Anti-rheumatic property and physiological safety of KMU-11342 in in vitro and in vivo models

Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disorder characterized by joint destruction due to synovial hypertrophy and the infiltration of inflammatory cells. Despite substantial progress in RA treatment, challenges persist, including suboptimal treatment responses and adverse effects associated with current therapies. This study investigates the anti-rheumatic capabilities of the newly identified multi-protein kinase inhibitor, KMU-11342, aiming to develop innovative agents targeting RA. In this study, we synthesized the novel multi-protein kinase inhibitor KMU-11342, based on indolin-2-one. We assessed its cardiac electrophysiological safety using the Langendorff system in rat hearts and evaluated its toxicity in zebrafish in vivo. Additionally, we examined the anti-rheumatic effects of KMU-11342 on human rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS), THP-1 cells, and osteoclastogenesis in RAW264.7 cells. KMU-11342 demonstrated the ability to inhibit LPS-induced chemokine inhibition and the upregulation of pro-inflammatory cytokines, cyclooxygenase-2, inducible nitric oxide synthase, p-IKKα/β, p-NF-κB p65, and the nuclear translocation of NF-κB p65 in RA-FLS. It effectively suppressed the upregulation of NLR family pyrin domain containing 3 (NLRP3) and caspase-1 cleavage. Furthermore, KMU-11342 hindered the activation of osteoclast differentiation factors such as RANKL-induced TRAP, cathepsin K, NFATc-1, and c-Fos in RAW264.7 cells. KMU-11342 mitigates LPS-mediated inflammatory responses in THP-1 cells by inhibiting the activation of NLRP3 inflammasome. Notably, KMU-11342 exhibited minimal cytotoxicity in vivo and electrophysiological cardiotoxicity ex vivo. Consequently, KMU-11342 holds promise for development as a therapeutic agent in RA treatment.


Introduction
Rheumatoid arthritis (RA) is characterized by joint damage stemming from the degradation of the extracellular matrix by infiltrating immune cells [1].The pathogenesis of RA involves various cells, including activated macrophages, neutrophils, and synoviocytes.Moreover, chondrocytes and fibroblasts, T cells, and B cells, contribute to the production of chemokines and inflammatory cytokines, disrupting immune quilibrium and leading to damage in cartilage and bone [2].Hence, RA's to variable and often unsatisfactory drug responses patients [4].Therefore, developing effective new streatments for RA remains a formidable challenge.
In this study, we developed the novel multiple protein kinase inhibitor, KMU-11342, we assessed its electrophysiological safety and toxicity in vivo and ex vivo, and showcased its anti-rheumatic potential by revealing its anti-inflammtory mechanisms in vitro studies, in primary human RA fibroblast-like synoviocytes (RA-FLS) and by hindering osteoclastogenesis in RAW264.7 cells.

Isolating and culturing fibroblast-like synoviocytes from primary human rheumatoid arthritis (RA-FLS)
RA-FLS were sourced from the Division of Rheumatology, Department of Internal Medicine, under the supervision of Dr. Chang-Nam Son at Keimyung University.Primary cell isolation was conducted using tissues obtained from patients undergoing bone replacement surgery to address damage incurred during the procedure, specifically those with osteoarthritis.All patients provided prior informed consent for the utilization of their tissues, and the study was conducted in accordance with the guidelines of Keimyung University Dongsan Hospital [IRB No. 2020-11-031], as approved by the Keimyung Academic Review Board.Tissues, encompassing fat, ligaments, and cartilage, were meticulously dissected by skilled technicians within the institution.Subsequently, the dissected tissues were placed in a 100-mm dish filled with cell-culture phosphate-buffered saline (PBS) and thoroughly cleansed with DMEM (Welgene, Gyeongsan, Korea).The osteoarthritic tissues were then carefully crushed into 2-3 mm pieces, and 4 mg/mL type-1 collagenase (Worthington Biochemical, Freehold, NJ, USA) was introduced, followed by incubation under 5% CO 2 at 37 °C for 4 h.After centrifugation (1,200 rpm, 10 min), the isolated cells were resuspended in DMEM supplemented with 10% FBS, 2 mmol/L glutamine, penicillin (100 U/ mL), and streptomycin (100 mg/mL), and subsequently placed in rotation within a 75 cm 2 flask.Following a day, the adherent cells were maintained in a controlled environment of 5% CO 2 at 37 °C in DMEM containing 10% FBS.The culture medium was replenished every three days.Upon reaching 90 -95% confluency, the medium was diluted at a 1:3 ratio with fresh culture medium, and the cells were serially cultivated before being preserved in a nitrogen tank.designated concentrations of KMU-11342 at 24 h-post fertilization, HPF.KMU-11342's toxicity was assessed based on morphological changes in the embryos, employing a modified version of the fish embryo acute toxicity (FET) test ( OECD Guideline 236 (No, 2013)).Paramenters such as hatchability, body length, average heart rate per minute, and the visual appearance of treatment and solvents were meticulously analyzed.Photographic documentation was performed using a light microscope (Carl Zeiss Stereo microscope DV4, Seoul, Republic of Korea) to accurately quantify viable zebrafish.

