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. 2023 Jul 27;80(8):230. doi: 10.1007/s00018-023-04881-x

Discovery of a selective NLRP3-targeting compound with therapeutic activity in MSU-induced peritonitis and DSS-induced acute intestinal inflammation

Yinghua Zhou 1,#, Zhongjin Yang 1,#, Yitao Ou 1, Haowei Cai 1, Zhuorong Liu 1, Geng Lin 1, Shuli Liang 1, Lei Hua 1, Yuyun Yan 1, Xiuxiu Zhang 1, Ruiwen Wu 1, Aiping Qin 1, Wenhui Hu 1,✉, Ping Sun 1,✉
PMCID: PMC11073129  PMID: 37498355

Abstract

The aberrant activation of the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is known to contribute to the pathogenesis of various human inflammation-related diseases. However, to date, no small-molecule NLRP3 inhibitor has been used in clinical settings. In this study, we have identified SB-222200 as a novel direct NLRP3 inhibitor through the use of drug affinity responsive target stability assay, cellular thermal shift assay, and surface plasmon resonance analysis. SB-222200 effectively inhibits the activation of the NLRP3 inflammasome in macrophages, while having no impact on the activation of NLRC4 or AIM2 inflammasome. Furthermore, SB-222200 directly binds to the NLRP3 protein, inhibiting NLRP3 inflammasome assembly by blocking the NEK7 − NLRP3 interaction and NLRP3 oligomerization. Importantly, treatment with SB-222200 demonstrates alleviation of NLRP3-dependent inflammatory diseases in mouse models, such as monosodium urate crystal-induced peritonitis and dextran sulfate sodium-induced acute intestinal inflammation. Therefore, SB-222200 holds promise as a lead compound for the development of NLRP3 inhibitors to combat NLRP3-driven disease and serves as a versatile tool for pharmacologically investigating NLRP3 biology.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00018-023-04881-x.

Keywords: Directly bind, Inflammatory bowel disease, Inflammasome, Peritonitis, SB-222200

Introduction

The nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) is a cytosolic sensor that responds to various pathogen-associated molecular patterns and host-derived danger-associated molecular patterns. These include extracellular adenosine triphosphate (ATP), monosodium urate crystals (MSU), cholesterol crystals, unsaturated fatty acids, amyloid-β, islet amyloid polypeptide, and nigericin [1, 2]. When activated, NLRP3 binds to the adapter protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC). This binding recruits the effector protein pro-caspase-1, resulting in the formation of a protein complex called the NLRP3 inflammasome [2]. The auto-activated caspase-1 then mediates the proteolytic processing of pro-IL-1β and pro-IL-18 into their mature subunits [2, 3]. Furthermore, caspase-1 drives the cleavage of Gasdermin D (GSDMD) into the N-terminus of GSDMD (GSDMD-NT). GSDMD-NT oligomerizes in the plasma membrane, forming pores that facilitate pyroptosis and the release of cytosolic contents, including IL-1β, IL-18, and lactate dehydrogenase (LDH) [4]. The activation of NLRP3 inflammasome plays a crucial part in the host immune response and inflammation.

Aberrant activation of the NLRP3 inflammasome has been implicated in the pathogenesis of various human diseases, such as gout, inflammatory bowel disease (IBD), type 2 diabetes, atherosclerosis, non-alcoholic fatty liver disease, silicosis, and Alzheimer’s disease [5–10]. Mutations in the NLRP3 gene can result in spontaneous activation of the NLRP3 inflammasome and are associated with cryopyrin-associated periodic syndromes, a group of rare auto-inflammatory disorders [11]. Therefore, inhibiting the activation of the NLRP3 inflammasome may offer a potential therapeutic approach for these conditions, which currently lack effective clinical interventions. NLRP3 inflammasome inhibition can be achieved through both indirect and direct means. Indirect inhibition involves regulating the upstream signals of inflammasome activation, such as inhibiting K+ efflux (e.g., β-hydroxybutyrate), clearing mitochondrial reactive oxygen species (e.g., ciclopirox), and protecting lysosome membrane (e.g., disulfiram) [12–14]. Direct inhibition involves targeting the NLRP3 protein to directly block the activation of the NLRP3 inflammasome. MCC950 is currently the most well-characterized inhibitor of NLRP3. It functions by blocking NLRP3 ATPase activity and inflammasome activation through its interaction with the Walker B motif within the NACHT domain of NLRP3 [15]. Zhou and colleagues have also reported that CY-09, tranilast, and oridonin can bind to the NACHT domain of NLRP3, thereby suppressing NLRP3 inflammasome activation [16–18]. Directly targeting NLRP3 itself may offer a more specific approach compared to regulating upstream signals of NLRP3 activation. MCC950 has demonstrated beneficial effects in various mouse models of NLRP3-driven diseases, such as rheumatoid arthritis, Alzheimer’s disease, psoriasis, IBD, and non-alcoholic fatty liver disease [8, 19–22]. Although MCC950 was evaluated in phase II clinical trials for rheumatoid arthritis, elevated serum liver enzyme levels were observed, indicating liver toxicity [23].

