antibiotic-antimycotic solution (Welgene Inc., Gyeongsan, Korea), and the culture fluid was
replaced every 3 days. When 90%–95% of the bottom of the flask was filled with fibroblasts,
the medium was diluted 1:3 with fresh culture.
4.5. Kinase-Profiling Analysis
To test the specificity of a novel multi-protein kinase inhibitor KMU-1170, a kinase-
profiling service was conducted by Eurofins Cerep S.A. (Celle-Lévescault, France). The
kinase-profiling analysis was performed with 1 µM of compound and a concentration
of ATP at the Km value for each individual kinase and kinase substrate according to
Eurofin’s protocols.
4.6. Cell Viability Assay
The cell viability analysis was performed using XTT assay (Welgene Inc., Gyeongsan,
Korea). Briefly, 2
×
105 THP-1 cells were plated in 96-well plates for 24 h. Cells were
treated with various concentrations of KMU-1170 and incubated under 5% CO2 at 37 °C
for 24 h. Subsequently, 0.5 mg/mL XTT solution was added to the culture solution and
then incubated under 5% CO2 at 37 °C for 3 h. Measurement of optical density (OD) was
performed using a microplate reader (BMG Labtech, Ortenberg, Germany) at a 450 nM
wavelength.

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4.7. Western Blotting Analysis
THP-1 cells (2
106 cells/well in 60-mm dishes) and FLS (2
×
×
106 cells/well in
100-mm dishes) were plated for western blotting analysis. After culturing for 24 h, cells
were lysed in RIPA buffer (20 mM HEPES and 0.5% Triton X-100, pH 7.6) and super-
natant fractions were collected. Protein concentrations were measured using a BCA assay
kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein were elec-
trophoresed in SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare
Life Science, Pittsburgh, PA, USA). Subsequently, the membranes were incubated with
blocking buffer (0.05% Tween 20 with 5% non-fat dry milk in Tris-buffered saline (TBS))
for 1 h and then incubated for 24 h with appropriately diluted primary antibodies against
specific target proteins. Next, the membranes were washed with TBS with Tween 20, and
then incubated at room temperature for 1 h with the appropriate secondary antibodies. The
specific proteins were detected with ECL western blotting kit (EMD Millipore, Darmstadt,
Germany) according to the manufacturer’s protocols, and the signal strength was measured
using a Chemi Image System Fusion FX (Vilber Lourmat, Collégien, France). Protein bands
were quantified using Image-J software. Quantitative analysis of band density using the
Image-J is shown as the mean
±
standard deviation (SD) of three independent experiments.
4.8. RNA Isolation and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Real
Time Quantitative PCR (qPCR)
Total cellular RNA was isolated using TRIzol Reagent (Thermo Fisher Scientific,
Waltham, MA, USA). Each RNA was quantified using a NanoDrop 1000 (Thermo Fisher
Scientific, Waltham, MA, USA). Each cDNA was obtained from 1 µg of the isolated RNA
using Moloney murine leukemia virus reverse transcriptase (Gibco-BRL, Gaithersburg,
MD, USA) according to the manufacturer’s instructions. The primer sequences used in the
study are shown in Table 2. For PCR, DNA polymerase was used with primers targeting
IL-1β, TNF-α, and IL-6. The amplified PCR products were separated by electrophoresis
on a 2% agarose gel and detected under ultraviolet light. For qPCR, by using the specific
primer and SYBR GREEN Premix (Toyobo, Osaka, Japan), qPCR was performed on the
LightCycler® 480 real-time PCR system (Roche Diagnostics, Mannheim, Germany). The
experiments were performed three times, and the threshold cycle number (Ct) of each
target mRNA was normalized to β-actin. The delta-delta Ct values of each genes are
indicated as the mean ± SD of three independent experiments.
Table 2. Primer sequences for PCR and Real Time PCR.
0
0
Primers
Sequences (5 → 3 )
CTG TCT GGT TCC TAC GTC ACC
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
iNOS
CCC ACG TTA CAT GGG AGG ATA
ACC TTG AAG GAG TCA GGC ATG AG
TGT TCG GTG TCC AGT TCC AAT A
ATC ACA GGC TTC CAT TGA CC
TAT CAT CTA GTC CGG AGG GG
CCT TGG GCC TCA AGG AAA A
CTC CAG CTG TAG AGT GGG CTT A
GGA GAA GGG TGA CCG ACT CA
CTG CCC AGA CTC GGC AA
COX-1
COX-2
IL-1β
TNF-α
IL-6
ATG GCA CAG TAT CTG GAG GAG
TAA GCT GGA CTC ACT CTC GGA
AAT CTG GCA CCA CAC CTT CTA
ATA GCA CAG CCT GGA TAG CAA
β-actin