Cell cultivation and osteoclastogenesis
Cell cultures were maintained continuously throughout the duration of experiment.The THP-1 cell line, consisting of human monocytic leukemia cells, and the RAW264.7 cell line, comprising murine macrophage cells, were the primary cells used in this study.THP-1 cells were plated at densities tailored to each specific experiment and incubated for 24 h with 100 nM PMA in the medium.Both cell lines, THP-1 and RAW264.7 were obtained from the Korea Cell Line Bank (Seoul, Korea) and cultured in Roswell Park Memorial Institute (RPMI1640; Welgene, Gyeongsan, Korea) and Dulbecco's Modified Eagle Medium (DMEM; GIBCO, BRL, Grand Island, NY, USA) respectively.The culture medium was supplemented with a 1% antibacterial-antifungal solution (GIBCO, BRL, Grand Island, NY, USA) and 10% fetal bovine serum (FBS; GIBCO, BRL, Grand Island, NY, USA) containing of penicillin and streptomycin.Conditions within the incubator were regulated at 95% humidity and 37 °C, with a constant 5% CO 2 .Cells were sub-cultured weekly or bi-weekly.For the exploration of osteoclast differentiation, RAW264.7 cells were seeded at density of 4 × 10 4 cells/well in 12-well plates.Differentiation was induced over a 5-day period with α-minimum essential medium (MEM; Welgene, Gyeongsan, Korea) supplemented with 50 ng/mL RANKL.The culture medium was refreshed every three days.Groups undergoing treatment were pre-treated with KMU-11342 at concentrations of 0.01, 0.25, and 0.5 µM for 1 h prior to RANKL administration.

Kinase-profiling analysis
The kinase inhibitor KMU-11342, underwent kinase-profiling at Eurofins Cerep S.A. (Celle-Lévescault, France).The profiling was conducted in alignment with the manufacturer's established protocol.In the analyses of individual kinase and their substrates, the compound was used at a concentration of 1 µM/mL, alongside ATP at

Left ventricle pressure (LVP) and electrocardiogram (ECG) recordings
LVP, was measured by placing a water-filled latex balloon attached to a metal cannula, inside the left ventricle via the pulmonary vein, which was then connected to a pressure transducer (EMKA Technologies).ECG readings were taken using two surface electrodes (EMKA Technologies) positioned against the epicardium.One electrode was placed on the right ventricle near the atrium-ventricle ring, and the other on the left ventricle.All hemodynamic parameters were captured and analyzed using iox2 software (EMKA Technologies) for a duration of 10-15 min before and after reperfusion with varying concentrations of KMU-11342 in the buffer.QTc intervals in milliseconds were computed using Bazett's formula (QTcB = QT/RR 1/2 , Fridericia: QTcF = QT/RR 1/3 ).KMU-11342 administered in escalating concentrations from 0.1 to 5 µM.The study protocols were reviewed and sanctioned by the Institutional Animal Care and Use Committee (IACUC) of the Korea Institute of Toxicology (KIT) under the code RS21006.

Toxicology test in zebrafish
Zebrafish (Danio rerio) were sourced from the Zebrafish International Resource Center (University of Oregon, Eugene, OR, USA) and maintained in accordance with the guideline [5].In briefly, the fish were fed standard diets of feed, and raw brine substituted shrimp.These fish were cultured in UV-treated water under the following conditions.The aquatic environment was strictly regulated: temperature, at 28.5 °C ± 1 °C; pH, 6.9-7.5; and a circadian, rhythm of a 14 h light/10 h dark cycle.Post natural mating, zebrafish embryos were collected rinsed thoroughtly, and carefully inspected.The embryos were then exposed to boiling for 10 min at 95 °C before loading onto a polyacrylamide gel.Equal amounts of protein were subjected to electrophoresis in 10% SDS-polyacrylamide gels and subsequently transferred to nitrocellulose membranes.Following a blocking step with 5% skim milk for 1 h, the membranes were incubated for 24 h with appropriately diluted primary antibodies specific to the target proteins of interest.Post-incubation, the membranes were washed with Tris-buffered saline/Tween buffer and then subjected to a 1 h incubation at room temperature with HRPconjugated anti-IgG secondary antibodies.Protein bands were visualized using enhanced chemiluminescence, and the signal intensity was quantified with a Chemi Image System (Fusion Fx7, Vilber Lourmat, Collégien, France).Notably, β-actin served as the internal control for the Western blot analysis.

RNA isolation and quantitative real time PCR
To analyze gene expression related to pro-inflammatory cytokines and chemokines at the mRNA level through realtime quantitative PCR (qPCR).THP-1 cells were cultured by uniformly seeding a consistent cell count in 6-well dishes, each receiving an identical number of cells, and treated with KMU-11342 at concentrations of 0.1, 0.5, and 1 µM/mL for 1 h.Subsequently, the cells were stimulated with LPS for a duration of 6 h.Total RNA was extracted from each sample using TRIzol solution (Invitrogen, San Diego, CA, USA), following the manufacture's protocol.Subsequently, 1 µg of the extracted and quantified RNA underwent reverse transcription, employing deoxynucleoside triphosphate (dNTP), buffer, dithiothreitol, RNase inhibitor, and SuperScript II reverse transcriptase.The resultant cDNA was subjected to RT-PCR using specific primers, and the outcomes were analyzed.The primers utilized in this study are detailed in Table 1.