To date, there have been no clinical applications of NLRP3-targeting inhibitors for the treatment of inflammation-related diseases in humans. Therefore, there is an urgent need to identify NLRP3 inhibitors that can directly bind to NLRP3. In this study, we have discovered a new direct NLRP3 inhibitor named SB-222200 ((S)-3-Methyl-2-phenyl-N-(1-phenylpropyl)-4-quinolinecarboxamide). SB-222200 has been shown to bind directly to NLRP3, inhibiting NLRP3 inflammasome assembly, caspase-1 activation, and IL-1β production. Notably, SB-222200 has demonstrated its ability to alleviate NLRP3-dependent inflammatory diseases in mouse models, including MSU-induced peritonitis and dextran sulfate sodium (DSS)-induced acute intestinal inflammation.

Materials and methods

Reagents and antibodies

SB-222200 (#HY-15722), Talnetant (#HY-14552), and Tranilast (#HY-B0195) were obtained from MCE (USA). Lipopolysaccharide (LPS, #L2630) and ATP (#74,804-12-9) were purchased from Sigma-Aldrich (USA). Nigericin (#tlrl-nig-5), MSU (#tlrl-msu), poly (dA:dT) (#86,828-69-5), Flagellin from Salmonella typhimurium (FLA-ST) Ultrapure (#tlrl-epstfla-5), and Pam3CSK4 (#tlrl-pms) were bought from InvivoGen (USA). Disuccinimidyl suberate (#C100015-0100) was bought from Sangon Biotech (China). Recombinant human NLRP3 protein (#CSB-EP822275HU) was purchased from CUSABIO (China). Imiquimod (#T0134) was bought from TargetMol (USA). Macrophage colony-stimulating factor (M-CSF) (#416-ML-050) was obtained from R&D (USA). FBS (#FSP500) was purchased from ExCell (China). DMEM (#CR-12800) and RPMI-1640 (#CR-31800) were procured from Cienry (China). Opti-MEM (#31,985,070) and Trypsin–EDTA (#25,200,072) were provided by Gibco (USA). pEnCMV-NEK7 (human)-3 × FLAG-SV40-Neo (#P33002) and pCMV-NLRP3(human)-3 × Myc-SV40-Neo (#P36334) were brought from Miaolingbio (China). Penicillin/Streptomycin (#15,140,122) was obtained from ThermoFisher (USA). DSS (#216,011,080) was bought from MP Biomedicals (USA). Lactate dehydrogenase (LDH) cytotoxicity assay kit (#C0016), Cell counting kit-8 (CCK-8) reagent (#C0037), MQAE (#S1082), RIPA Lysis Buffer (#P0013C), PEI (#C0539), Anti-Flag Magnetic Beads (#P2115), and Anti-Myc Magnetic Beads (#P2118) were obtained from Beyotime (China). IL-1-beta Mouse Uncoated Elisa Kit (#88-7013-77), IL-6 Mouse Uncoated Elisa Kit (#88-7064-77), and TNF-alpha Mouse Uncoated Elisa Kit (#88-7324-77) were obtained from ThermoFisher. MitoSOX Red mitochondrial superoxide indicator (#M36008) was obtained from Invitrogen (USA). Protein A/G plus agarose beads (#sc-2003) were purchased from SantaCruz (USA). Anti-IL-1β (#AF-401-NA) was from R&D Systems. Anti-caspase-1 (#AG-20B-0042) and anti-NLRP3 (#AG-20B-0014-C100) were procured from AdipoGen (USA). Anti-β-actin (#P30002) was obtained from Abmart (China). Anti-Flag Antibody (#ab205606), Anti-Myc Antibody (#ab206486), Anti-NEK7 (#ab133514), and anti-GSDMD (#ab209845) were from Abcam (USA). Anti-ASC (#67824S) was obtained from CST (USA).

Cell culture and treatment

J774A.1 cells (Jennio Biotech; China) were cultured in DMEM supplemented with 10% FBS. Bone marrow-derived macrophages (BMDMs) were isolated from the tibiae of C57BL/6 J mice (6–8 weeks) and cultured in RPMI-1640 supplemented with 10% FBS, 20 ng/mL M-CSF, 2 mM L-glutamine, and 1% penicillin–streptomycin.

