Scaffold compound L971 exhibits anti‐inflammatory activities through inhibition of JAK/STAT and NFκB signalling pathways

Abstract JAK/STAT and NFκB signalling pathways play essential roles in regulating inflammatory responses, which are important pathogenic factors of various serious immune‐related diseases, and function individually or synergistically. To find prodrugs that can treat inflammation, we performed a preliminary high‐throughput screening of 18 840 small molecular compounds and identified scaffold compound L971 which significantly inhibited JAK/STAT and NFκB driven luciferase activities. L971 could inhibit the constitutive and stimuli‐dependent activation of STAT1, STAT3 and IκBα and could significantly down‐regulate the proinflammatory gene expression in mouse peritoneal macrophages stimulated by LPS. Gene expression profiles upon L971 treatment were determined using high‐throughput RNA sequencing, and significant differentially up‐regulated and down‐regulated genes were identified by DESeq analysis. The bioinformatic studies confirmed the anti‐inflammatory effects of L971. Finally, L971 anti‐inflammatory character was further verified in LPS‐induced sepsis shock mouse model in vivo. Taken together, these data indicated that L971 could down‐regulate both JAK/STAT and NFκB signalling activities and has the potential to treat inflammatory diseases such as sepsis shock.

tyrosine kinase/ signal transducer and activator of transcription (JAK/STAT) and nuclear factor kappa B (NFκB) signalling to exert their biological effects in sepsis shock. When cells are activated with exogenous stimuli such as endotoxin, gram-positive bacterial products (eg peptidoglycans and lipoteichoic acid), cytokines (eg TNFα and IL1) and other physical and chemical stressors, STATs and NFκB become activated in the cytoplasm and then translocate into the nucleus where they activate transcription of many inflammatory mediators. 4 Besides, certain pathogen recognition receptors (PRRs) sensing stimuli rapidly activate NFκB, Interferon regulatory factor 3 and 7 (IRF3/7), leading to secretion of cytokines and chemokines including IL6 and interferons, which in turn activate JAK/STAT signalling. Moreover, some cytokine transcription is modulated by NFκB and STAT complex. 5 Therefore, dual targeting JAK/STAT and NFκB might provide more efficient antiinflammatory effects.
As classical anti-inflammatory therapy utilizing non-steroidal anti-inflammatory drugs and cytochrome c oxidase 1/2 (COX1/2) inhibitors exhibits a wide spectrum of potential risks, the advanced generation of novel anti-inflammatory drugs primarily acting against pro-inflammatory mediator production are being developed. Among these biological targets, JAK/STAT and NFκB signalling molecules represent the most promising pathways for achieving optimal therapeutic response with minimal side effects. 6 Numerous compounds acting directly on the NFκB protein complexes or NFκB-related signalling pathways were developed including plant-derived agents (extracts and essence), steroidbased compounds and several small-molecule mediators jointly composing a large therapeutic group. Some of small-molecular NFκB inhibitors are approved and launched into clinical practice to treat various inflammatory diseases such as inflammatory bowel disease, rheumatoid arthritis and non-specific inflammation. 7 There are also several small molecule inhibitors targeting JAK kinase and STAT function, which are already launched on the market or currently evaluated in different clinical trials against various inflammatory conditions. 8,9 As inhibitors targeting these two pathways both exhibited similar anti-inflammatory applications, and these two pathways frequently crosstalk with each other in various conditions, [10][11][12] synergetic drug combination or drugs with dual inhibition activity might be more efficient for the treatment of inflammatory disease such as sepsis shock. In a very recently reported study, combination of sitagliptin and tofacitinib significantly ameliorates adjuvant induced arthritis via modulating the interaction between JAK/STAT and NFκB signalling, 13 verifying the feasibility of dual targeting drug or drug combination development.
In the current study, a dual STAT and NFκB-based luciferase drug screening system was constructed to screen the compounds which meet this criterion. Total of 18 840 small molecular compounds were screened using this reporter system, and a scaffold compound named L971-0101 (L971) was identified as lead compound targeting both JAK/STAT and NFκB signalling. It can potently inhibit constitutive and cytokine-induced activation of STAT1, STAT3 and NFκB and their downstream gene expression in both cancer cells and macrophages. To comprehensively uncover L971 function, the high-throughput RNA sequencing was employed in macrophages treated with LPS or LPS+L971. The analysis results clearly demonstrated that L971 significantly downregulates LPS-induced JAK/STAT and NFκB signalling activities, immune cell activation and inflammatory diseases such as sepsis shock. As an anti-inflammatory compound, L971 was proved to alleviate LPS-induced sepsis shock mouse model in vivo, suggesting its therapeutic potential for sepsis treatment.