Immunofluorescence staining
RA-FLS and THP-1 cells (1 × 10 3 ) were cultured on 8-chamber glass slides for 24 h.The cells were then treated with KMU-11342 at concentrations of 0.1, 0.5, and 1 µM/mL for 1 h, followed by LPS stimulation for 6 h.The cells were meticulously washed with PBS, fixed using 4% formaldehyde, and permeabilized with 0.2% Triton X-100 in PBS.To block nonspecific binding, the cells were incubated with bovine serum albumin (BSA) for 1 h.Thereafter, they were incubated with primary antibodies at 4°C for 24 h.Subsequent steps involved incubation with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Thermo Scientific, Wilmington, DE, USA) and 4'-6-diamidino-2-phenylindole (DAPI) the Km value.In brief, each kinase assay involbed a reaction mixture comprising 8 mM/L MOPS at pH 7.0, 0.2 M EDTA, 50 µM/L EAIYAAPFAKK, and 10 mM/L acetate.
[Υ-33P] ATP was incorporated tailored to specific activity and concentration requirements, was introduced, and the ensuing response to the added Mg/ATP mixture was precisely measured.The reaction was allowed to proceed at room temperature for 40 min.Subsequently, phosphoric acid 0.5% was introduced to halt the reaction.Then, 10 µL of the reaction mixture was applied to filter paper, which was sequentially rinsed with methanol and phosphoric acid, followed by drying.The dried samples were subjected to scintillation counting for 4 min, utilizing a P30 filter matrix.

Measurement of cytotoxicity
The intracellular toxicity induced by the synthetic compound KMU-11342 was scrutinized through the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2 H-tetrazolium-5-carboxanilide (XTT) assay.THP-1 and RAW264.7 cells (1 × 10 5 cells) were seeded in 96-well plates, treated with varying concentrations of KMU-11342, and incubated in a controlled culture environment at 37 °C for 24 h.Following this incubation period, the culture solution was augmented with XTT solution, and cell survival was evaluated on the subsequent day.The XTT, utilized at a concentration of 0.5 mg/ mL, underwent reaction under identical culture conditions for 2 h.Post-reaction, the absorbance of the supernatant was quantified at 450 nm.

Analysis of western blotting
To validate the modulation of expression levels in inflammation-related signaling proteins elicited by KMU-11342, Western blotting was systematically conducted.RA-FLS and THP-1 cells were seeded at densities of 1 × 10 6 cells/well, and 2 × 10 6 cells/well, respectively, in 100 mm and 60 mm dishes.Following a 24 h culture period, the cells were treated with KMU-11342 at concentrations of 0.1, 0.5, and 1 µM/mL for 1 h, and then stimulated with LPS for 6 h.Total protein was extracted from the treated using RIPA buffer solution (Cell Signaling, Technology, Beverly, MD, USA).Protein concentrations were determined using a bicinchoninic acid (BCA) assay kit (Thermo Scientific, Wilmington, DE, USA).The supernatants of the cell culture media were precipitated using a trichloroacetic acid (TCA), according to the protocol by Luis Sanchez (2001).After the precipitation step, SDS-PAGE was performed, wherein the samples were dissolved in 20 µL of 2× or 4× sample buffer (with or without β-mercaptoethanol), followed by Tartrate-resistant acid phosphatase staining (TRAP) and F-actin ring staining RAW264.7 cells were seeded in 12-well culture dishes and treated with 50 ng/mL RANKL or pre-treated with KMU-11342 (at concentrations of 0.01, 0.1, and 0.25 µM/mL) for 1 h.Following a five-day cultivation period, the cells underwent TRAP staining using a leukocyte acid phosphatase kit (TaKaRa.Bio, Kyoto, Japan) follosing the manufacturer's instructions.The cultured cells were fixed in a fixation solution for 20 min at room temperature and subsequent washing with distilled water.Microscopic examination identified cells with three or more nuclei as multinuclear osteoclasts.For F-actin ring staining, RAW264.7 cells were again plated in a 12-well culture dish and stimulated with 50 ng/mL RANKL for 6 days.Staining was accomplished using Phalloidin Cru-zFluor™488 Conjugate (Santa Cruz, CA, USA).Following a wash with PBS, cells were fixed in 4% neutral buffered formalin for 20 min at room temperature.Subsequent steps involved cell permeabilization for 10 min with 0.1% Triton X-100 in PBS, blocking with 1% BSA for 1 h at room temperature, and incubation with the stain antibody (1:5000 dilution) for 30 min.After triple washing with PBS, the stained cells were captured through imaging using a fluorescence microscope.

Bone resorption assay
The osteoclasts were generated using the forementioned method and cultured on Osteo Assay Surface multi-well plates (CSR-BRA-24KT, Cosmo Bio co.LTD, Japan) for 7 days.Various concentrations of KMU-11342 were introduced to the cultures from day 1.After 7 days, the plates were washed with saline and a solution of 5% sodium hypochlorite solution was applied for 5 min to detach the cells.The resorption areas were visualized under a microscope.Comparative analysis of each group was performed using ImageJ software.