Prior to treatment with the compounds, J774A.1 cells were primed with LPS (1 μg/mL, 5 h) and then stimulated with NLRP3 activators nigericin (10 μM, 1 h) or ATP (4 mM, 0.5 h). BMDMs were pretreated with LPS (100 ng/mL, 3 h), followed by treatment with the compounds for 1 h, and finally stimulated with NLRP3 activators nigericin (5 μM, 0.5 h), ATP (4 mM, 0.5 h), imiquimod (IMQ; 900 μM, 2 h), MSU (200 μg/mL, 8 h), or SiO2 (50 μg/mL, 8 h). For NLRC4/AIM2 inflammasome activation, BMDMs were primed with LPS (100 ng/mL, 3 h) before treatment with the compounds for 1 h, followed by incubation with FLA-ST (2.5 µg/mL, 14 h), or Poly dA:dT (0.25 µg/mL, 14 h) transfected with Lipofectamine 3000. To activate the noncanonical NLRP3 inflammasome, BMDMs were pretreated with Pam3CSK4 (1 μg/mL, 5 h) before treatment with SB-222200, followed by transfection with LPS (2 μg/mL, 16 h) using Lipofectamine 3000.

Cytokine analysis by enzyme-linked immunosorbent assay (ELISA)

Cytokines IL-1β, IL-6, and TNF-α were detected in cell culture supernatants, mouse peritoneal lavage fluid, and tissues using ELISA kits, following the instructions provided by the manufacturer.

Western blot

Mouse colon tissue samples or cells were lysed in RIPA lysis buffer containing protease inhibitors for 30 min on ice. The lysates were then mixed with protein-loading buffer and separated using a 12% sodium dodecyl sulfate–polyacrylamide gel. The resulting proteins were subsequently transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% milk for 1 h at room temperature, the PVDF membranes were incubated overnight at 4 °C with specific primary antibodies. Secondary antibodies were then added and incubated at room temperature for 1 h. The reaction signals were detected using enhanced chemiluminescence (ECL), and the greyscale analysis of the bands was performed using Image J.

LDH release assay

J774A.1 cells were subjected to the NLRP3 inflammasome activation assay. The supernatants were collected and the activity of LDH was measured using an LDH assay kit.

Cell counting kit-8 assay

J774A.1 cells were plated in 96-well plates at a density of 85–90% and treated with varying concentrations of SB-222200 for either 2 or 24 h. After incubation, a CCK-8 solution was added for 1 h and the absorbance was measured at 450 nm using an Epoch2 microplate reader (BioTek Instruments, USA).

Cellular thermal shift assay (CETSA)

After treatment, the cells were detached with 0.25% trypsin and collected by centrifugation at 800 g for 5 min. The cells were then washed twice with PBS and re-suspended in PBS containing protease inhibitors. The cell suspension was divided into 6 equal aliquots and lysed by repeated freezing and thawing with liquid nitrogen after heating for 3 min at temperatures of 43, 45, 50, 55, 60, and 65 °C. The supernatants were carefully collected by centrifugation at 3000 g for 25 min and subsequently analyzed by western blotting.

Drug affinity responsive target stability (DARTS) assays

After treatment, cells were lysed in an IP lysis solution containing protease inhibitors for 30 min. The supernatants were then centrifuged at 14,000 g for 10 min at 4 °C. After determining the protein concentration, 40 μg of protein lysate was incubated with pronase (0.04 mg/mL) for 10 min at room temperature. The reaction was stopped by adding 20 × protease inhibitors and analyzed by immunoblotting.

ASC oligomerization assay

After treatment, BMDMs were lysed in an IP lysis buffer containing protease inhibitors on ice for 30 min. The lysate was then centrifuged for 10 min at 4 ℃, 12,000 rpm. The resulting pellets were crosslinked for 30 min by adding 4 mM disuccinimidyl suberate. After centrifugation, the crosslinked precipitates were re-suspended in loading buffer, boiled for 10 min, and then analyzed by immunoblotting.

ASC speck staining assay

LPS-primed BMDMs cells were treated with SB-222200 for 1 h and then stimulated with nigericin for 0.5 h. Subsequently, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.1% Triton X-100 for another 30 min. Following incubation with a rabbit monoclonal anti-ASC antibody, the cells were incubated with Alexa Fluor 560-conjugated secondary antibody and DAPI. Images were captured using a fluorescence microscope and processed using Image-Pro Plus software. The percentage of ASC speck-positive cells was determined by normalizing the number of speck-positive cells to the number of nuclei.

NLRP3 oligomerization assay

The oligomerization of NLRP3 was assessed using the SDD-AGE assay. J774A.1 cells were seeded in 6-well plates and cultured overnight. After treatment, the cells were lysed with IP lysate containing protease inhibitors for 30 min and then centrifuged at 300 g for 3 min at 4 ℃ to obtain the total cell lysate. 20 μg of protein was added to the SDS-PAGE protein-loading buffer and boiled for 10 min, followed by immunoblotting analysis. Additionally, 60 μg of protein was added to the native non-reduced protein-loading buffer and loaded onto a vertical 1.3% agarose gel. After electrophoresis in electrophoresis buffer (1 × TBE and 0.1% SDS) at a constant pressure of 80 V on ice for 1 h, the proteins were transferred to PVDF membranes for immunoblotting.