| Antibodies and reagents
Primary antibodies used in this study were detailed in Table S1.

| Cell lines and cell culture
HeLa, DU145, A549 and THP-1 cells were obtained from the American Type Culture Collection (ATCC). HeLa, peritoneal macrophages and SKA-Ⅱ cells were incubated in Dulbecco's modified Eagle's medium (DMEM), and DU145 and THP-1 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium. These media were supplemented with 10% foetal bovine serum (FBS, Gibco), penicillin (100 IU/ml) and streptomycin (100 mg/ml), and the cells were maintained at 37°C in humidified incubators containing 5% CO 2 . All the cell lines were authenticated by STR profiling and tested without mycoplasma contamination.

| Primary peritoneal macrophage isolation
Detailed protocol to harvest primary mouse peritoneal macrophage was reported previously. 14 Briefly, 38.5 g of the BBL ™ Thioglycollate Medium Brewer Modified powder (BD Biosciences, Cat. 211716) was dissolved in 1 L of purified water with frequent agitation, autoclaved at 121°C for 15 minutes and stored at 4°C for at least 3 months. Mice were injected intraperitoneally with 1 ml of aged thioglycolate. Seventy-two hours later, mice were killed, and peritoneal macrophages were harvested by flushing peritoneal cavity.

| Animals
Male C57BL/6 mice (SPF degree, 6 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. They were maintained in the specific-pathogen-free laboratory animal room

| Luciferase reporter assay
SKA-Ⅱ cells (1 × 10 4 /well) were seeded into white 96-well plates (Corning) and incubated overnight at 37℃ in an incubator containing 5% CO 2 . These cells were then treated with the either vehicle or L971 at the indicated concentrations for 24 hours. Luciferase activity was determined using Promega luciferase kits (Cat. E2510) and detected by a SpectraMax ® L microplate reader (Molecular Devices).

| Western blotting
Immune cells or tumour cells treated with 2.5, 5, 7.5 and 15 μM of L971 with or without cytokine treatment: 20 ng/ml IL6, 50 ng/ ml IFNβ and 20 ng/ml TNFα for 10 minutes, or 100 ng/ml LPS for 0.5 hours. Cells were washed twice with cold PBS and harvested in lysis buffer containing protease and phosphatase inhibitors.
Total of 20 μg protein lysates were resolved by SDS-PAGE electrophoresis gel and transferred onto nitrocellulose membranes (GE Healthcare, Cat. 10600034). After blocking with nonfat milk solution (with 0.5‰ Tween-20), the membranes were probed with primary antibodies at 4℃ overnight and then incubated with horseradish peroxidase-conjugated secondary antibodies for 2 hours at room temperature. Immune complexes were detected with an Immobilon™ western chemiluminescence horseradish peroxidase substrate (Millipore, Cat. WBKLS0500) and photographed with a Tanon 5200 imaging system. The quantification was managed by ImageJ software.

| Real-time PCR measurement
Cells were treated the same as described in 2.7 Western blotting.

| Mouse sepsis model
Mice were randomly divided into different groups and intraperitoneally (i.p.) treated with vehicle (10% HS-15 + 2% DMSO in PBS buffer) or L971 at 2.5 or 5 mg/kg. Twelve hours later, all the mice were challenged i.p. with 10 mg/kg LPS. Body temperature and mouse survival were monitored.

