Anti-Inflammatory Effects of the Novel PIM Kinase Inhibitor KMU-470 in RAW 264.7 Cells through the TLR4-NF-κB-NLRP3 Pathway

PIM kinases, a small family of serine/threonine kinases, are important intermediates in the cytokine signaling pathway of inflammatory disease. In this study, we investigated whether the novel PIM kinase inhibitor KMU-470, a derivative of indolin-2-one, inhibits lipopolysaccharide (LPS)-induced inflammatory responses in RAW 264.7 cells. We demonstrated that KMU-470 suppressed the production of nitric oxide and inducible nitric oxide synthases that are induced by LPS in RAW 264.7 cells. Furthermore, KMU-470 inhibited LPS-induced up-regulation of TLR4 and MyD88, as well as the phosphorylation of IκB kinase and NF-κB in RAW 264.7 cells. Additionally, KMU-470 suppressed LPS-induced up-regulation at the transcriptional level of various pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6. Notably, KMU-470 inhibited LPS-induced up-regulation of a major component of the inflammasome complex, NLRP3, in RAW 264.7 cells. Importantly, PIM-1 siRNA transfection attenuated up-regulation of NLRP3 and pro-IL-1β in LPS-treated RAW 264.7 cells. Taken together, these findings indicate that PIM-1 plays a key role in inflammatory signaling and that KMU-470 is a potential anti-inflammatory agent.


Introduction
PIM kinases are a family of serine/threonine kinases that were named after they was identified as proviral integration sites of the Moloney murine leukemia virus [1]. The PIM kinases regulate angiogenesis, immunity, and tumor progression [2][3][4], and consist of three isoforms: PIM-1, PIM-2, and PIM-3. Recent studies demonstrated that PIM-1 plays an important role in inflammatory responses [5][6][7]. These results suggest that PIM-1 has promising potential as an anti-inflammatory target.
Inflammatory responses rely on intracellular signaling pathways and various molecular interactions. When macrophages are stimulated with lipopolysaccharides (LPS), large molecules that are a major component of the outer membrane of gram-negative bacteria, by binding to a potent immune receptor, Toll like receptor 4 (TLR4), it induces activation of nuclear factor kappa-B (NF-κB) [8] and inflammatory responses, including production and release of pro-inflammatory cytokines [9]. Recruitment of the myeloid differentiation factor (MyD88) to the plasma membrane is crucial for the intracellular inflammatory signal cascade by TLR4 [10]. This cascade results in the Int. J. Mol. Sci. 2020, 21, 5138 2 of 16 downstream activation of NF-κB and mitogen-activated protein kinase (MAPK), which further activates the extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and MAPK p38 pathways [11]. Inflammasomes, cytosolic multiprotein complexes, are responsible for caspase-1-mediated cleavage of pro-inflammatory cytokines, such as interleukin-1β (IL-1β), and consist of NLRP3, the adaptor molecule apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC) and pro-caspase-1 [9]. Moreover, activation of the NLRP3 inflammasome is regulated downstream of NF-κB [9].
In the present study, we synthesized a novel PIM kinase inhibitor, a derivative of indolin-2-one, named KMU-470, and explored its anti-inflammatory properties in LPS-treated RAW 264.7 cells. We also investigated the potential of PIM-1 as an anti-inflammatory target using PIM-1 siRNA.
In the present study, we synthesized a novel PIM kinase inhibitor, a derivative of indolin-2-one, named KMU-470, and explored its anti-inflammatory properties in LPS-treated RAW 264.7 cells. We also investigated the potential of PIM-1 as an anti-inflammatory target using PIM-1 siRNA.