Immunoprecipitation

After treatment, J774A.1 cells were lysed using IP lysate containing protease inhibitors for 30 min on ice. The supernatant was collected by centrifugation at 12,000 rpm for 10 min at 4 ℃. Specific primary antibodies were added and incubated overnight at 4 °C on a shaker. Then, 20 μL of protein A/G plus agarose beads was added to the aforementioned supernatant and incubated for 8 h at 4 °C. Immune complexes were washed with PBS, and then protein-loading buffer was added. The mixture was boiled for 10 min before detection using immunoblotting.

Transfection of plasmids

HEK-293 T cells were seeded in a 10 cm culture dish and incubated overnight at 37 °C. Polyethylenimine linear (PEI) was mixed with plasmid (plasmid: PEI = 1:2.5), incubated for 20 min, and added to the culture dish. SB-222200 was added 8 h after transfection, and samples were collected 24 h after transfection.

Detection of mitochondrial ROS

After treatment, J774A.1 cells were incubated with 5 μM Mitosox Red for 30 min in the dark, and the cellular fluorescence intensity was measured at 510/580 nm.

Detection of intracellular chloride ion

The J774A.1 cells were seeded in 24-well plates and cultured overnight at 37 °C. Following treatment with LPS and SB-222200, nigericin was added. Nigericin stimulation was terminated at 0, 5, 10, 15, and 20 min. The supernatant was then removed, and ultrapure water was added. The supernatants were incubated at 37 °C for 15 min. Subsequently, the lysates were transferred to EP tubes and centrifuged at 8000 rpm for 5 min at 4 °C. The supernatant was added to a black 96-well plate and mixed with MQAE to detect the fluorescence.

Surface plasmon resonance (SPR) analysis

For SPR experiments, the Biacore T200 (GE Healthcare; USA) was used. The running buffer was 10 × PBS (Sangon Biotech, #I308FD0152; China). All steps were performed at 25 °C. After determining the optimal pH value required for protein-coupled immobilization, the ligand–protein NLRP3 was immobilized to the CM5 chip (Cytiva, #10,324,467) through amino coupling. The analyte compounds were then injected after gradient dilution to obtain the final results.

Molecular docking

The molecular docking between SB-222200 and NLRP3 protein was assessed using DOCK 6.9 through the Docking Server (https://cloud.yinfotek.com/). The crystal structure of the human NLRP3 protein (PDB: 6NPY) was obtained from the Protein Data Bank.

Mice

Male C57BL/6 mice (20–24 g, 6–8 weeks old) were obtained from Guangdong Experimental Animal Center in China. The mice were housed in a controlled environment with a 12 h light/dark cycle and ad libitum access to food and water. All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Use Ethics Committee of Guangzhou Medical University (GY2020-035, 2018-126).

MSU-induced peritonitis in mice

The Male C57BL/6 mice were randomly divided into the 6 groups (n = 5): the blank group, model group, SB-222200 low-dose group (10 mg/kg), SB-222200 medium-dose group (15 mg/kg), SB-222200 high-dose group (20 mg/kg), and MCC950 group (5 mg/kg). The treated groups received intraperitoneal injections of SB-222200 or MCC950, dissolved in an aqueous solution containing 2% dimethyl sulfoxide, 20% PEG300, and 78% saline. The blank and MSU groups received intraperitoneal injections of the same volume of vehicle. Peritonitis was induced 1 h later by intraperitoneal injection of MSU (3 mg MSU crystals in 0.3 mL sterile PBS). Four hours later, the mice were sacrificed, and the peritoneal cavity was washed with 1 mL of cold PBS to collect the peritoneal lavage fluid. The levels of IL-1β and IL-6 in the peritoneal lavage fluid, liver, and spleen were measured using ELISA kits.

DSS-induced colitis in mice

Male C57BL/6 mice were randomly assigned to 4 groups (n = 7): the blank group, the model group, SB-222200 low-dose group (10 mg/kg), and SB-222200 high-dose group (20 mg/kg). The colitis model group received oral administration of a 2.5% DSS aqueous solution for 7 days (days 2–8). SB-222200 was dissolved in an aqueous solution containing 2% dimethyl sulfoxide, 20% PEG300, and 78% saline, and injected intraperitoneally from days 1 to 8. The degree of hematochezia was monitored daily from Day 1. On day 9, the mice were sacrificed, and colon tissues were collected to measure colon length. The tissues were then sectioned at a thickness of 4 μm and stained with hematoxylin & eosin (H&E) for routine histology examination to assess the injury score. The grading of histological damage was determined based on the criteria described by Dann and colleagues, with some modifications [24].

Statistical analysis

The data are presented as mean ± standard error of the mean (SEM). The unpaired Student's t-test was used to compare two groups of data, while one-way ANOVA with Dunnett's post hoc test was used to compare multiple groups of data (GraphPad Prism 9 software). Statistical significance was defined as p < 0.05(*), p < 0.01(**), p < 0.001(***), and p < 0.0001(****).

