New use of NU6300

Abstract

The application provides a new use of NU6300 and belongs to the technical field of medicine.The application provides a new use of NU6300 in inhibiting pyroptosis.Furthermore, the application verifies that NU6300 has a therapeutic effect on colitis by inhibiting pyroptosis, significantly reducing DSS-induced epithelial and mucosal damage, crypt expansion and goblet cell depletion, and verifies that NU6300 has a protective effect on LPS-induced sepsis by inhibiting pyroptosis, and the protective effect of NU6300 on LPS-induced sepsis is superior to that of NSA.It is proved that NU6300 can play an important role in the treatment of diseases mediated by pyroptosis and has good clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, specifically relating to a new application of NU6300. Background Technology

[0002] Pyroptosis is an important innate immune response, widely involved in the development of autoimmune diseases, infectious inflammatory diseases, neurological diseases, and atherosclerotic diseases. As a newly discovered form of programmed cell death, pyroptosis differs from other forms of cell death, such as apoptosis and autophagy, in both morphology and mechanism. Once pyroptosis occurs, cells swell, the nucleus undergoes significant condensation, protrusions form on the cell surface before rupture, and multiple pores form on the cell membrane, disrupting cell membrane integrity. This leads to the dissolution and release of cellular contents and pro-inflammatory molecules, ultimately resulting in inflammation. Therefore, pyroptosis is a crucial driver of intracellular inflammasome activation.

[0003] Pyroptosis occurs via both classical and non-classical pathways. The classical pathway relies on the caspase-1 pathway. Stimulated by signals from pathogens, bacteria, etc., the adaptor protein ASC binds to pro-caspase-1, recruiting and activating caspase-1. This activates inflammatory factors such as IL-18 and IL-1β, and cleaves the N-terminus of GSDMD (a 242-amino acid protein mainly expressed on the surface of immune cells and small intestinal mucosal epithelial cells), which then binds to phospholipids on the cell membrane, forming pores and releasing their contents, thus inducing pyroptosis. The non-classical pathway is the caspase-4, 5, and 11-dependent pathway, directly activated by bacterial LPS, etc. Activated caspases 4, 5, and 11 cleave GSDMD and indirectly activate caspase-1, triggering pyroptosis. Therefore, GSDMD can serve as a specific substrate for caspases (caspase-1, -4, -5, and -11) and as an effector molecule for lysis and highly inflammatory forms of pyroptosis. Upon activation, GSDMD-N binds to phosphorylated phosphatidylinositol, unique to eukaryotic cell membranes, and cardiolipin, unique to prokaryotic cell membranes, and oligomerizes through charge-charge interactions to form molecular channels. This allows mature IL-1β and IL-18 to pass through, disrupting the normal osmotic barrier of the cell membrane. The influx of water and ions leads to cell enlargement, swelling, and rupture, thereby triggering pyroptosis. However, excessive or inappropriate pyroptosis can be highly pathological and contribute to the development of various diseases. Therefore, GSDMD is a key executor of pyroptosis and is closely related to the occurrence and development of many diseases.

[0004] In-depth research on pyroptosis can help find new therapeutic targets for diseases. Due to the key role of GSDMD in inflammasome-related pyroptosis, GSDMD targets have become ideal and new targets for therapeutic intervention.

[0005] Recent studies have confirmed the strategy of directly inhibiting GSDMD to suppress pyroptosis in diseases. Mechanistically, this is achieved by preventing GSDMD cleavage and oligomerization of the GSDMD-NT fragment, thereby reducing pyroptosis pore formation. However, currently only a few direct or indirect small molecule inhibitors are available, such as necrotizing sulfonamides, LDC7559, magnesium, disulfiram, and dimethyl fumarate. These inhibitors can prevent GSDMD cleavage or block GSDMD-NT oligomerization to alleviate pyroptosis. To date, no GSDMD-specific inhibitors have been successfully developed clinically. Therefore, further research and development of novel, more specific GSDMD-targeting inhibitors are crucial, providing new insights and a window of opportunity for the clinical treatment of pyroptosis diseases.

[0006] NU6300 is a covalently irreversible and ATP-competitive CDK2 (cyclin-dependent kinase 2) inhibitor with the following structural formula:

[0007] CDK2 plays an important role in the regulation of the eukaryotic cell cycle and transcription, and participates in physiological processes such as transcription, metabolism, neural differentiation and development, as well as in the regulation of various diseases.

[0008] However, there are currently no reports of NU6300 inhibiting inflammasome activation and pyroptosis. Summary of the Invention

[0009] The purpose of this invention is to provide a new application for the NU6300, the structural formula of which is as follows:

[0010]

[0011] This invention provides a use of NU6300 as a pyroptosis inhibitor.

[0012] The inhibitor is a reagent that inhibits pyroptosis caused by NLRP3 inflammatory activation; preferably, the inhibitor is a reagent that inhibits NLRP3 activation; preferably, the inhibitor is a reagent that inhibits cytoplasmic swelling and plasma membrane rupture.

[0013] The reagent is a reagent that inhibits the GSDMD protein; preferably, the reagent is a reagent that binds to the Cys 191 position of GSDMD.

[0014] The inhibitor is a reagent that inhibits GSDMD oligomerization and membrane pore formation.

[0015] The present invention also provides the use of NU6300 in the preparation of medicaments for the treatment and / or prevention of pyroptosis-mediated diseases.