| Transcriptome sequencing
Mouse peritoneal macrophages were isolated and treated with or without L971 (15 μM) for 0.5 hours, followed by vehicle or LPS (100 ng/ml) challenge for additional 4 hours. Total RNA was isolated using TRIzol (Thermo Fisher Scientific). After extracting the total RNA of the sample and digesting the DNA with DNase, the magnetic beads with Oligo (dT) were used to enrich eukaryotic mRNA.
cDNA library construction and sequencing were performed using the Illumina Hiseq Xten platform. After passing the quality inspection, raw data (raw reads) were processed using Trimmomatic. The reads containing ploy-N and the low-quality reads were removed to obtain clean reads. Then, the clean reads were mapped to reference genome using Hisat2. 17 The FPKM value of each gene was calculated using cufflinks, 18 and the read counts of each gene were obtained by htseq-count. 19 Differentially expressed genes (DEGs) were identified using the DESeq. P-value <.05 and fold change >2 or fold change <0.5 were set as the threshold for significantly differential expression. Hierarchical cluster analysis of DEGs was performed to explore genes expression pattern. KEGG pathway enrichment analysis, 20 PPI 21 and IPA 22 analysis of DEGs were, respectively, performed.

| Statistical analysis
All the histograms and line charts were made by GraphPad Prism 8.0.1. Results were graphed as mean ± SEM. Statistical significance was calculated by a two-tailed Student's t test for comparisons.
Significant differences are indicated as * P-value <.05.

| L971 was identified as an inhibitor of JAK/STAT and NFκB signalling
To identify compounds that can effectively inhibit JAK/STAT and NFκB signalling, we constructed a luciferase reporter plasmid which contains both STAT and NFκB-binding elements ( Figure 1A) and stably transfected into human lung cancer cell line A549 to establish the dual target reporter system SKA-ΙΙ. Similarly to previously published method, [23][24][25] we established a high-throughput drug screen workflow ( Figure 1B) and total of 18 840 small molecules were screened ( Figure 1C). Among them, 924 hit compounds exhibited more than 50% inhibition activities compared with vehicle at 24 hours whereas BP-1-102 26 and Brevilin A 25 which are known to inhibit STAT3 activity, shows 76% and 84% inhibition activity in our reporter system. These compounds were further filtered by searching PubMed database, 27 and compounds previously reported to directly or indirectly interact with JAK/STAT and NFκB signalling were omitted. After that, total of 256 compounds were selected for further EC 50 determination of luciferase activity and cytotoxicity (data not shown). L971, a novel scaffold compound from Targetmol mini scaffold library, was identified as one of the most potent inhibitors in the reporter system ( Figure 1C). From the PhysChem predictions, 28  in HeLa cells ( Figure S1B). Meanwhile, L971 could also inhibit IκB degradation induced by TNFα ( Figure 1G) and IKK phosphorylation induced by LPS ( Figure S1E) in human monocytic leukaemia THP-1 cells, indicating its inhibitory activity for NFκB signalling.
These data suggest that L971 could function as a dual inhibitor of JAK/STAT and NFκB signalling pathways.

| L971 inhibits JAK/STAT and NFκB signalling and downstream gene expression in peritoneal macrophage
As JAK/STAT and NFκB signalling pathways serve as regulatory hubs that coordinate immune and inflammatory responses, 11  F I G U R E 1 L971 was identified as an inhibitor of STAT and NFκB signalling pathways by a STAT and NFκB-based luciferase drug screening system. A, Schematic view of the luciferase reporter plasmid, N represents C or T. B, Workflow of high-throughput screening procedure. C, SKA-II cells were seeded into 96-well plates (1 × 10 4 /well) and cultured overnight. Cells were then treated with 18 840 individual compounds at 20 μM for 24 h, and luciferase activities were determined. The two black dotted lines represent the relative luciferase value of compound/vehicle treatment ranging from 0.5 to 2. D, The chemical structure, molecular weight and SMILES of L971. E, SKA-II cells were seeded as in (C) and treated with vehicle or L971 at 0.9375, 1.875, 7.5, 15 and 30 μM. Luciferase activities after 24 h were determined after 24 h. F, A549 cells were treated with vehicle or L971 at 2.5, 5, 7.5 and 15 μM for 2 h. Whole cell lysates were processed for Western blot analysis and probed with anti-pTyr705-STAT3 and anti-STAT3 antibodies. Tubulin was used as a loading control. G, THP-1 cells were treated for 2 h with vehicle or L971 as in (F), followed by TNFα stimulation (20 ng/ml, 10 min). Whole cell lysates were processed for Western blot analysis and probed with anti-IκBα antibodies. Tubulin was used as a loading control