Results

SB-222200 specifically inhibits the activation of NLRP3 inflammasome

To investigate the impact of compounds that hinder the activation of the NLRP3 inflammasome, we conducted experiments using the murine macrophage cell line J774A.1 cells. These cells were initially primed with LPS and subsequently stimulated with various NLRP3 agonists. Among the compounds tested, SB-222200 (Fig. 1a) was found to effectively reduce the release of IL-1β, a marker of NLRP3 inflammasome activation, when challenged with nigericin (Fig. 1b) or ATP (Fig. 1c). Furthermore, SB-222200 demonstrated the ability to decrease IL-1β secretion in BMDMs (Fig. 1d and e). To confirm the inhibitory effect of SB-222200 on NLRP3 inflammasome activation, we assessed the impact of SB-222200 on caspase-1 cleavage and IL-1β release through immunoblotting. As depicted in Fig. 1f, SB-222200 effectively suppressed the release of cleaved caspase-1 and IL-1β in the supernatant, while not affecting the expression levels of pro-caspase-1, pro-IL-1β, NLRP3, and ASC. Additionally, we observed that SB-222200 blocked GSDMD activation (Fig. 1g) and LDH release (Fig. 1h), indicating its ability to inhibit NLRP3-dependent pyroptosis. Moreover, we conducted a cell viability assay to assess the cytotoxicity of SB-222200, and the results demonstrated no cytotoxic effects at concentrations below 25 μM (Fig. S1 in the Supporting Information).

Fig. 1.

Fig. 1

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SB-222200 inhibits the activation of NLRP3 inflammasome. a Structure of SB-222200. LPS-primed J774A.1 cells were incubated with SB-222200 for 1 h and then stimulated with nigericin (b) or ATP (c) for 1 h. The supernatants were collected and the concentration of IL-1β was measured by ELISA. LPS-primed BMDMs were incubated with SB-222200 for 1 h. After stimulation with nigericin (d) or ATP (e), the supernatants were collected and IL-1β concentrations were measured by ELISA. f LPS-primed J774A.1 cells were incubated with SB-222200 for 1 h and then stimulated with nigericin for 1 h. The expressions of IL-1β, caspase-1, NLRP3, and ASC were detected by Western blotting. g The expression of GSDMD was detected by Western blotting. h Cell death was evaluated by detecting LDH release in the cell supernatants. Data are presented as n = 5 (B, C, D, E, H) as mean ± SEM, ****p < 0.0001

To determine whether SB-222200 is a universal inhibitor for the NLRP3 inflammasome, we activated the cells using other agonists, such as MSU, SiO2, and IMQ. Pre-treatment with SB-222200 effectively blocked the release of IL-1β triggered by these agonists (Fig. 2a). Additionally, SB-222200 treatment inhibited the noncanonical NLRP3 activation induced by cytosolic LPS (cLPS) in BMDMs and J774A.1 cells (Fig. 2a and b). To assess the specificity of SB-222200’s effect of on NLRP3 inflammasome activation, we examined its impact on the AIM2 and NLRC4 inflammasomes. The results demonstrated that SB-222200 did not affect the activation of the AIM2 inflammasome (Fig. 2c) or the NLRC4 inflammasome (Fig. 2d). Taken together, these findings provide evidence that SB-222200 selectively inhibits NLRP3 inflammasome activation.

Fig. 2.

Fig. 2

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SB-222200 is a specific inhibitor of NLRP3 inflammasome. a LPS or Pam3CSK4-primed BMDMs were incubated with SB-222200 (10 μM) for 1 h, and then the supernatants were collected after stimulation with a series of stimuli including nigericin, ATP, IMQ, MSU, SiO2, and cLPS. IL-1β levels were measured by ELISA. b Pam3CSK4-primed J774A.1 cells were incubated with SB-222200 for 1 h, then LPS was transfected using Lipo3000. The supernatants were collected to detect the concentration of IL-1β with ELISA. LPS-primed BMDMs were incubated with SB-222200 for 1 h and then stimulated with Poly dA: dT (c) or FLA-ST (d) for 14 h. The supernatants were collected and IL-1β concentrations were measured by ELISA. Data are presented as n = 5 and represent mean ± SEM. *p < 0.05, ****p < 0.0001, ns not significant