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4.9. Immunofluorescence Staining
THP-1 cells (1
103) were cultured in 8-chamber glass slides for 24 h, treated for
1 h with KMU-1170 at 1 mol/L, and then stimulated with LPS for 6 h. The cells were
×
µ
washed with phosphate-buffered saline (PBS), fixed with 4% formaldehyde, permeabilized
using 0.2% Triton X-100 in PBS, and incubated for 1 h with bovine serum albumin to block
nonspecific binding. Subsequently, the cells were incubated with primary antibodies at
4 ◦C for 24 h and then with FITC-conjugated goat anti-mouse IgG (Thermo Fisher Scien-
tific, Waltham, MA, USA) and 40,6-diamidino-2-phenylindole (Thermo Fisher Scientific,
Waltham, MA, USA; for nuclear staining), after which the stained cells were examined
under a fluorescence microscope.
4.10. Statistical Analysis
Quantitative results are presented as the mean and SDs. The data were analyzed by a
one-way ANOVA and post-hoc comparisons (Student-Newman-Keuls) using the Statistical
Package for Social Science 26.0 software (SPSS Inc.; Chicago, IL, USA). A p value less than
or equal to 0.05 was considered significant.
Author Contributions: Conceptualization, H.S.B. and S.K.; methodology, H.S.B., V.S.H., and, J.L.;
formal analysis, H.S.B.; investigation, H.S.B., and V.S.H.; resources, S.H.K., J.L.; data curation, H.S.B.,
V.S.H., J.L., and S.K.; writing—original draft preparation, H.S.B. and J.L.; writing—review and editing,
H.S.B., V.S.H., S.H.K., J.L., and S.K.; visualization, H.S.B., J.L., and S.K.; supervision, J.L., and S.K.;
project administration, V.S.H., S.H.K., J.L., and S.K.; funding acquisition, S.K. All authors have read
and agreed to the published version of the manuscript.
Funding: This study was supported by grants from the National Research Foundation of Korea
(grant funded by the Korea Government Ministry of Science, ICT and Future Planning; grant nos.
2014R1A5A2010008 and NRF-2019R1I1A3A01063114).
Institutional Review Board Statement: The study was conducted according to the guidelines of the
Declaration of Helsinki, and approved by the Institutional Review Board of Keimyung University
Dongsan Medical Center on 20 November 2020 (IRB No. 2020-11-031).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: The data used in the current study are available from the corresponding
authors upon reasonable request.
Acknowledgments: We would like to thank all the members of our research group for their enthusi-
astic participation in this study.
Conflicts of Interest: The authors have no conflict of interest to declare.
Abbreviations
FLS
LPS
iNOS
COX
TLR
IL
fibroblast-like synoviocyte
lipopolysaccharide
inducible nitric oxide synthase
cyclooxygenase
toll-like receptor
interleukin
TNF
tumor necrosis factor
NLRP3 the NOD-, LRR- and pyrin domain-containing protein 3
ASC
JAK
FDA
BTK
the adaptor molecule apoptosis-associated speck-like protein containing a CARD
Janus-associated kinase
the US Food and Drug Administration
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

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IKK
OA
inhibitor of NF-κB kinase
osteoarthritis
HRP
FBS
IRB
horseradish peroxidase
fetal bovine serum
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
Ct
SPSS
quantitative PCR
the threshold cycle number
the Statistical Package for Social Science
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