[0016] The drug is used to treat and / or prevent autoimmune diseases, infectious inflammatory diseases, neurological diseases, or atherosclerotic diseases.

[0017] The medications for treating and / or preventing autoimmune diseases include medications for treating and / or preventing colitis.

[0018] The drug for treating and / or preventing colitis is a drug that reduces epithelial and mucosal damage, crypt dilatation, and goblet cell depletion; preferably, the drug reduces DSS-induced epithelial and mucosal damage, crypt dilatation, and goblet cell depletion.

[0019] The medications for treating and / or preventing infectious inflammatory diseases include medications for treating and / or preventing sepsis.

[0020] The drug for treating and / or preventing sepsis is a drug that reduces the spleen index; preferably, the drug is a drug that reduces the increase in the spleen index caused by LPS-induced sepsis.

[0021] The colitis mentioned is ulcerative colitis.

[0022] This invention discovers that the small molecule NU6300 can inhibit pyroptosis. Furthermore, this invention combines PI uptake and staining with transmission electron microscopy to observe the pyroptosis phenotype, and further identifies targets through target stability analysis of drug affinity response (DARTS) and proteomic analysis. Subsequently, key regulatory targets are verified through DARTS analysis, cell thermal transfer assay (CETSA), and biomembrane interference assay (BLI). Oligomerization analysis and the influence of membrane pore formation are further combined to elucidate the mechanism of pyroptosis inhibition. Finally, the role of NU6300 in pyroptosis-related animal models of colitis and sepsis is evaluated.

[0023] This invention investigates the role of NU6300 in regulating pyroptosis, providing a new window for the treatment of inflammatory diseases. Simultaneously, this invention reveals the molecular mechanism by which NU6300 targets GSDMD to inhibit inflammatory cell pyroptosis, offering new ideas and strategies for developing novel small-molecule inhibitors of inflammatory pyroptosis.

[0024] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0025] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0026] Figure 1 NU6300 specifically inhibits pyroptosis. (A) Chemical structure of NU6300. (B and C) After LPS induction, cells were infected with NU6300 (0.5, 1, and 2 μM), and THP-1 cells and BMDMs were stimulated with poly(dA:dT) (0.5 μg / mL) (D) or Flagellin (250 ng / mL) (E) for 6 h. Cytotoxicity was then detected by LDH assay. (F) PMA-induced differentiated THP-1 cells and BMDMs were pre-incubated with Pam3CSK4 (400 μg / mL) for 3 h, treated with NU6300, and then transfected with LPS (1.5 μg) overnight. Cytotoxicity was determined by LDH assay. (G) Before nigericin stimulation of LPS-induced THP-1 cells and BMDMs, cells were treated with NU6300 (2 μM), z-VAD-fmk (20 μM), NSA (10 μM), or Dis (20 μM) to assess cytotoxicity. The figures are shown as mean ± SEM, n = 3. Statistical analysis was performed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001; no statistically significant difference was found in ns.

[0027] Figure 2 NU6300 specifically inhibits NLRP3 inflammatory pyroptosis. (A and B) THP-1 cells and BMDMs were stimulated with LPS (1 μg / mL) and NU6300 (0.5, 1, and 2 μM) for 40 min, then induced with nigericin (10 μM) for 35 min, and stained with PI (2.5 μg / mL). Fluorescence microscopy images were taken and the PI uptake ratio was calculated. Scale bar, 50 μm (A); kinetic analysis of PI uptake (B). (C) HT-29 cells were pretreated with NU6300 (2 μM) or NSA (10 μM) for 1 h, then stimulated with 25 ng / mL TNFα (T), 400 nM MSMAC mimic (S), and 20 μM z-VAD-fmk (Z) for 24 h. Cell viability was detected by CCK8 assay. (D) Transmission electron microscopy was used to observe the morphology of THP-1 cells after NU6300 treatment and LPS and nigericin induction. Scale bar, 1μm.

[0028] Figure 3NU6300 directly interacts with human GSDMD. (A) DARTS experiments were performed on LPS-stimulated THP-1 cells. Cells were lysed with NP40 buffer, then incubated with DMSO or NU6300 (1000, 100, and 10 μM) for 50 min, followed by digestion with different concentrations of streptomycin (1:500, enzyme-to-protein ratio) for 30 min, and the reaction was terminated with a phosphatase inhibitor. Samples were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining. (B and C) Immunoblot analysis of THP-1 cells (B) or purified GSDMD protein (C) in DARTS experiments was performed, with the target protein normalized to the control protein to obtain the relative expression of GSDMD protein. (D and E) CETSA experiments were performed on LPS-induced THP-1 cell lysates or purified GSDMD protein after incubation with NU6300 (10 and 20 μM) at different temperatures. GSDMD protein expression was normalized using unheated samples. (F) The binding of GSDMD to NU6300 was assessed by BLI analysis, and equilibrium binding signals (Req) were plotted based on analyte concentrations. Data are expressed as mean ± SEM, n = 3. Statistical analysis was performed using one-way or two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001; ns, no significant difference.