Identification of KMU-470 as A Novel PIM Kinase Inhibitor and Analysis of Inflammatory Kinase
KMU-470 was synthesized from 5-bromoindoline-2,3-dione as shown in Figure 1. The compound 1 was obtained by reduction and borylation. Suzuki coupling reaction of compound 1 with pyrazine derivative, 2, yielded compound 3. The Knoevenagel condensation with 1H-imidazole-4-carbaldehyde gave KMU-470. To confirm KMU-470 as a PIM kinase inhibitor, the PIM kinase inhibitory activity of the compound was investigated. In addition, the IC50 values of KMU-470 were compared with those of PIM-447 and AZD-1208, known PIM kinase inhibitors. KMU-470 inhibited PIM-1, PIM-2, and PIM-3 activities with IC50 values of 5.6, 220, and, 6.9 nM, respectively (Table 1). However, KMU-470 showed inhibitory activities against other kinases, including inflammatory kinases, when it was tested at concentrations of 10 and 1 μM. In particular, percent activities of MAPK1, Yes, and Lyn at 1 μM concentration of KMU-470 were found to be 6, 22, and 30, respectively ( Table 2).   To investigate the role of PIM-1 in LPS-induced inflammatory responses in RAW 264.7 cells, we used PIM-1 siRNA and a pan-PIM kinase inhibitor. PIM-1 siRNA inhibited LPS-induced pro-IL-1β and phosphorylation of Bad, the substrate of PIM kinase (Figure 2A). Considering KMU-470 as a PIM kinase inhibitor, we next compared the anti-inflammatory potency between KMU-470 and PIM-447, a well-known pan-PIM kinase inhibitor in RAW 264.7 cells. A concentration of 1 µM KMU-470 significantly inhibited LPS-induced up-regulation of IL-1β mRNA expression in RAW 264.7 cells, whereas the same concentration of PIM-447 did not ( Figure 2B).

KMU-470 Inhibits LPS-Induced NO Production and iNOS Expression in RAW 264.7 Cells
To measure the influence of KMU-470 on NO production, we performed NO measurement in RAW 264.7 cells. KMU-470 suppressed LPS-induced NO production in RAW 264.7 cells ( Figure 3A). In order to investigate the relevance of iNOS protein and the mechanism of NO suppression, the expression levels of iNOS protein were determined. KMU-470 attenuated LPS-induced up-regulation of iNOS protein in RAW 264.7 cells ( Figure 3B

KMU-470 Inhibits LPS-Induced NO Production and iNOS Expression in RAW 264.7 Cells
To measure the influence of KMU-470 on NO production, we performed NO measurement in RAW 264.7 cells. KMU-470 suppressed LPS-induced NO production in RAW 264.7 cells ( Figure 3A). In order to investigate the relevance of iNOS protein and the mechanism of NO suppression, the expression levels of iNOS protein were determined. KMU-470 attenuated LPS-induced up-regulation of iNOS protein in RAW 264.7 cells ( Figure 3B

KMU-470 Suppressed LPS-Induced TLR4 and MyD88 Expression in RAW 264.7 Cells
Next, we determined the effect of KMU-470 on the LPS-induced TLR4 and MyD88 expression in RAW 264.7 cells. KMU-470 suppressed up-regulation of TLR4 and MyD88 protein by LPS ( Figure 4).

KMU-470 Inhibits LPS-Induced Phosphorylation of MAPKs and Activation of NF-κB in RAW 264.7 Cells
LPS induced phosphorylation of ERK, JNK, and p38 in a time dependent manner, whereas KMU-470 significantly inhibited LPS-induced phosphorylation of JNK and ERK. However, p38 showed no change in phosphorylation level ( Figure 5A

KMU-470 Inhibits LPS-Induced NLRP3 Inflammasome Activation and Up-Regulation of Pro-IL-1β
We further investigated whether KMU-470 affected the NLRP3 inflammasome signal. KMU-470 showed a marked decrease in expression of NLRP3, ASC, and pro-IL-1β proteins compared with that of the LPS alone group. However, pro-caspase-1 expression was not changed by KMU-470 ( Figure 6A).  The relative optical densities of MAPKs, p-IKKα/β, and p-NF-κB bands were analyzed by the Image-J program. *** p < 0.001.

KMU-470 Inhibits LPS-Induced NLRP3 Inflammasome Activation and Up-Regulation of Pro-IL-1β
We further investigated whether KMU-470 affected the NLRP3 inflammasome signal. KMU-470 showed a marked decrease in expression of NLRP3, ASC, and pro-IL-1β proteins compared with that of the LPS alone group. However, pro-caspase-1 expression was not changed by KMU-470 ( Figure  6A).