Mitochondrial bioenergetics assay
Mitochondrial respiration and extracellular acidification rate (ECAR) were assessed using the Seahorse XF96e Extracellular Flux analyzer (Seahorse Biosciences, North Billerica, MA, USA).RAW264.7 cells were pretreated with KMU-11342 for 1 h, stimulated with 100 ng/mL LPS for 2 h, and then treated sequentially in the following sequence: The Oxygen consumption rate (OCR) was then measured after sequentially after the addition of 1 µM oligomycin (complex V inhibitor) at baseline, 0.5 µM, and 1 µM carbonyl cyanide 4-(trifluoromethoxy) for nuclear staining (Vector Laboratories, Burlingame, CA, USA).Finally, the stained cells were meticulously examined under a fluorescence microscope to capture and analyze the immunofluorescence patterns.CA, USA), according to the manufacturer's instructions.Analysis of mtDNA content to obtain the mtDNA/nDNA ratio was used the real time qPCR.Different genes are used to evaluate the relative copy number of mtDNA and nDNA.We determined mitochondrial genes using 16 S ribosomal RNA and cytochrome b, and the nuclear encoded genes using hexokinase 2.

Results
Cardiotoxicity and safety profiling of a multi-protein kinase inhibitor, KMU-11342 in zebrafish in vivo and rat heart ex vivo model KMU-11342 was synthesized from 5-bromoisatin as shown in Fig. 1A.Compound 1 was derived through reduction and Miyaura borylation reaction, and its Suzuki coupling with the pyrazine derivative, 2, yielded compound 3.The final product, KMU-11342, was formed via Knoevenagel condensation of compound 3 with 1H-imidazole-4-carbaldehyde.
We further investigated the inhibitory effect of KMU-11342 on inflammatory kinases.Kinase panel screening demonstrated that KMU-11342 inhibited the activities of various kinases, including IKKα(h), IKKβ(h), JAK1(h), JAK2(h), JAK3(h), MAPK1(h), JNK3(h), TAK1(h), and TYK2(h) with percentages of inhibition at 1 µM concentration being 98, 53, 76, 79, 99, 97, 94,71, and 93%, respectively (Table 2).These results underscore KMU-11342's potent inhibitory activity against inflammatory protein kinases.As drug-induced cardiotoxicity is a significant factor in the discontinuation or withdrawal of commercial drugs from the market [6], in vivo LVP assays are crucial for ensuring cardiac safety during drug development [7].To evaluate the potential cardiotoxicity of KMU-11342, we employed the langendorff heart system for LVP, and electrocardiogram (ECG) analysis.Our analysis of cardiotoxicity factors including heart phenylhydrazone (fCCP, electron transport chain separator), and ultimately 1 µM rotenone (complex III inhibitor) and 1 µM antimycin A (complex I inhibitor).OCR measurements were taken in 8 wells for each condition at three distinct time points across the four evaluation.Alternatively, an XF cell mito stress test (Agilent Technologies) was performed according to the manufacturer's instructions.All OCR data were normalized to total protein per well measured using the Bradford assay.

Mitochondrial DNA copy number
Total DNA was extracted from RAW264.7 cell samples using the DNeasy Blood and Tissue kit (Qiagen, Valencia, cardiotoxicity induced by KMU-11342 (Fig. 1C).We also assessed the toxicity of KMU-11342 in zebrafish embryos.Observations showed no significant alterations in key phenotypes of embryos exposed to KMU-11342 up to a concentration of 1 µM.Image analysis demonstrated no changes in brain growth, eye size, yolk sac swelling, or dorsal curvature of the body axis in a dose-dependent rate, developed pressure, maximal rates of contraction and relaxation (dP/dtmax and dP/dtmin), and ECG waveform parameters RR-interval (RR-I), QT-interval (QT-I), QT correction Bazett (QTcB-I) and Fridericia (QTcF-I), revealed that KMU-11342 did not affect any hemodynamic parameters in LVP analysis (Fig. 1B).Moreover, ECG analysis did not indicate any dose dependent Human RA-FLS (Fig. 3C-F).Further investigation confirmed the anti-inflammatory efficacy of KMU-11342 was undertaken.Pre-treatment of both human RA-FLS and THP-1 cells with KMU-11342 resulted in the inhibition of LPS-induced up-regulation of pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6 (Fig. 3G-N).

KMU-11342 attenuated LPS-induced activation of MAPKs, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) pathway in human RA-FLS and THP-1 cells
COX-2 and iNOS are pivotal in mediating inflammatory response.The pre-treatment with KMU-11342 curtailed the LPS-induced up-regulation of COX-2 and iNOS in both human RA-FLS and THP-1 cells (Fig. 2).However, KMU-11342 did not suppress the protein expression level of COX-1 (Fig. 2C, F), indicating a selective inhibitory effect of KMU-11342 on COX-2.Subsequently, we explored whether KMU-11342 could inhibit MAPKs, which are upstream regulators of the pro-inflammatory cytokines, COX-2, and iNOS.The results indicated that KMU-11342 effectively inhibited the LPS-stimulated activation of JNK, but not of ERK and p38, in THP-1 cells (Fig. 2G).These findings underscore the potential of KMU-11342 in selectively modulating key inflammatory pathways, offering insights into its therapeutic relevance in mitigating inflammation-associated responses.