[0029] Figure 4NU6300 binds to Cys191 on GSDMD. (A) MS / MS map of human GSDMD peptide FSLPGATCLQGEGQGHLSQK modified on Cys191 after GSDMD and NU6300 incubation. (B) LPS-induced THP-1 cells were stimulated with nigericin (10 μM) after 1 h of pre-incubation with NU6300 (0.5, 1, and 2 μM) and NAC (500 μM) for LDH detection. Data are expressed as mean ± SEM, n = 3. (C) GSDMD-N (p30) was transfected into HEK-293T cells to evaluate the inhibitory effect of NU6300 on pyroptosis. After 4 h of transfection, the medium was replaced with NU6300 (2.5–20 μM) and cultured for another 16 h for LDH detection. (D) HEK-293T cells were transfected with p30, p30-C191A, p30-C268A, or p30-C191A / C268A, followed by LDH analysis. (E) HEK-293T cells transfected with p30, p30-C191A, p30-C268A, or p30-C191A / C268A were treated with or without NU6300 (2.5, 5, and 10 μM) before LDH analysis. Data are expressed as mean ± SEM, n = 6. Statistical analysis was performed using one-way or two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001; ns, no significant difference.

[0030] Figure 5 NU6300 disrupted GSDMD oligomerization and pore formation. (A) p30-transfected HEK-293T cells were treated with NU6300 (2 μM) for 16 h, and p30 oligomerization was analyzed after cross-linking with disuccinimide succinate. (B) THP-1 cells or BMDMs were stimulated with LPS, pretreated with NU6300 (0.5, 1, and 2 μM) for 40 min, and stimulated with nigericin (10 μM) for 35 min before GSDMD oligomerization was analyzed. (C) LPS-induced THP-1 cells were incubated with NU6300 (2 μM) or z-VAD-fmk (20 μM) before nigericin stimulation. Cells were stained with GSDMD antibody and DAPI, and then observed and photographed under a confocal fluorescence microscope. Scale bar, 10 μm.

[0031] Figure 6NU6300 feedback inhibits the activation of the NLRP3 inflammasome. (AD) Oligomerization of ASCs in THP-1 cells or BMDMs was detected by a bis(succinimide) crosslinking assay. Cells were induced with NU6300 (2 μM) using LPS (1 μg / mL) or Pam3CSK4 (400 μg / mL), followed by transfection with poly(dA:dT) (0.5 μg / mL) (A) and flagellin (250 ng / mL) (B) for 6 h, or transfected overnight with cLPS (1.5 μg / mL), or induced with nigericin for 35 min. (EG) LPS-stimulated THP-1 cells were cultured with NU6300 (2 μM), z-VAD-fmk (20 μM), NSA (10 μM), or Dis (20 μM) before nigericin stimulation. Immunoblotting of culture supernatant (Sup) or whole-cell lysate (WCL) was used to analyze the lysis of pro-caspase-1, GSDMD, and pro-IL-1β in treated cells, as well as the maturation and release of caspase-1 and IL-1β (E). Caspase-1 activity was measured using Caspase-Glo 1 reagent (F), and IL-1β release was detected by ELISA (G). Figures show mean ± SEM, n = 3. Statistical analysis was performed using one-way ANOVA, ***P < 0.001.

[0032] Figure 7 NU6300 alleviated DSS-induced colitis in mice. C57BL / 6 mice were induced with colitis by 3.25% DSS for 6 days, then given normal drinking water for 5 days, and intraperitoneally injected daily with NU6300 (5, 10, and 20 mg / kg) and NSA (20 mg / kg) for 5 consecutive days (A). (B) Body weight recorded. (C) Mouse DAI score. (D) Colon length. (E) H&E staining to observe colonic pathological changes. Scale bar, 200 μm. (F) ELISA to detect IL-1β and TNFα levels in colonic tissue. (G) Western blotting to detect the expression of caspase-1 and GSDMD proteins cleaved in the colon. The values ​​are shown as mean ± SEM (n = 6). Statistical analysis was performed using one-way ANOVA. *P < 0.05, ***P < 0.001; ns, no significant difference.

[0033] Figure 8NU6300 has a protective effect against LPS-induced sepsis in mice. BALB / c mice (n=10) were intraperitoneally injected with normal saline (Normal), NU6300 (5 and 10 mg / kg), or NSA (20 mg / kg) for 1 h, followed by LPS (8 mg / kg) (A). (B) Survival curves were analyzed using the log-rank (Mantel-Cox) test (n=10). (CD) 4 h after drug and LPS stimulation, serum IL-1β (C) and TNFα levels were detected by ELISA (D), and the spleen index (E) was calculated (n=6). Data are presented as mean ± SEM and analyzed using one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001; ns, no significant difference. Detailed Implementation

[0034] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0035] PMA, short for Phorbol-12-myristate-13-acetate, is a commonly used phorbol ester. PMA is a tumor promoter that can promote tumor formation in mouse skin. THP-1: Human monocytic leukemia cells. BMDM: Mouse bone marrow macrophages. LPS: Lipopolysaccharide. Poly(dA:dT): Inflammasome inducer. Flagellin: Salmonella typhimurium flagellin. LDH: Lactate dehydrogenase. HT29 cells: Human colorectal adenocarcinoma cells. NSA: Necrosulfonamide, a pyroptosis inhibitor. Nigericin. TNF-α: A pleiotropic pro-inflammatory cytokine. Second mitochondria-derived activator of caspase (SMAC): SMAC mimic (z-VAD-fmk: apoptosis inhibitor). EZ-LinkNHS-LC-LC-Biotin is a long-chain, NHS ester-activated biotinylated reagent that can be used for general intracellular labeling.

[0036] The NSA used in this invention was purchased from TargetMol, model / part number T7129.

[0037] The DSS used in this invention was purchased from Yeasen, model / part number 60316ES60.

[0038] LPS used in cell experiments was purchased from Sigma, model / catalog number L2654.