Discussion
Members of the PIM kinase family are known as oncogenes, and many studies have been conducted on the role of PIM in tumorigenesis [12]. Recently, studies on the function of the PIM kinase family in the inflammatory response have been attracting attention. PIM-1, one of the three PIM kinases, is significantly induced by cigarette smoke in RAW 264.7 cells [7], and the inhibition o PIM-1 in a dextran sodium sulfate colitis mice model improved colitis by reducing the excessive activity of macrophages and the immune response of Th1 and Th17 [5]. In addition, PIM-1 kinase inhibitors effectively prevent the generation of cytokines, which cause allergic inflammation of the airways [6]. These findings highlight the importance of PIM-1 kinase in inflammatory responses However, the exact mechanisms by which PIM kinases influence the inflammatory process remain unknown. In the present study, we developed a novel PIM kinase inhibitor and investigated its anti inflammatory mechanism in RAW 264.7 cells.

Discussion
Members of the PIM kinase family are known as oncogenes, and many studies have been conducted on the role of PIM in tumorigenesis [12]. Recently, studies on the function of the PIM kinase family in the inflammatory response have been attracting attention. PIM-1, one of the three PIM kinases, is significantly induced by cigarette smoke in RAW 264.7 cells [7], and the inhibition of PIM-1 in a dextran sodium sulfate colitis mice model improved colitis by reducing the excessive activity of macrophages and the immune response of Th1 and Th17 [5]. In addition, PIM-1 kinase inhibitors effectively prevent the generation of cytokines, which cause allergic inflammation of the airways [6]. These findings highlight the importance of PIM-1 kinase in inflammatory responses. However, the exact mechanisms by which PIM kinases influence the inflammatory process remain unknown. In the present study, we developed a novel PIM kinase inhibitor and investigated its anti-inflammatory mechanism in RAW 264.7 cells.
Indolin-2-one is considered as a privileged scaffold for the discovery of kinase inhibitor [13]. Antitumor drugs such as Sunitinib and Intedanib are representative kinase inhibitors using an indolin-2-one scaffold [13]. Most PIM kinase inhibitors utilize the ε-amino group of Lys-67 of PIM-1 kinase as a key interaction point. For example, PIM-447 and INCB053914, under clinical trials I and II, use the pyridine ring [14,15], and AZD-1208 uses the thiazolidine-2,4-dione [16] for this purpose. Previously we reported that a series of compounds, synthesized using indoln-2-one for a scaffold and the imidazole ring for the interaction with the ε-amino group of Lys-67 of PIM-1 kinase, shows PIM kinase inhibitory activities with submicromolar IC 50 values for all PIM-1, PIM-2, and PIM-3 kinases [17]. It was also found that the introduction of a pyrazine ring substituted with an aminoalkyl moiety, which was expected to make hydrogen bond interactions with PIM kinases, to an appropriate scaffold improved the potency of the compound against PIM kinases [18,19]. Therefore, considering the above three factors, we designed a new series of PIM kinase inhibitors that inhibits PIM kinase by competitively binding to the ATP binding pocket of PIM kinase. Indolin-2-one was used as a key scaffold, imidazole was introduced for the interaction with the ε-amino group of Lys-67 (PIM-1), and the pyrazine ring substituted with an aminoalkyl moiety was attached to the indolin-2-one ring to increase the potency of compound. We obtained potent PIM kinase inhibitors with single-or double-digit nanomolar IC 50 values for all three PIM kinases. During screening for the anti-inflammatory activity of the PIM kinase inhibitors, KMU-470 was selected. It strongly inhibited the activities of PIM-1 and also PIM-3 kinase, with IC 50 values of 5.6 nM and 6.9 nM, respectively (Table 1). However, this could be problematic for targeting if the inhibitor is applied to a multi-cellular system because KMU-470 is a multi-kinase inhibitor affecting the activity of several different kinases (Table 2).
First, the role of PIM-1 in the LPS-mediated inflammatory response was evaluated using PIM-1 siRNA. As shown in Figure 2A, Pim-1 siRNA inhibited LPS-induced up-regulation of pro-IL-1β and p-Bad in RAW 264.7 cells. As shown in Figure 1B, KMU-470 suppressed LPS-induced up-regulation of pro-IL-1β even at a concentration of about 1/10 that of PIM-447, a pan-PIM kinase inhibitor. This may result from KMU-470 having inhibitory activity on various inflammation-related kinases ( Table 2), but it suggests that the anti-inflammatory effect of KMU-470 is superior to that of PIM-447.