KMU-11342 inhibits LPS-stimulated inflammatory mediator production in human RA-FLS and THP-1 cells
To assess the potential cytotoxic effects of KMU-11342 in THP-1 cells, an XTT assay was conducted.The results indicated that KMU-11342 exhibited no cytotoxicity at concentrations up to 1 µM (Fig. 3A).Further, to gauge the potential of KMU-11342 as an RA therapeutic, we compared its anti-inflammatory activity with various other RA and anti-inflammatory agents noting the potential superiority of KMU-11342.Specifically, THP-1 cells pre-treated with 1 µM KMU-11342 exhibited inhibited LPS-induced up-regulation of IL-1β, a response not observed even at higher concentrations of other drugs tested (Fig. 3B).Additionally, KMU-11342 exhibited a significant inhibitory effect on the LPS-stimulated up-regulation of chemokine mRNA levels, including CXCL10, CCL2/MCP-1, CCL3/MCP-1α, and CCL4/MCP-1β, in Fig. 3 Inhibitory effect of KMU-11342 on LPS-mediated up-regulation of inflammatory mediators in human RA-FLS and THP-1 cells.

KMU-11342 upregulates the gene expression involved in mitochondria biogenesis pathway and increases mitochondrial DNA (mtDNA) copy number
To determine the impact of KMU-11342 on energy metabolism during macrophage-mediated inflammatory responses, mitochondrial oxygen consumption rates were measured in macrophages using Seahorse extracellular analyzer.Inflammation induced by LPS compromised basal/maximal respiratory rates, protein leakage, spare respiration capacity, and mitochondrial ATP production (Fig. 6A).Conversely, KMU-11342 preserved mitochondrial function by promoting maximal cellular respiration (Fig. 6A, B).These results suggest that KMU-11342, known for its anti-inflammatory properties, also contributes to mitochondrial function preservation.The LPS-treated group displayed a metabolic shift from a quiescent state to aerobic pathway under stress conditions.However, the KMU-11342-treated group exhibited only a minor slight shift in energy metabolism from quiescent to an aerobic pathway (Fig. 6C).Moreover, the potential role of KMU-11342 in glycolysis regulation was that KMU-11342 may inhibit the activation of NLRP3 inflammasome and the recruitment of ASCs, although it does not affect protein expression.Consequently, these findings illustrate that KMU-11342 impedes the LPSmediated activation of the TAK1-NF-κB-NLRP3 inflammasome pathway.

KMU-11342 attenuates receptor activator of nuclear factors κB ligand (RANKL)-induced osteoclast differentiation, up-regulation of osteoclastogenesis-associated factors, and activation of TAK1/MAPKs/NF-κB Signal pathways
The persistent progression of rheumatoid arthritis results in the inevitable destruction of bones and cartilage.So, we investigated whether the inhibitory effect of KMU-11342 on osteoclast differentiation, a process pivotal to bone resorption, and explore its influence on F-actin ring formation-an essential indicator of bone resorption.Osteoclast differentiation was induced in RAW264.7 cells using RANKL (50 ng/mL) over a period of 5 days.Immunofluorescence analysis demonstrated a dose-dependent suppression by KMU-11342 of the formation of TRAP-positive polynuclear osteoclasts and the extent of F-actin ring formation (Fig. 5A).Furthermore, KMU-11342 treatment significantly dose-dependently reduced the count of TRAP-positive osteoclasts (Fig. 5C) and exhibited no cytotoxicity up to a concentration of 0.5 µM in RAW264.7 cells (Fig. 5B).The bone resorption assays indicated that KMU-11342 decreased The visualized ASC speck formation using immunofluorescence analysis with anti-ASC (red) antibody in THP-1 macrophages pretreated with KMU-11342 (0.5 and 1 µM) for 1 h followed by treatment with LPS (1 µg/ml) for 2 h and ATP (2mM) for 30 min.Arrows indicate ASC specks.DAPI (blue) was used as a nuclear marker.Scale bar: Subsequently, the influence of KMU-11342 mitochondrial biogenesis genes was investigated by examining genes related to this process, including Peroxisome proliferatoractivated receptor gamma coactivator 1-alpha (PGC-1α), explored by assessing it effect on glycolytic actibity in LPSexposed macrophages.KMU-11342 was found to counteract the LPS-induced reduction in glycolysis and suppress glycolytic reserve in RAW264.7 macrophages (Fig. 6D).
innovative medications continues due to the limited efficacy of adverse effects of existing treatments [9].A deeper understanding of RA pathogenesis is crucial for enhancing treatment effectiveness, particularly in managing pathological aspects such as bone erosion and regulating the inflammatory signaling pathways of implicated immune cells [9].In this study, we introduced a novel multi-protein kinase inhibitor, KMU-11342, showcasing its potential in mitigating inflammation in various immune cells implicated in RA pathogenesis and in inhibiting osteoclastogenesis.Additionalyy, KMU-11342's drug development prospects are further supported by its demonstrated low cytotoxicity in vivo studies using zebrafish and its electrophysiological safety confirmed through ex vivo studies on rat hearts.KMU-11342, a 3,5-disbustituted indolin-2-one derivative of 3-((1H-imidazol-5-yl)methylene)-5-(6-(cyclopentylamino)pyrazin-2-yl)indolin-2-one, emerged from screening our proprietary kinase inhibitor library for anti-inflammatory.The indolin-2-one scaffold is pivotal in kinase.Inhibitor discovery, as evidenced by its role in the development of analog such as sunitinib and nintedanib, used in cancer and idiopathic pulmonary fibrosis treatments, respectively [10].Morever, indolin-2-one-based anti-inflammatory inhibitors have been reported in recent literature [11][12][13][14].Significantly, KMU-11342 exhibited potent inhibition of protein kinases associated with inflammation (Table 2).Cardiovascular toxicity is remains pivotal challenge in the development and clinical application of drugs, including kinase inhibitors [15].Consequently, a thorough assessment of potential torsade de Points (TdP), a severe form of ventricular arrhythmia associated with delayed ventricular repolarization is crucial for ensuring the safety of new therapeutics [16,17].
The international conference on Harmonisation (ICH) has released S7B and E14 guidelines for comprehensive cardiotoxicity evalutions, such as LVP and ECG to assess the risk of arrhythmia induction [17,18].In this study, the electrophysiological safety of KMU-11342 was validated through ECG, and LVP analyses (Fig. 1A, B).While traditional models for drug toxicity evaluation have predominantly used higher animals zebrafish have emerged as a viable vertebrate model for initial drug screening, assessing developmental and general toxicity [19].Consequently, zebrafish were employed in this study to ascertain the developmental toxicity of KMU-11342.The results indicated minimal cardiotoxicity and non-lethal effects on zebrafish embryos, including the absence of pathological changes in major organs during development.Recent evidence spanning over the past decade emphasizes the critical role of Toll-like receptors (TLRs) in the onset of various rheumatic diseases [20].Reports have particularly highlighted the involvement of TLRs in RA pathogenesis [21] with TLR4 being notably overexpressed in the inflamed synovium of RA patients PGC-1β, citrate synthase (CS), and mitochondrial transcription factor A (TFAM).KMU-11342 markedly reversed the LPS triggered down-regulations of genes associated with mitochondrial biogenesis (Fig. 6E-H).Furthermore, the study evaluated the association between KMU-11342 treatment and changes in total mitochondrial mass, revealing that KMU-11342 alleviated the LPS-induced decrease in the mtDNA copy numbers ratio (Fig. 6I, J).