[0039] LPS used in animal experiments was purchased from Sigma, model / item number L2880.

[0040] Example 1: The inhibitory effect of NU6300 on pyroptosis

[0041] I. Experimental Methods

[0042] 1. Selective effects of NU6300 on classical and non-classical pyroptosis.

[0043] After PMA induction, human monocytic leukemia cells (THP-1 cells) and adherent mouse bone marrow macrophages (BMDM cells) were induced with LPS for 3 h, then treated with NU6300 for 40 min. They were then transfected with Poly(dA:dT) to activate the AIM2 inflammasome and Flagellin to activate the NLRC4 inflammasome and treated for 6 h, respectively. The cell supernatant was collected and the release of LDH was measured. Alternatively, Pam3CS4K was used to induce the cells for 3 h, followed by LPS transfection and non-classical inflammation for 16 h. The cell supernatant was collected and the release of LDH was measured.

[0044] 2. Analysis of the effect of PI staining on PI uptake

[0045] After stimulating and inducing cells according to experimental requirements, the supernatant was discarded, and PI dye was added and incubated at 37°C for 15 min. DAPI staining was then added for 10 min, followed by PBS washing and observation using a fluorescence microscope. Cells were also seeded in 96-well plates, treated with Nigericin as before, and then PI dye was added. Kinetic analysis was performed every 5 min to monitor changes in fluorescence intensity 2 h after staining.

[0046] 3. Transmission electron microscopy analysis

[0047] THP-1 cells differentiated from PMA induced by LPS were treated with NU6300 (2 μM) for 40 min, followed by induction with nigericin (10 μM) for 35 min. After incubation, cells were washed with PBS and collected, then fixed with 0.5% glutaraldehyde at 4°C for 5 min, followed by centrifugation at 12000 rpm for 15 min. The cell pellet was then fixed in 3% glutaraldehyde. Ultrathin sections were imaged and observed using a JEM-1400PLUS transmission electron microscope.

[0048] 4. Detection of the role of necroptosis

[0049] Human colorectal adenocarcinoma cells (HT29 cells) were seeded into 96-well plates (8000 cells / well). After 24 hours, the cells were pretreated with NU6300 or NSA (pyroptosis inhibitor) for 1 hour, followed by treatment with TNFα (T), SMAC mimic (S) and z-VAD-fmk (Z) for 24 hours. Cell viability was then measured using CCK8 assay.

[0050] 5. Immunofluorescence analysis of the effect of NU6300 on GSDMD well formation

[0051] First, the cleaned slides were dried and placed in 24-well plates. After cooling, THP-1 cells were differentiated using PMA and seeded into the wells. After treating the cells according to the experimental requirements, pre-cooled methanol (-20℃) was added, and the cells were fixed at -20℃ for 20 min. The slides were washed with PBS for 2 min, and the process was repeated 3 times. 5% BSA was added along the wall for blocking at room temperature for 1 h. After washing with PBS, GSDMD primary antibody was added, and the slides were incubated at 4℃ overnight. After washing with PBS, secondary antibody containing Hochest (staining agent) was added and incubated for 1 h. After washing with PBS 3 times, the slides were removed and allowed to stand for several minutes until the moisture disappeared. The slides were then mounted with an anti-fluorescence quencher and stored. The cell membrane pores were observed by photographing with a confocal microscope.

[0052] 6. Target Stability of Drug Affinity Response (DARTS) Experimental Analysis of NU6300's Potential Targets

[0053] THP-1 cells were seeded into 10cm dishes. After processing the cells according to experimental requirements, the culture medium was discarded, and the cells were washed with PBS. 600-800 μL of NP-40 lysis buffer was added, and the cells were lysed on ice for 30 min. Centrifugation was then performed at 13,000 rpm for 15 min at 4°C. The supernatant was collected and incubated with NU6300 or DMSO (dimethyl sulfoxide) at room temperature for 50 min. Subsequently, protease was added, and lysis was performed for 30 min. A phosphatase inhibitor was then added, and the cells were incubated at room temperature for 10 min. After sample processing, SDS-PAGE (polyacrylamide gel electrophoresis) analysis was performed. Coomassie brilliant blue staining was used, and strips with pharmacological activity were excised and sent for mass spectrometry analysis to identify potential target proteins.

[0054] 7. Cellular thermal displacement analysis (CETSA) to examine the target of action.

[0055] As above, DARTS analysis was combined with Western blot analysis. Based on the data of possible target proteins, the corresponding primary antibody was selected and incubated overnight, followed by incubation with the corresponding secondary antibody to screen for target proteins. Then, CETSA analysis was performed: cells induced by LPS for 3 hours were lysed with RIPA lysis buffer and centrifuged at 13,000 rpm for 15 minutes at 4°C. After collecting the supernatant, the cells were aliquoted and incubated with drugs or DMSO for 1 hour. Then, the cells were heated for 5 minutes using a PCR instrument and cooled to room temperature for 3 minutes. Western blot was used to detect the expression of target proteins at different temperatures.

[0056] 8. DARTS analysis validated the association between NU6300 and the target protein.

[0057] To determine the concentration of GSDMD protein, aliquots were made into 22.5 μL tubes, and 2.5 μL of NU6300 or DMSO were added. The mixture was incubated at room temperature for 50 min. Then, a protease was added, and the mixture was lysed for 30 min. Following this, a phosphatase inhibitor was added, and the mixture was incubated at room temperature for 10 min. Western blot analysis was then performed on the processed samples.