iNOS is an enzyme that generates NO by converting L-arginine to L-citrulline in cells. Excess amounts of NO induce the expression of inflammatory cytokines. Consequently, the resulting inflammatory reactions can cause tissue damage, gene mutation, and nerve cell damage [20,21]. LPS promotes the production of iNOS and NO in RAW 264.7 cells, resulting in a pronounced inflammatory reaction [22,23]. A recent study has shown that LPS-induced increase of iNOS expression led to the generation of large amounts of NO, resulting in physiological dysfunction [24]. As shown in Figure 3, KMU-470 significantly decreased the generation of NO and iNOS protein expression. Accordingly, KMU-470 is considered to have anti-inflammatory effects because NO generation can be suppressed by suppressing the expression or activity of iNOS.
TLRs recognize pathogens or specific molecular components of pathogens in order to initiate the immune response [25]. Among them, TLR4 is activated by the LPS displayed on the outer membrane of Gram-negative bacteria, resulting in the activation of specific intracellular pathways, receptor dimerization, and the subsequent recruitment of a variety of adapter molecules, such as MyD88 [26]. We showed that KMU-470 inhibited LPS-induced TLR4 and MyD88 protein expression ( Figure 4). LPS activates macrophages by binding to TLR4, and the LPS-initiated signaling cascade leads to activation of MAPKs and NF-κB signaling pathways [27]. It was also reported that the activation of NF-κB may occur via the MAPK signaling pathway [28,29]. These studies suggest that blocking the phosphorylation and activation of MAPKs is an important way to alleviate LPS-induced inflammation [30]. In addition, the LPS stimulus triggers activation of TLR4-MyD88, which activates NF-κB through phosphorylation and degradation of IκBα [31,32]. As shown in Figure 5, KMU-470 inhibited LPS-induced phosphorylation of IKK α/β and NF-κ B in RAW 264.7 cells.
Recent studies have found that NOD-like receptors (NLRs) are important mediators in inflammatory diseases [37,38]. In particular, one of the NLRs, known as NLRP3 inflammasome, is a complex composed of NLRP3, ASC, and caspase-1, which regulates inflammatory cytokines [39]. In this study, KMU-470 inhibited LPS-induced up-regulation of NLRP3 and ASC in RAW 264.7 cells, suggesting that KMU-470 may also inhibit the inflammasome function of pro-IL-1 β cleavage.
PIM kinase inhibitors, such as AZD-1208 from Astrazeneca and PIM-447 from Novartis, have been evaluated for their potential in the pharmaceutical market of the anti-cancer field [15,40]. However, there are no reports of PIM kinase inhibitors entering the anti-inflammatory pharmaceutical market. As shown in Figure 2, it was revealed that PIM-1 kinase plays an important role in the inflammatory signaling pathway. In addition, KMU-470 was found to have inhibitory effects against multiple kinases including PIM kinase, and it was confirmed that KMU-470 has better anti-inflammatory efficacy than AZD-1208 or PIM-447, which has good PIM kinase selectivity [15,16]. These results suggest the possibility of KMU-470 entering the anti-inflammatory pharmaceutical market. Moreover, this may lead to the development of candidate compounds in the anti-inflammatory pharmaceutical market through appropriate optimizing processes such as pharmacokinetics, pharmacodynamics, in vivo study, and clinical study.
In conclusion, this study demonstrated that PIM-1 plays an important role in the inflammatory signaling associated with pro-IL-1 β up-regulation by LPS, and the novel PIM kinase inhibitor, KMU-470, has anti-inflammatory properties by regulating the TLR4/MyD88/p-IKKα/β/p-NF-κB signaling pathway and NLRP3 inflammasome (Figure 7), suggesting that this inhibitor may be developed as a potent anti-inflammatory agent.
However, there are no reports of PIM kinase inhibitors entering the anti-inflammatory pharmaceutical market. As shown in Figure 2, it was revealed that PIM-1 kinase plays an important role in the inflammatory signaling pathway. In addition, KMU-470 was found to have inhibitory effects against multiple kinases including PIM kinase, and it was confirmed that KMU-470 has better anti-inflammatory efficacy than AZD-1208 or PIM-447, which has good PIM kinase selectivity [15,16]. These results suggest the possibility of KMU-470 entering the anti-inflammatory pharmaceutical market. Moreover, this may lead to the development of candidate compounds in the antiinflammatory pharmaceutical market through appropriate optimizing processes such as pharmacokinetics, pharmacodynamics, in vivo study, and clinical study.
In conclusion, this study demonstrated that PIM-1 plays an important role in the inflammatory signaling associated with pro-IL-1 β up-regulation by LPS, and the novel PIM kinase inhibitor, KMU-470, has anti-inflammatory properties by regulating the TLR4/MyD88/p-IKKα/β/p-NF-κB signaling pathway and NLRP3 inflammasome (Figure 7), suggesting that this inhibitor may be developed as a potent anti-inflammatory agent.