Discussion
Rheumatoid arthritis (RA) stands as a prevalent, persistent autoimmune disorder marked by chronic inflammation of the joints, frequently leading to gradual joint deterioration and functional impairment [1].Although various drug have been developed to counteract RA [8], the quest for underscore the necessity for further drug development in RA treatment.KMU-11342 exhibited superior antiinflammatory efficacy compared to celecoxib, ibrutinib, methotrexate, and tofacitinib (Fig. 3B).While additional research is essential, these findings position KMU-11342 as a promising drug candidate for RA treatment.RA manifests as a systemic inflammatory joint disorder, characterized by the intricate interplay of pathophysiological processes encompassing the activation of RA-FLS, osteoclastogenesis, and synovial membrane inflammation [39].Notably, FLS contributes to many pathological aspects of RA, such as synovitis, pannus growth, and ultimately cartilage/bone destruction, by promoting them [40].Pro-inflammatory cytokines and chemokines, integral to TLRs signaling, intricately contribute to the pathogenesis of RA, serving as pivotal mediators released by RA-FLS in response to cytokines and TLR ligands [21,40].Therefore, controlling FLS could be a promising target for treating RA, as it may lead to an appropriate therapeutic effect for the disease.As shown in Fig. 3C -F, in human RA-FLS, KMU-11342 inhibited the LPS-induced production of various chemokines, such as CXCL10, CCL2, CCL3, and CCL4, which are highly expressed in RA [41].Moreover, macrophages central to RA pathophysiology, produce pro-inflammatory cytokines that contribute to chronic inflammation, tissue damage, and pain in affected joints [42].KMU-11342 effectively suppressed the LPS-induced upregulation of IL-1β, IL-6, and TNF-α in both RA-FLS and THP-1 cells.These findings strongly indicate the anti-inflammatory properties of KMU-11342.NF-κB, a nuclear transcription factor, orchestrates the expression of diverse inflammatory mediators, encompassing inducible enzymes like COX-2 and iNOS, as well as TNF-α, IL-1β, IL-6, and various cell-adhesion molecules [43].As shown in Fig. 4B -E, KMU-11342 effectively curtailed LPS-induced phosphorylation of IKKα/β and impeded the phosphorylation and subsequent nuclear translocation of NF-κB p65 in both RA-FLS and THP-1 cells.NLRP3 expression, regulated by NF-κB binding to the NLRP3 promoter [44], and the NLRP3 inflammasome, implicated in RA development, play a pivotal role in caspase-1 activation, which mediates the maturation and secretion of IL-1, a proinflammatory cytokine [45,46].KMU-11342 attenuated the synergistic effect of LPS and ATP on caspase-1 activation and NLRP3 upregulation in THP-1 cells (Fig. 4F).When NLRP3 inflammasome is activated, the adapter protein ASC assembles into large protein complexes known as ASC specks or pyroptosomes [47], which is considered a typical hallmark of inflammasome activation.In other words, the activation of NLRP3 inflammasome is essential for the oligomerization of ASC, which [22].TLR4 signaling is mediated through the recruitment of specific adaptor molecules and occurs via pathways including mitogen-activated protein kinases (MAPKs), nuclear factor kappa B (NF-κB), and janus family tyrosine kinase (JAK)-STAT [23][24][25].Understanding and modulating these pathways thereby controlling their overactivation can significantly contreibute to the treatment of inflammatory diseases.Accordingly, we investigated the anti-inflammatory mechanisms of KMU-11342 within an LPS-induced TLR4-mediated inflammatory activation.Prostaglandins (PGs), derived from arachidonic acid, are lipid mediators of inflammation that play significant role in the pathogenesis of RA and are considered potential therapeutic targets [26].COXs are crucial enzymes in synthesizing PGH 2 , the precursor of various PGs.There are two primary isoforms in mammals, COX-1 and COX-2 [27].Significantly, numerous studies have shown elevated expression of COX-2 in the synovial tissues of RA patients [28][29][30].Furthermore, COX-2 selective inhibitors represent a novel therapeutic approach, offering effectively control of inflammation and pain with fever side effects, such as gastrointestinal irritation [31].Additionally, the NOS/nitric oxide signaling pathway has been increasingly implicated in RA pathogenesis [32].KMU-11342 demonstrated inhibition of LPS-induced up-regulation of COX-2 and iNOS in both human RA-FLS and THP-1 cells (Fig. 2A-F).MAPKs are pivotal regulators of pro-inflammatory cytokine synthesis and are present in the synovial tissues of RA patients [33].TAK1, and MAP3K7, key mediators in innate and adaptive immune responses, integrate inflammatory signals mediated by cytokines, TLR, and T and B cell receptors [34,35].In this investigation, KMU-11342 was observed to inhibit the LPSinduced phosphorylation of JNK and TAK1 in Figs.2G and 4A, respectively.These findings suggesting its potential as an anti-inflammatory agent targeting key inflammation regulators.
Recent developments in anti-inflammatory pharmacotherapy have introduced drugs targeting diverse inflammatory signaling pathways [36].Methotrexate, for instance, has been a cornerstone in RA treatment since the 1980s.Agents blocking pro-inflammatory cytokines, such as TNF-α inhibitors (adalimumab, etanercept, certolizumab, infliximab, and golimumab), interleukin-1 (IL-1) antagonists (anakinra), and IL-6 inhibitors (toxylizumab), has become a standard practice [37].Moreover, tofacitinib, a small molecule inhibitor targeting JAK1, JAK3, and JAK2, marked a significant milestone as the kinase inhibitor approved in the United States [38].However, the long-term administration of biological agents for RA poses increased risks of cancer and infections, along with drug reactions and various other issues associated with drug resistance and chronic use.These challenges extensive investigations to date, the identification of pivotal therapeutic targets crucial for the clinical efficacy of targeted drugs in rheumatoid arthritis (RA), including cytokines with diverse and multifaceted expression activities and kinases involved in upstream immune synapses or signaling pathways, holds significance [62].Our study strongly asserts that KMU-11342, despite being a multifunctional kinase, exhibits substantial potential for the treatment of RA.