[0058] 9. Thermal displacement analysis (TSA) was used to verify the link between NU6300 and the target protein.

[0059] GSDMD protein concentration was determined by aliquoting 22.5 μL into each tube and adding 2.5 μL of NU6300 or DMSO. The mixture was then incubated at room temperature for 1 h. Subsequently, the sample was heated for 30 min using a PCR instrument, cooled to room temperature for 3 min, and then denatured in a metal bath with electrophoresis loading buffer before Western blot analysis to detect the expression of the target protein at different temperatures.

[0060] 10. Biomembrane interferometry (BLI) to verify the direct link between NU6300 and the target protein.

[0061] GSDMD protein was dissolved in PBS to a concentration of 100 μg / mL and biotinylated using EZ-Link NHS-LC-LC-Biotin. After pre-wetting the SA biosensor with PBS to record a baseline, biotinylated GSDMD from a 96-well black F-plate was directly immobilized onto the SA biosensor. Compound NU6300 was diluted to an appropriate concentration with PBS (containing 2.5% DMSO and 0.02% Tween 20) to a final volume of 200 μL / well. An equal volume of PBS (containing 2.5% DMSO and 0.02% Tween 20) was added to each well as a control. The four main steps—loading for 300 s, baseline for 60 s, association for 120 s, and dissociation for 120 s—were repeated cyclically. The assay was performed using ForteBio. DataAcquisition and DataAnalysis software collects and analyzes data.

[0062] 11. Verification of whether NU6300 inhibits GSDMD expression using pyroptosis inhibitors: THP-1 cells were seeded in 48-well or 6-well plates, initiated with LPS for 3 h, treated with NU6300 or the Caspase-1 inhibitor z-VAD-fmk for 40 min, induced with nigericin for 35 min, and the cell supernatant was collected for ELISA analysis to detect IL-1β expression; the cells in the 6-well plates were lysed with RIPA lysis buffer, and the protein was extracted for Western blot detection of GSDMD protein cleavage.

[0063] 12. The effect of NU6300 on ASC oligomerization

[0064] After PMA-induced differentiation of THP-1 cells, LPS was used for 3 hours, followed by NU6300 treatment for 40 minutes and then nigericin stimulation for 35 minutes. Cells were lysed with NP-40 lysis buffer and cross-linked with disuccinimidyl suberate. The polymerization of ASCs was detected.

[0065] 13. Caspase-1 activity assay

[0066] Caspase-1 activity in cultured cells was detected using the Caspase-Glo 1 inflammasome detection kit. THP-1 cells were cultured at 2 × 10⁶ cells / cells. 5 Cells were seeded at 1000 / mL in 96-well plates, differentiated overnight with PMA, initiated with LPS, incubated with DMSO or NU6300, and then induced with nigercin. After induction, the culture medium was removed, 100 μL of Caspase-Glo 1 reagent was added, and the mixture was gently mixed. After incubation at room temperature for 1 h, luminescence was measured using a Biotek Synergy plate reader.

[0067] II. Experimental Results

[0068] 1. NU6300 specifically inhibits pyroptosis.

[0069] NU6300 ( Figure 1 A) Dose-dependent inhibition of LPS and nigericin-induced THP-1 and BMDM cell death, IC50 50 The values ​​were 0.8899 μM and 0.9321 μM. Figure 1 B and C). Transfection with poly(dA:dT) to activate AIM2 or flagellin to activate the NLRC4 classical inflammasome significantly led to the death of NU6300 THP-1 cells and BMDMs. Figure 1D and E). In addition to classical activation, NU6300 also directly inhibited the activation of non-classical inflammasomes in cells after prestimulation with Pam3CSK4 followed by LPS transfection. Figure 1 F) indicates that pyroptosis is a common downstream consequence of inflammasome signaling under strong stimulation. Generally, caspase-1-dependent pyroptosis is due to activation of the NLRP3 inflammasome. To determine if this is the case, this invention treated LPS-induced cells with NU6300, or the pan-caspase inhibitor z-VAD-fmk, and the pyroptosis inhibitors Disulfiram (Dis) and Necrosulfonamide (NSA) prior to nigericin stimulation, and assessed the effect by LDH release. Results showed that NU6300 (2 μM) had similar inhibitory effects on THP-1 cells and BMDMs (primary bone marrow-derived macrophages) as pyroptosis inhibitors (F). Figure 1 G).

[0070] Experimental results show that NU6300 has an inhibitory effect on pyroptosis.

[0071] 2. Inhibitors of pyroptosis induced by NLRP3 inflammatory activation

[0072] PI staining and PI uptake kinetics analysis revealed that NU6300 improved cell death and membrane permeability in THP-1 cells and BMDMs after nigericin stimulation. Figure 2 A and B). Interestingly, in HT-29 cells, the combination of TNF-α (T), the pan-caspase inhibitor z-VAD-fmk (Z), and a second mitochondrial-derived caspase activator (Smac) mimic (S) induced the degree of necroptosis, while NSA, as an inhibitor of necroptosis, was blocked, and no protective effect was observed after NU6300 treatment. Figure 2 C). Transmission electron microscopy revealed that NU6300 improved the characteristic cytoplasmic swelling and plasma membrane rupture associated with pyroptosis. Figure 2 D). These data indicate that NU6300 specifically inhibits the occurrence of NLRP3 inflammatory pyroptosis.