General Information
Microwave assisted reactions were performed using a CEM Discover BenchMate. Reaction completions were monitored on E. Merck silica gel F254 TLC plates. Purification of the synthesized compounds was performed by flash column chromatography using Merck Silica Gel 60 (230-400 mesh). The synthesized compounds were characterized by an 1 H NMR on Bruker AVANCE 400 ( 1 H: 400 MHz) and JEOL ECA 500 ( 1 H: 500 MHz) spectrometers and their chemical shift δ values were measured in ppm with TMS as a standard reference. 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 (0.10 g, 0.41 mmole), hydrazine hydrate (0.035 g, 1.1 mmole), and ethanol (EtOH, 1.5 mL). The mixture was irradiated for 10 min at 100 • C by applying 100 W. After addition of sodium hydroxide (0.053 g, 1.3 mmole), the mixture was irradiated for 10 min at 90 • C by applying 100 W. The mixture was acidified with 6 M hydrochloric acid (HCl). The precipitate formed by addition of cold water was obtained by filtration and washed with 6 M HCl and then with water. Drying under vacuum gave 0.07 g of 5-bromoindolin-2-one in 75% yield, which was used for the next step without further purification.

Biochemical IC 50 Value Determination
The recombinant PIM kinase, human 5-FAM-labelled Bad peptide substrate, and ATP were prepared in a reaction buffer containing 10 mM HEPES (pH 7.2), 10 mM MgCl 2 , 0.05% NaN 3 , 0.01% Triton X-100, and 2 mM DTT. Kinase reactions were performed in 384-well black flat bottom polystyrene plates (Corning 3820, Corning, NY, USA). Reagents were added to the plate as follows: 2.5 µL compound, 2.5 µL PIM kinase, 2.5 µL ATP, and 2.5 µL 5-FAM-labelled Bad peptide substrate (a final concentration of 100 nM). The final concentrations of ATP in the kinase reactions were 30 µM, 5 µM, and 20 µM for PIM1, PIM2, and PIM3, respectively. Serial dilutions of the compound (1:3) were prepared over a range from 10 µM to 0.0005 µM in reaction buffer (final reaction concentration of 1% DMSO). Kinase reactions were incubated at room temperature for 90 min, and then the reactions were stopped by the addition of 20 µL of IMAP binding reagent (solution containing 75% Buffer A: 25% Buffer B, and a 1 in 600 dilution of beads). After quenching, the plates were incubated at room temperature for 2 h, and then the fluorescence polarization measurements were carried out on an Infinity F200 (Tecan, Männedorf, Zürich, Switzerland). The percentage of inhibition was calculated for each compound concentration and the data were fitted to the equation of a 4-parameter logistic curve. IC 50 values were obtained using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, USA).