Conclusion
In summary, this study explored the anti-rheumatic properties of KMU-11342 by inhibiting the TLR4/NF-κB/NLRP3 inflammasome signaling in LPS-stimulated RA-FLS and THP-1 cells, and by attenuating osteoclast differentiation.Importantly, KMU-11342 exhibited minimal in vivo cytotoxicity and ex vivo electrophysiologic cardiotoxicity.Hence, the findings suggest that KMU-11342 holds promise as a prospective therapeutic drug for rheumatoid arthritis.
generates speck formation [48].As shown in Fig. 4G, KMU-11342 inhibited inflammasome activation as represented by ASC speck formation.These findings suggest that KMU-11342, inhibiting NF-κB activity, holds potential as a therapeutic agent for the RA treatment.
It also suppressed the RANKL-induced enhancement of NFATc-1 and MMP-9 mRNA and protein levels (Fig. 5H, I, K, L) and decreased Cathepsin K, TRAP, and OSCAR mRNA levels (Fig. 5G, J), which are specific markers of osteoclast differentiation.Furthermore, KMU-11342 impeded the phosphorylation of TAK1, a precursor of NF-κB, vital for RANKL-induced osteoclastogenesis (Fig. 5M).Notably, the stimulation of TLR and pro-inflammatory cytokines within the MAPK pathway influences bone cell adhesion, migration, fusion, survival, and bone resorption [57].As depicted in Fig. 5N, KMU-11342 mitigated the RANKL-induced phosphorylation of ERK, JNK, and p38.Mitochondria serve as an important site of intracellular energy production, and the effects of drugs on mitochondrial function are relevant in determining drug efficacy and toxicity [58].Moreover, mitochondrial biogenesis is orchestrated by master regulators encoded genes of the nucleus and mitochondria, such as PGC-1α, PGC-1β, and TFAM, which mediate mtDNA replication [59].In particular, PGC-1α is a major regulator of mitochondrial biogenesis, and While TFAM is primarily expressed within the nucleus, it exerts its regulatory actions within the mitochondrial compartment [60].And the mtDNA copy number frequently serves as a reliable marker for assessing mitochondrial content [61].Under the conditions of LPS-induced inflammatory response, the administration of KMU-11342 resulted in both a decrease and an increase in the levels of GC-1, CS, and TFAM.Furthermore, KMU-11342 demonstrated the ability to elevate DNA copy number.These findings strongly imply that KMU-11342 exerts a stimulating effect on mitochondrial biogenesis.In