SB-222200 suppresses NLRP3 inflammasome assembly

Next, we investigated the mechanism by which SB-222200 suppresses the activation of NLRP3 inflammasome. SB-222200 is known to inhibit neurokinin-3 receptor (NK3R) [25]. Therefore, we examined whether it suppressed NLRP3 inflammasome activation by inhibiting NK3R activity. However, our results showed that talnetant, an analog of SB-222200 with NK3R-inhibitory activity, did not affect NLRP3-dependent IL-1β release (Fig. S2 in the Supporting Information). This indicates that SB-222200 blocked NLRP3 activation in an NK3R-independent manner. We then investigated the effect of SB-222200 on the priming step of NLRP3 activation. Pre-treatment with SB-222200 did not inhibit TNFα release and NF-κB activation (Fig. S3a and b in the Supporting Information), suggesting that SB-222200 did not affect the priming step. Additionally, we examined whether SB-222200 influenced mitochondrial ROS production and ion efflux (chloride and potassium), which are important upstream signaling events responsible for NLRP3 activation [26, 27]. Our results showed that SB-222200 could not inhibit nigericin-induced mitochondrial ROS production and intracellular chloride efflux (Fig. S4a and b in the Supporting Information). SB-222200 also blocked the IMQ-stimulated activation of the NLRP3 inflammasome (Fig. 2a), which is considered to be independent of potassium efflux [28]. These findings suggest that SB-222200 may have no effects on the upstream signaling of NLRP3 inflammasome activation.

NEK7 is an essential partner that interacts with NLRP3, leading to the assembly and activation of the NLRP3 inflammasome [29]. Following stimulation with nigericin, the endogenous interaction between NEK7 and NLRP3 was strengthened (Fig. 3a). Pre-treatment with SB-222200 decreased the NEK7 − NLRP3 interaction (Fig. 3a). We then examined whether SB-222200 could affect NLRP3 oligomerization and the interaction between NLRP3 and ASC. As expected, SB-222200 inhibited NLRP3 oligomerization (Fig. 3b) and the endogenous interaction between NLRP3 and ASC (Fig. 3c) in macrophages challenged with nigericin. After that, we evaluated ASC oligomerization and ASC speck formation, which are critical steps for caspase-1 cleavage. The results demonstrated that SB-222200 suppressed disuccinimidyl suberate-crosslinked ASC (Fig. 3d) and reduced the proportion of ASC speck-positive cells (Fig. 3e and f). These findings indicate that SB-222200 interferes with the assembly of the NLRP3 inflammasome.

Fig. 3.

Fig. 3

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SB-222200 suppresses NLRP3 inflammasome assembly. a LPS-primed J774A.1 cells were incubated with SB-222200 for 1 h and then stimulated with nigericin for 1 h. Endogenous NEK7 binding to NLRP3 was detected by co-immunoprecipitation assays. b Oligomerization of NLRP3 was measured with the SDD-AGE system. c Endogenous ASC binding to NLRP3 was detected by co-immunoprecipitation assays. d After cross-linking with disuccinimidyl suberate, ASC oligomerization was analyzed by Western blot. e Representative images of ASC specks detected by immunofluorescence after LPS-primed BMDMs were incubated with SB-222200 for 1 h and then stimulated with nigericin. Scale bar: 100 μm. f Percentage of cells shown in e with ASC specks. Data are presented as n = 3 and represent mean ± SEM. ***p < 0.001

SB-222200 directly binds to NLRP3

Because SB-222200 is a broad-spectrum inhibitor of NLRP3 and inhibits NLRP3 − NEK7 interaction, we hypothesized that SB-222200 may directly bind to NLRP3. Our results showed that SB-222200 effectively suppressed the degradation of NLRP3, as evidenced by the DARTS assay (Fig. 4a) and CETSA (Fig. 4b). Moreover, the SPR assay revealed a high affinity interaction between SB-222200 (KD = 351.8 nM) (Fig. 4c) and NLRP3, similar to the positive control MCC950 (KD = 71.5 nM) (Fig. S5 in the Supporting Information). To further validate these results, we conducted transfection experiments using Myc-tagged NLRP3 and Flag-tagged NEK7 in HEK-293 T cells. Notably, SB-222200 treatment significantly suppressed the direct interaction between NEK7 and NLRP3 (Fig. 4d). Additionally, we utilized docking software (DOCK 6.9) to explore the molecular mechanism underlying the SB-222200–NLRP3 interaction. Our analysis indicated that the binding of SB-222200 to NLRP3 may be attributed to the formation of hydrogen bonds involving residues, such as ILE257, VAL262, LEU270, ILE285, HIE286, PHE297, TRP320, ILE328, and ARG335 (Fig. S6 in the Supporting Information). Collectively, these findings suggest that SB-222200 directly binds to NLRP3, inhibits NEK7 − NLRP3 binding, and subsequently blocks the activation of the NLRP3 inflammasome.

Fig. 4.