[0073] Experimental results showed that NU6300 improved the characteristic cytoplasmic swelling and plasma membrane rupture associated with pyroptosis, and NU6300 specifically inhibited the occurrence of NLRP3 inflammatory pyroptosis.

[0074] 3. NU6300 directly targets the GSDMD protein.

[0075] To identify potential therapeutic targets, this invention performed a Drug Affinity Target Stability (DARTS) experiment on protein lysates from THP-1 cells. A significantly stronger protective band of approximately 50 kDa was observed in the protein extracts from NU6300-treated cells. Figure 3 A). Specifically, GSDMD is considered the direct and ultimate executor of pyroptosis, initiating pyroptosis with a weight of 53 kDa. As expected, GSDMD achieved high scores in shotgun proteomics based on peptide mass fingerprinting combined with tandem mass spectrometry, indicating that NU6300 may bind directly to GSDMD. To further demonstrate the specificity of the NU6300-GSDMD interaction, this invention definitively identified the direct interaction between GSDMD and NU6300 from DARTS samples by immunoblotting and Coomassie brilliant blue staining. Indeed, the addition of NU6300 effectively protected GSDMD from protease degradation in a dose-dependent manner, as evidenced by the increased protease concentration in THP-1 cell and BMDM lysates. Figure 3 B). It is worth noting that GSDMD purified by streptococcal protease-mediated degradation also has a similar protective effect. Figure 3 C). Further cell thermal displacement experiments confirmed that NU6300 significantly enhanced the thermostability of GSDMD protein in THP-1 cell lysate and purified GSDMD protein in a dose-dependent manner, indicating a close interaction between NU6300 and GSDMD. Figure 3 (D and E). Real-time binding / dissociation of NU6300 and biotinylated GSDMD was monitored using biomembrane interferometry. Analysis showed that the specific binding effect of NU6300 to GSDMD was 29.1 μM, and the Kon and Kdis rates were 1.51 × 10⁻⁶, respectively. 3 (1 / Ms) and 4.39×10 -2 (1 / s)( Figure 3 F), consistent with the data above. The above results confirm that GSDMD is the direct target of NU6300.

[0076] Experimental results show that GSDMD protein is the direct target of NU6300.

[0077] 4. The NU6300 is directly integrated with GSDMD's Cys191.

[0078] Vinyl sulfone is a newly emerging electrophilic reagent for thiol reactions. It can covalently react with thiols of Cys residues via nucleophilic aromatic substitution Michael addition reactions and is recognized as an irreversible inhibitor of cysteine ​​proteases. N-acetylcysteine ​​(NAC) contains a highly active Cys residue, which is inactivated by thiol-reactive compounds. Notably, the effect of NU6300 on recombinant human GSDMD was investigated by liquid chromatography-tandem mass spectrometry (LC-MS / MS), revealing significant modification of Cys191. Trypsin fragment analysis showed that half of the symmetrical NU6300 molecule was attached to the thiol group. Figure 4 A). Furthermore, competitive binding experiments showed that NAC pre-incubation significantly reversed the inhibitory effect of NU6300 on nigericin-induced pyroptosis in THP-1 cells. Figure 4 B) suggests that NU6300 may target reactive Cys. GSDMD is a key ligand protein involved in NLRP3 inflammasome-induced pyroptosis, containing GSDMD N and C domains. Once cleaved, the N-terminal fragment of GSDMD (p30) inserts into the cell membrane, leading to pore formation and triggering pyroptosis. To investigate the mechanism by which NU6300 inhibits GSDMD, the N-terminal and C-terminal cleavage fragments of GSDMD were expressed in human embryonic kidney cells (HEK-293T). Cell death analysis showed that NU6300 concentration-dependently ameliorated pyroptosis induced by transfection of the p30 fragment in HEK-293T cells (human embryonic kidney cells). Figure 4 C). Further analysis was performed using cysteine-to-alanine (Cys to Ala) mutations at several conserved human cysteine ​​residues (C38, C56, C191, C268) within the p30 fragment. As expected, the mutation at c191 on p30 significantly reduced cell death compared to wild-type or full-length GSDMD, consistent with LC / MS analysis results, while the mutation at C268 also showed reduced cytotoxicity. Figure 4 D). To determine whether the C191A mutant affects the ability of NU6300 to inhibit pyroptosis, p30, p30-C191A, p30-C268A, and p30-C191A / C268A were expressed in HEK-293T cells. This invention observed that NU6300 reduced pyroptosis in HEK-293T cells expressing p30 and p30-C268A, while no statistically significant difference was observed in p30-C191A and p30-C191A / C268A. Figure 4 E). In summary, Cys191 is an important target for covalent modification, and NU6300 directly binds to Cys191 on GSDMD.

[0079] Experimental results show that NU6300 directly binds to Cys191 on GSDMD.

[0080] 5. NU6300 disrupts GSDMD oligomerization and membrane pore formation.

[0081] The assembly of the classic NLRP3 inflammasome activates caspase-1, leading to widespread cleavage of the porogen GSDMD and subsequent oligomerization of GSDMD-N, which is responsible for executing pyroptosis. To determine whether NU6300 works by blocking p30 oligomerization, this invention examined GSDMD oligomers in p30-containing HEK-293T cells. NU6300 significantly blocked the formation of p30 oligomers. Figure 5 A). Furthermore, NU6300 also significantly inhibited GSDMD oligomerization upon stimulation of THP-1 cells and BMDMs with LPS and nigericin. Figure 5 B). Considering that p30 oligomers form large oligomeric membrane pores on the plasma membrane, ultimately leading to pyroptosis, the effects of NU6300 and z-VAD-fmk on pore formation stimulated by LPS and nigericin were examined using GSDMD staining. Figure 5 C) further confirms this.