Fig. 2
Fig. 2 Inhibitory effect of KMU-11342 on LPS-induced up-regulation of COX-2 and iNOS expression in human RA-FLS and THP-1 cells.(A, B) In human RA-FLS; total RNA was extracted, and used to evaluate the mRNA expression levels of COX-2 and iNOS, respectively ** p < 0.01, # p < 0.001 compared with LPS alone treated group.(C) Whole cell lysates were isolated and used to measure the protein expression levels of COX-1, COX-2 and iNOS by Western blotting.(D, E) In THP-1 cells; total RNA was extracted, and used to evaluate the mRNA expression levels of COX-2, respectively.* p < 0.05 Fig.3 Inhibitory effect of KMU-11342 on LPS-mediated up-regulation of inflammatory mediators in human RA-FLS and THP-1 cells.(A) Cytotoxic effect of KMU-11342 in THP-1 cells was performed by XTT assay.# p < 0.001 compared with control group.Cell were differentiated into macrophages for 24 h using PMA (100 nM), then the cells were treated with different doses of KMU-11342 (0.01, 0.1, 0.5, 1, 5, and 10 µM) for 24 h.(B) THP-1 cells were differentiated into macrophages for 24 h using PMA (100 nM), and treated with LPS (1 µg/mL) for 6 h after pre-treatment with KMU-11342 (1 µM), celecoxib (25 µM), ibrutinib (5 µM), methotrexate (1 µM) and tofacinib (1 µM) for 1 h.Whole cell lysates were isolated and used to measure the protein expression levels of pro-IL-1β by Western blot analysis.(C-F) Human RA-FLS cells were treated with LPS (1 µg/mL) for 6 h after pre-treatment with different doses of KMU-11342 (0.1, 0.5, and 1 µM) for 1 h.Total RNA was extracted, and used to evaluate the mRNA expression levels of CXCL10, CCL2, CCL3, and CCL4, respectively.# p < 0.001 compared with the LPS only group.(G-N) Human RA-FLS and THP-1 cells were pre-treatment with the indicated concentrations of KMU-11342 (0.1, 0.5, and 1 µM) for 1 h and stimulated with LPS (1 µg/mL) for 6 h.(G-I) In human RA-FLS; total RNA was extracted, and used to evaluate the mRNA expression levels of IL-1β, TNF-α and IL-6, respectively # p < 0.001 compared with LPS alone treated group.(I) Whole cell lysates were isolated and used to measure the protein expression levels of IL-1β, TNF-α and IL-6 by Western blotting.(J-M) In THP-1 cells; Total RNA was extracted, and used to evaluate the mRNA expression levels of IL-1β, TNF-α and IL-6, respectively ** p < 0.01, # p < 0.001 compared with LPS alone treated group.(N) Whole cell lysates were isolated and used to measure the protein expression levels of IL-1β, TNF-α and IL-6 by Western blotting.Values in the graph (A, C-I, K-M) indicate the mean ± SD of three independent experiments

Fig. 4
Fig.4 Inhibitory effect of KMU-11342 on activation of NF-κB signals and TAK1, MAPKs and NLRP3 inflammasome in human RA-FLS and THP-1 cells.RA-FLS and THP-1 cells were pre-treated with KMU-11342 for 1 h and stimulated with LPS (1 µg/mL) for 6 h.(A) THP-1 cells were stimulated with LPS (1 µg/mL) for the indicated time points.Whole cell lysates were isolated and used to measure the protein expression levels of p-TAK1 and TAK1 by Western blot analysis.(B) In human RA-FLS; whole cell lysates were isolated and used to measure the protein expression levels of p-IKKα/β, IKKα, p-NF-κB p65 and NF-κB p65 by Western blotting.(C) Human RA-FLS were stained with antibodies to NF-κB p65 (red) and DAPI (blue) and captured at ×200 using fluorescence microscope.(scale bar = 50 μm).(D) In THP-1 cells; whole cell lysates were isolated and used to measure the protein expression levels of p-IKKα/β, IKKα, p-NF-κB p65 and NF-κB p65 by Western blotting.(E) THP-1 cells were stained with antibodies to NF-κB p65 (green) and DAPI (blue) and captured at ×200 using fluorescence microscope.(scale bar = 50 μm).(F) THP-1 cells were stimulated with LPS (1 µg/mL) and ATP (2 mM) for additional 30 min.Cell lysate (Lysate) and media supernatant (Sup) were isolated and used to measure the protein expression levels of pro-IL-1β, IL-1β, pro-caspase-1, and caspase-1 for the Sup as well as NLRP3, ASC, procaspase-1, and pro-IL-1β for the Lysate by Western blot analysis.(G) The visualized ASC speck formation using immunofluorescence analysis with anti-ASC (red) antibody in THP-1 macrophages pretreated with KMU-11342 (0.5 and 1 µM) for 1 h followed by treatment with LPS (1 µg/ml) for 2 h and ATP (2mM) for 30 min.Arrows indicate ASC specks.DAPI (blue) was used as a nuclear marker.Scale bar: 50 μm

Table 1
Primer sequences for Real time PCR

Table 2
Kinase activity of 37 protein kinases upon treatment with 1µM