Fig. 4

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SB-222200 directly binds to NLRP3. LPS-primed J774A.1 macrophages were incubated with SB-222200 for 1 h, then stimulated with nigericin. DARTS (a) or CETSA (b) assay of NLRP3 in the cell lysates was performed. c Single-cycle kinetics analysis by SPR of the direct binding of SB-222200 to immobilized recombinant human NLRP3 protein. d Co-IP with Flag antibody and western blotting analysis to assess the NLRP3–NEK7 interaction in HEK-293 T cells transfected with the high-expression plasmid and treated with SB-222200 (20 μM) for 16 h

SB-222200 inhibits MSU-induced peritonitis in mice

In order to assess the potential of short-term treatment with SB-222200 in inhibiting NLRP3-dependent inflammation in vivo, we conducted an experiment using mice with MSU-induced peritonitis. Different doses of SB-222200 were administered one hour prior to intraperitoneal MSU challenge. Our findings demonstrate that treatment with SB-222200 resulted in a dose-dependent decrease in the production of IL-1β and IL-6 in the peritoneal fluid (Fig. 5a and b). Additionally, SB-222200 treatment led to a reduction in IL-1β levels in the liver and spleen (Fig. 5c and e), but had no effect on IL-6 production in these organs (Fig. 5d and f). These results indicate that SB-222200 may effectively inhibit the activation of NLRP3 in MSU-induced peritonitis in mice.

Fig. 5.

Fig. 5

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SB-222200 inhibits MSU-induced peritonitis in mice. Peritoneal IL-1β (a) and IL-6 (b) in mice treated with different doses of SB-222200 (0, 10, 15, and 20 mg/kg) or MCC950 (5 mg/kg) 1 h before i.p. MSU injection. The expression of IL-1β and IL-6 in mouse liver (c and d) or spleen (e and f). Data are presented as n = 5 and represent mean ± SEM.*p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant

SB-222200 alleviates DSS-induced acute experimental colitis in mice

Given that NLRP3 activation plays a crucial role in human IBD and DSS-induced experimental colitis [6, 30], we sought to investigate the potential of SB-222200 to combat IBD by inhibiting NLRP3-driven inflammation. To this end, we examined the effect of SB-222200 in a mouse model of acute colitis induced by DSS. Our results showed that treatment with SB-222200 significantly increased colon length (Fig. 6a and b) and reduced fecal blood index (Fig. 6c) compared to the DSS-induced model. In the DSS-treated mice, the colon tissue exhibited mucosal ulceration, loss of crypt, epithelial damage, and a significantly elevated colitis score. However, the SB-222200-treated groups showed markedly less histological damage (Fig. 6d and e). Furthermore, treatment with SB-222200 led to a decrease in the levels of IL-1β (Fig. 6f) and IL-6 (Fig. 6g) in the colons. Additionally, SB-222200 treatment resulted in a reduction in the levels of caspase-1 p20 and cleaved GSDMD in the colon (Fig. 6h), without affecting the expression levels of pro-IL-1β, NLRP3, and ASC (Fig. 6i). These findings indicate that SB-222200 may have the ability to inhibit the activation of the NLRP3 inflammasome.

Fig. 6.

Fig. 6

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SB-222200 alleviates DSS-induced experimental colitis in mice. C57BL/6 J mice were orally administrated with 2.5% DSS continuously for 7 days. SB-222200 injection was administrated through i.p. injection at the dose of 10 and 20 mg/kg per day for 8 days. a Representative photographs of the colons at the end of the experiment. b Colon length of mice from different groups. c Feces were collected from mice per day, and blood content was tested in fecal occult blood. d Mouse colon sections were examined by H&E staining. Scale bar, 200 µm. e Colitis score of each mouse in different groups. The expressions of IL-1β (f) and IL-6 (g) in mouse colon tissue. h Immunoblot analysis of caspase-1 and GSDMD in mouse colon tissue. i Immunoblot analysis of NLRP3, pro-IL-1β, and ASC in mouse colon tissue. Data are presented as n = 7 or 6 and represent mean ± SEM. *p < 0.01, ***p < 0.001, ****p < 0.0001

Discussion

The activation of the NLRP3 inflammasome is a significant contributor to the pathogenesis of various inflammatory diseases, making it a crucial therapeutic target. As our understanding of the role of the NLRP3 inflammasome in diseases has increases, efforts have been made to develop small-molecule inhibitors. In this study, we present findings that demonstrate SB-222200 as a potent, selective, and direct NLRP3 inhibitor. Our results show that SB-222200 inhibits ASC oligomerization and the interaction between NEK7 and NLRP3, and it may also directly bind to NLRP3. In vivo studies reveal that SB-222200 exhibits significant pharmacological efficacy in MSU-induced peritonitis and DSS-induced acute experimental colitis in mice. These findings suggest that SB-222200 could serve as a lead compound for the development of NLRP3 inhibitors to treat NLRP3-driven disease and as a versatile tool to pharmacologically interrogate NLRP3 biology.