[0082] Experimental results show that NU6300 can disrupt GSDMD oligomerization and membrane pore formation.

[0083] 6. NU6300 feedback regulation of NLRP3 inflammation activation

[0084] Given that ASCs bridge different sensor receptors, facilitating the propagation of inflammatory signals, and are essential for various inflammasome complexes, this invention explored the effect of NU6300 on ASC oligomerization in THP-1 and BMDM cells following NLRP3 inflammasome activation. As expected, NU6300 only slightly inhibited ASC oligomerization in AIM2, NLRC4, and non-classical inflammasomes. Figure 6 AC significantly attenuated ASC oligomerization in nigericin-triggered NLRP3 inflammasomes. Figure 6 D), but suggests that NU6300 may primarily inhibit NLRP3 inflammasome-mediated pyroptosis. This invention further investigated the effects of NU6300 and two other known GSDMD inhibitors (NSA and Dis) on early steps of the NLRP3 inflammasome-GSDMD signaling pathway, examining the expression of caspase-1, GSDMD, and IL-1β after nigericin activation of THP-1 cells. Importantly, all inhibitors showed significant inhibitory effects on the cleavage of pro-caspase-1, pro-IL-1β, and GSDMD. Figure 6E). Furthermore, as assessed by the Caspase-Glo 1 inflammasome assay, active caspase-1 was significantly inhibited by these inhibitors in cultured THP-1 cells. Figure 7 F). Similarly, the release of IL-1β in the cell culture supernatant was also blocked (F). Figure 6 (G). These results indicate that NU6300 has a feedback inhibitory effect on the NLRP3 inflammasome when it inhibits GSDMD, and that NU6300 can inhibit NLRP3 activation.

[0085] Experimental results show that NU6300 can inhibit NLRP3 activation.

[0086] Experimental results show that NU6300 covalently modifies Cys191 in GSDMD, hindering...

[0087] Advances in caspases have limited oligomerization, preventing pore formation induced by the N-terminal domain of GSDMD. This invention reveals that NU6300, along with two other GSDMD inhibitors (Dis and NSA), exhibits strong inhibitory activity against the NLRP3 inflammasome, suggesting a shared feedback inhibition of the NLRP3 inflammasome when GSDMD is inhibited. Furthermore, this invention demonstrates that NU6300 reduces the release of pro-inflammatory cytokines and provides protection in mouse models of colitis and sepsis. In summary, the results of this invention indicate that NU6300 is a novel inhibitor that directly targets GSDMD and holds promise as a potential intervention for inflammatory diseases.

[0088] The following experimental examples demonstrate the beneficial effects of the present invention.

[0089] Experimental Example 1: NU6300 Treatment of Colitis

[0090] I. Experimental Methods

[0091] 1. Laboratory animals

[0092] Male C57BL / 6 mice weighing 18–22g were randomly divided into 6 groups, with 6 mice in each group.

[0093] 2. Experimental modeling and intervention

[0094] This invention uses dextran sulfate sodium (DSS) to induce a colitis model.

[0095] Normal group: given regular drinking water for 11 days.

[0096] DSS group (control group): 3.25% DSS was given in drinking water for 6 days, followed by ordinary drinking water for 5 days.

[0097] NU6300 (5mg / kg): Administer 3.25% DSS in drinking water for 6 days, then administer regular drinking water for 5 days, and begin intraperitoneal injection of NU6300 (5mg / kg) for 5 consecutive days starting from day 7.

[0098] NU6300 (10 mg / kg): Administer 3.25% DSS in drinking water for 6 days, then administer regular drinking water for 5 days, and from day 7 onwards, administer NU6300 (10 mg / kg) intraperitoneally for 5 consecutive days.

[0099] NU6300 (20 mg / kg): Administer 3.25% DSS in drinking water for 6 days, then administer regular drinking water for 5 days, and from day 7 onwards, administer NU6300 (20 mg / kg) intraperitoneally for 5 consecutive days.

[0100] NSA group, positive control group (20 mg / kg): 3.25% DSS was given in drinking water for 6 days, normal drinking water for 5 days, and NSA (20 mg / kg) was injected intraperitoneally for 5 consecutive days starting from day 7.

[0101] 3. Result Evaluation Parameters

[0102] Daily weight loss was recorded throughout the experiment, and DAI was monitored based on three parameters: weight, fecal concentration, and occult blood detection. Mice were sacrificed on day 11, and colons were collected for colon length determination, H&E (hematoxylin-eosin) staining analysis, cytokine analysis, and Western blot analysis.

[0103] II. Experimental Results

[0104] Given that the pyroptosis executor GSDMD is activated during intestinal inflammation in a chemically induced colitis model.