| Transcriptome profile of LPS-induced proinflammatory response in peritoneal macrophage
To better elucidate LPS induced inflammatory response, we per-

| Anti-inflammatory effects of L971 were profiled by transcriptome sequencing
To  Figure 5C). Furthermore, in disease comparison, the majority of terms hindered by L971 treatment are related to inflammation diseases such as inflammatory response, rheumatic disease, inflammation of respiratory system component and sepsis ( Figure 5D).
All these data confirm that L971 could exert anti-inflammatory effects through JAK/STAT and NFκB signalling pathways, indicating the possible applications in inflammation-associated diseases like sepsis.

| L971 treatment ameliorates endotoxininduced septic shock in vivo
As a major cell wall component of gram-negative bacteria, LPS is the major endotoxin inducing a systemic inflammatory response and plays a central role in sepsis. 40 LPS-induced sepsis shock is a well-established animal model to mimic human sepsis pathology. 40 Moreover, sepsis is one of the diseases predicted to be alleviated by L971 ( Figure 5D). Based on the above assumption, we examined L971 protective efficiency in LPS induced mouse sepsis model in vivo. Upon a lethal dose of LPS injection (15 mg/ kg), mouse survival was dramatically improved by intraperitoneal infusion of L971 (5 mg/kg) ( Figure 6A) and survival rate rose accompanied with dose increasement ( Figure 6B). L971 also had an obvious effect on the recovery of mouse body temperature, which is conducive to the mouse survival in sepsis shock, 41 and amelioration especially dominates after 36 hours after LPS challenge ( Figure 6C). Furthermore, the body temperature at 36 hours was monitored with various doses of L971 treatment. The results show that even lower dose of L971 (2.5 mg/kg) is sufficient to protect the mice from lethal lesion ( Figure 6D). As shown in Figure 5C, activation of myeloid cells was predicted to be suppressed by L971.
Therefore, we measured the counts of neutrophil and monocyte in mouse whole blood, and these two myeloid populations decreased in L971 treated group as predicted ( Figure 6E,F). As G-CSF is crucial for neutrophil activation contributing to tissue damage and organ dysfunction during early sepsis, 42,43 we measured plasma G-CSF levels and data suggested that L971 could decrease the LPS induced G-CSF levels ( Figure 6G). Liver failure complication is recognized as one of the components that contribute to the severity of the sepsis shock. 44 Plasma alanine aminotransferase (ALT) level is positively correlated with liver cell injury and widely used in the measurement of liver damage. 45 ALT levels were potently reduced in L971 treated mice ( Figure 6H). Collectively, these findings suggested that L971 had protective effects in LPS-induced sepsis shock in vivo.

| D ISCUSS I ON
In recent years, the molecular mechanisms of inflammatory responses have been deeply elucidated. 46 The JAK/STAT and NFκB signalling pathways are considered to play important roles in the transmission of inflammatory signals. 4,5 Multiple drugs targeting these two pathways were developed, and some of them have already been approved for the treatment of immune diseases such as inflammatory bowel disease, rheumatoid arthritis and nonspecific inflammation. 6 As the frequent co-occurrence of these two pathways, drugs simultaneously targeting both pathways was predicted to be more efficient for the treatment of inflammatory diseases. 10,11 For this purpose, we constructed a dual luciferase   proteins, which provide a clue for further identification of L971 major targets.
In conclusion, we identified L971 as a novel anti-inflammatory scaffold compound targeting both JAK/STAT and NFκB signalling pathways. This is in line with the current trend of developing multitargeting anti-inflammatory drugs and has a great potential for further drug development.

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.