Kinase Profiling
To test the specificity of the novel PIM kinase inhibitor KMU-470, a kinase profiler service was conducted by Eurofins Cerep S.A. The screening was performed with 1 and 10 µM of compound and a concentration of ATP at the Km value for each individual kinase and kinase substrate according to Eurofin's protocols.

Cell Culture
RAW 264.7 murine macrophage cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). RAW 264.7 cells were cultured in DMEM (GIBCO, BRL, Grand Island, NY, USA) with 10% fetal bovine serum (Welgene Inc, Daegu, Korea) and 1% antibiotics. The cells were grown at 37 • C, 5% CO 2 in fully humidified air.

Measurement of NO Production
The amount of nitrite accumulated in the cell culture medium was measured directly using a Griess reagent as an indicator of NO production. RAW 264.7 cells were seeded 0.5 × 10 6 cells/well on 12-well plates for 24 h. The cells were treated with a 1 µM concentration of KMU-470 for 30 min before LPS stimulation; they were stimulated with three different concentrations of LPS at 50, 200, and 1000 ng/mL for 24 h. Cell culture medium (100 µL) was mixed with an equal volume of Griess reagent and incubated for 10 min at room temperature. The OD was measured by spectrophotometer (BMG Labtech, Ortenberg, Germany) at 540 nm. All data are shown as the mean ± standard deviation of three experiments.

Western Blot Analysis
RAW 264.7 cells were pre-treated with KMU-470 (0.5-1 µM/mL) for 24 h and stimulated with LPS (50 ng/mL) for 6 h. After stimulation, total cells were harvested, washed with PBS buffer, and then the total protein was extracted with a RIPA buffer (0.1 mM sodium orthovanadate, 137 mM NaCl, 15 mM EGTA, 0.1% Triton X-100, 25 mM MOPS, 15 mM MgCl 2 , 100 µM phenylmethylsulfonyl fluoride, and 20 µM leupeptin, adjusted to pH 7.2). The cell culture media supernatants were precipitated using TCA (trichloroacetic acid, protocol by Luis Sanchez 2001). A BCA protein assay kit (Thermo Scientific, Wilmington, USA) was used for the determination of the protein concentration. Cell lysates of equal protein concentrations were prepared in 5× sample buffer (ELPIS Bio Technology, Daejeon, Korea). Lysates were separated by 10% SDS-PAGE gel electrophoresis and then transferred onto Immobilon-P membranes (Millipore Corporation, Bedford, MA, USA). The membranes were rinsed and incubated with blocking buffer (5% non-fat milk in 1× TBS/0.1% Tween ® 20) for 1 h. Then, the membranes were incubated with primary antibodies overnight at 4 • C and then with horseradish peroxidase-conjugated secondary antibodies for 1 h. Immunoreactive proteins were detected using the ELC Western blot detection system kit (Millipore). Signal intensity was measured using the Chemi Image documentation system (Fusion Fx7, VILBER LOUTMAT). Protein expression levels were quantified using the Image J program.

Small Interfering RNA Transfection
The RAW 264.7 cells were seeded in six-well plates and transfected with PIM-1 siRNA (s10527, Life Technologies) or control siRNA using a transfection reagent (Santa Cruz biotechnology). All transfections were carried out with Lipofectamine RNAiMAX (Life Technologies). RAW 264.7 cells were transfected with 30-45 nM siRNA per well. After 72 h, cells were used for further experiments.

Statistical Analysis
All data are presented as the mean and standard error of mean (SEM) and experiments were repeated three times. Data analysis was performed using SPSS 25.0 software program. A p-value < 0.05 was considered significant. Acknowledgments: We would like to thank all the members of our research group for their enthusiastic participation in this study.

Conflicts of Interest:
The authors have no conflicts of interest to declare.