[0105] This invention further investigated the potential therapeutic effect of NU6300 on a DSS-induced colitis mouse model, in which C57BL / 6 mice were given 3.25% DSS for 6 consecutive days, followed by normal drinking water, and simultaneously injected intraperitoneally with NU6300 (5, 10, 20 mg / kg) and NSA (20 mg / kg) for 5 consecutive days. Figure 7 A). Notably, NU6300 demonstrated a significant dose-dependent reduction in weight loss and improvement in the severity of DSS-induced colitis, with the DAI score reflecting weight loss, diarrhea, and significant rectal bleeding. Figure 7 (B and C). Furthermore, administration of NU6300 significantly improved the hallmark symptom of DSS-induced colitis: shortening of the colon. Figure 7 D). Simultaneously, the aforementioned histological examination confirmed that NU6300 improves colonic inflammatory bodies, manifested as reduced DSS-induced epithelial and mucosal damage, crypt dilation, and goblet cell depletion. Figure 7E), even better than the NSA that has been reported to alleviate intestinal inflammation by inhibiting GSDMD-mediated pyroptosis.

[0106] ELISA analysis showed that NU6300 and NSA inhibited the expression of pro-inflammatory cytokines IL-1β and TNFα in the colonic region. Figure 7 F), Immunoblotting analysis showed decreased levels of caspase-1 and GSDMD-NT proteins. Figure 7 G).

[0107] Notably, compared with NSA administration, NU6300 significantly improved the severity of DSS-induced colitis, indicating that NU6300 has a good therapeutic effect in improving colitis.

[0108] Experimental results showed that NU6300 significantly reduced DSS-induced epithelial and mucosal damage, crypt dilation, and goblet cell depletion. NU6300's effect in alleviating intestinal inflammation by inhibiting GSDMD-mediated pyroptosis was superior to previously reported NSA, demonstrating good therapeutic efficacy in improving colitis.

[0109] Experimental Example 2: NU6300 has a protective effect against LPS-induced septicemia in mice.

[0110] I. Experimental Methods

[0111] 1. Laboratory animals

[0112] 22–25g C57BL / 6 mice were randomly divided into 6 groups, with 10 mice in each group.

[0113] 2. Experimental modeling and intervention

[0114] This invention uses an LPS-induced sepsis mouse model

[0115] NU6300 (5mg / kg): NU6300 (5mg / kg) was injected intraperitoneally first, followed by LPS (8mg / kg) 30 minutes later.

[0116] NU6300 (10 mg / kg): NU6300 (10 mg / kg) was injected intraperitoneally first, followed by LPS (8 mg / kg) 30 minutes later.

[0117] NSA (10 mg / kg): First, inject NSA (10 mg / kg) intraperitoneally, and 30 minutes later, inject LPS (8 mg / kg).

[0118] LPS group (8mg / kg): No drug was injected; LPS (8mg / kg) was injected together with the experimental group.

[0119] 3. Result Evaluation Methods

[0120] For survival analysis, mice (n=10 / group) were monitored every 12 hours after drug administration for 96 hours. To detect cytokines (n=6 / group randomly selected), blood was collected 4 hours after LPS stimulation and centrifuged at 4000 rpm for 15 min. Cytokine concentrations were determined by ELISA. For the spleen index, the spleen was removed and weighed immediately 4 hours after LPS stimulation. The spleen index was calculated using the following formula: Spleen index (g / g) = Spleen / Body weight × Body weight × 100%.

[0121] II. Experimental Results

[0122] Given the good protective effect of GSDMD inhibitors in LPS-induced sepsis mouse models, this invention further evaluates the potential of NU6300 in this regard.

[0123] Balb / c mice were intraperitoneally injected with NU6300 (5 and 10 mg / kg) or NSA (10 mg / kg) 1 hour before LPS (8 mg / kg) injection. Figure 8 A).

[0124] Survival curves showed that mice administered NU6300 and the GSDMD inhibitor NSA effectively protected against LPS-induced sepsis-induced death. Figure 8 B).

[0125] Four hours after LPS stimulation, both doses of NU6300 significantly increased serum concentrations of IL-1β and TNFα. Figure 8 C and D). Furthermore, compared to LPS-stimulated mice, the spleen index in the NU6300 treatment group was significantly reduced ( Figure 8 E) The spleen index at NU6300 concentration of 5 mg / kg was lower than that at NSA concentration of 10 mg / kg, indicating that NU6300 has a superior protective effect against LPS-induced sepsis compared to NSA. This invention is consistent with previous studies on GSDMD inhibitors, confirming the protective effect of NU6300 against LPS-induced sepsis.

[0126] Experimental results show that NU6300 has a protective effect against LPS-induced sepsis, and that the protective effect of NU6300 against LPS-induced sepsis is superior to that of NSA.

[0127] This invention demonstrates, based on data from mouse models of colitis and sepsis, that NU6300 is effective in treating and preventing inflammatory diseases.

[0128] In summary, this invention provides a novel use for NU6300 in inhibiting pyroptosis. Furthermore, this invention verifies that NU6300 significantly reduces DSS-induced epithelial and mucosal damage, crypt dilation, and goblet cell depletion in colitis by inhibiting pyroptosis, and also verifies the protective effect of NU6300 against LPS-induced sepsis by inhibiting pyroptosis, with NU6300 showing superior protective efficacy against LPS-induced sepsis compared to NSA. This demonstrates that NU6300 can play an important role in the treatment of pyroptosis-mediated diseases and has significant clinical application value.

Claims

1. Use of NU6300 in the preparation of medicaments for the treatment and / or prevention of colitis, wherein the structural formula of NU6300 is as follows: .

2. The use as described in claim 1, characterized in that, The medications used to treat and / or prevent colitis are those that reduce epithelial and mucosal damage, crypt dilation, and goblet cell depletion.

3. The use as described in claim 2, characterized in that, The drug is used to alleviate DSS-induced epithelial and mucosal damage, crypt dilation, and goblet cell depletion.

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