Currently, numerous compounds have demonstrated potent inhibitory activity against the activation of the NLRP3 inflammasome and have been tested in animal models. However, most of these compounds target upstream signals of the inflammasome and have other unavoidable biological effects, such as parthenolide and its derivatives [31, 32]. Our study revealed that SB-222200 directly binds to the NLRP3 protein, inhibiting inflammasome assembly and the subsequent maturation of caspase-1 and release of IL-1β. The protein NEK7 is an interacting partner of NLRP3 that is required for its stabilization and activation [29]. In our study, SB-222200 inhibited the direct interaction between NEK7 and NLRP3, suggesting that it binds directly to NLRP3 and disrupts the NEK7 − NLRP3 interaction, thereby blocking subsequent activation of the NLRP3 inflammasome. Similarly, Zhou et al. reported that oridonin could also inhibit the interaction between NLRP3 and NEK7 by directly binding to NLRP3 [17]. However, the binding sites of SB-222200 on NLRP3 have not been confirmed, which should be considered a limitation of this study. Future studies should include experimental identification of binding sites, such as site-directed mutagenesis of residues.

SB-222200 is an orally active and blood–brain barrier penetrant antagonist of NK3R, which modulates monoaminergic and amino acid neurotransmission in the central nervous system [25, 33]. In order to investigate whether SB-222200 blocks NLRP3 activation by inhibiting NK3R, we examined the inhibitory effect of another NK3R inhibitor, talnetant, on the activation of NLRP3 inflammasome. Our results demonstrated that talnetant was unable to reduce NLRP3-dependent IL-1β release, suggesting that NK3R is not involved in the inhibition of NLRP3 inflammasome. Therefore, further medicinal chemistry studies are required to optimize SB-222200 in order to develop a lead compound that specifically inhibits NLRP3 inflammasome without affecting NK3R.

IBD is a chronic inflammatory disease in the gastrointestinal tract, including Crohn’s disease and ulcerative colitis [34]. However, the currently available drugs, such as sulfasalazine, azathioprine, 5-aminosalicylic acid, and glucocorticoid, are not considered satisfactory [35, 36]. Several studies have indicated that the activation of the NLRP3 inflammasome is implicated in the development of IBD [6, 20, 37]. Our study demonstrated that SB-222200 effectively alleviated acute IBD in mice induced by DSS by inhibiting NLRP3 inflammasome. Consistent with our findings, MCC950 and tranilast were also reported to reduce intestinal damage and increase colon length in DSS-induced IBD mice [20, 38]. Another limitation of our study is that we have not yet verified the efficacy of SB-222200 in chronic intestinal inflammation. In future studies, we plan to investigate the impact of SB-222200 on chronic colitis models and other diseases associated with chronic inflammation, such as high-fat diet-induced diabetes and non-alcoholic fatty liver disease. Considering the involvement of the NLRP3 inflammasome in the progression of gout, Alzheimer’s disease, atherosclerosis, and other inflammation-related diseases, SB-222200 or its derivatives could potentially be utilized for the development of new therapeutics targeting the NLRP3 inflammasome for these conditions.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 1427 KB) (1.4MB, docx)

Abbreviations

ASC

Apoptosis-associated speck-like protein containing a caspase-recruitment domain

BMDMs

Bone marrow-derived macrophages

CCK-8

Cell counting kit-8

CD

Crohn's disease

CETSA

Cellular thermal shift assay

DARTS

Drug affinity responsive target stability

DSS

Dextran sodium sulfate

ELISA

Enzyme-linked immunosorbent assay

GSDMD

Gasdermin D

IBD

Inflammatory bowel disease

IL-1β

Interleukin-1β

LDH

Lactate dehydrogenase

MSU

Monosodium urate crystals

NLRP3

Nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3

NK3R

Neurokinin-3 receptor

SPR

Surface plasmon resonance analysis

UC

Ulcerative colitis

Author contributions

YZ, ZY, WH, and PS designed experiments; YZ, ZY, YO, HC, ZL, GL, SL, LH, YY, XZ, RW, and AQ performed research; AQ and WH contributed reagents and research tools; all authors analyzed data; YZ, and PS wrote the paper; WH and PS designed and supervised the project. All authors read and approved the final manuscript.

Funding

This study was sponsored by School of Pharmaceutical Sciences in GMU (Grant No. 02–410-2206314), Plan on enhancing scientific research in GMU (Grant No. 02–410-2302372XM), Guangzhou Municipal Science and Technology Project (CN) (No. 202102080450), the Innovative Team Project of Ordinary Universities in Guangdong Province (Grant No. 2022KCXTD022), and National Natural Science Foundation of China (Grant No. 81872743).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Conflict of interest

The authors declare no competing financial interest.

Ethical approval and consent to participate

All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Use Ethics Committee of Guangzhou Medical University (GY2020-035, 2018-126).

Consent for publication

All authors agree for publication.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Yinghua Zhou and Zhongjin Yang equally contributed to this work.

Contributor Information

Wenhui Hu, Email: huwenhui@gzhmu.edu.cn.

Ping Sun, Email: sun_ping@gzhmu.edu.cn.

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Associated Data

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Supplementary Materials

Supplementary file1 (DOCX 1427 KB) (1.4MB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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