Application of small molecule compound SFII in preparation of drugs for resisting pyroptosis and GSDMD inhibitor with pyroptosis resisting activity

Abstract

The invention discloses an application of a small molecule compound SFII in preparation of drugs for resisting pyroptosis and GSDMD inhibitors with pyroptosis resisting activity. The small molecule compound Scutellaria flavone II (SII) extracted from Scutellaria baicalensis roots plays an important role in preparation of drugs for inhibiting pyroptosis inflammation so as to prevent or treat atherosclerosis. Experiments prove that the active pharmaceutical ingredient SFII has a definite effect on promoting atherosclerosis by virtue of Gasdermin D and GSDMD mediated macrophage pyroptosis, is convenient to obtain, can be used for preparing drugs for inhibiting GSDMD protein activity to resist pyroptosis and exerting an anti-inflammatory effect so as to prevent or treat atherosclerosis, and has a remarkable effect. The small molecule compound SFII provides a new treatment strategy for atherosclerosis, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of chemical biology, and mainly relates to the use of a small molecule compound SFII in the preparation of a drug for resisting cell pyroptosis and a GSDMD inhibitor with anti-pyroptosis activity, including the use of a small molecule compound SFII in the preparation of a drug for resisting cell pyroptosis and exerting an anti-inflammatory effect to prevent or treat atherosclerosis, and discloses a GSDMD inhibitor prepared based on the drug. Background Art

[0002] Atherosclerosis (As) and the resulting coronary atherosclerotic heart disease (CHD) are the most common cardiovascular diseases and one of the main causes of death in humans. Atherosclerosis, characterized by vascular wall dysfunction and plaque formation, is the main pathological process in the formation and development of CHD. Chronic nonspecific inflammation is a key factor in the occurrence and development of atherosclerosis. Therefore, the treatment strategy based on inflammatory response has important scientific significance and clinical application value in the study of AS.

[0003] In the AS model, macrophage inflammatory pyroptosis is associated with the expansion of the necrotic core and plaque instability. Gasdermin-D (GSDMD) is a newly discovered executive protein molecule that causes cell pyroptosis. Recent studies have shown that GSDMD and APOE double knockout mice (GSDMD - / - APOE - / - ) also confirmed that GSDMD knockout can alleviate the progression of atherosclerosis. These findings suggest that GSDMD-mediated pyroptosis in macrophages is involved in the formation of atherosclerotic plaques. Therefore, it is of great significance to explore the mechanism of GSDMD and atherosclerosis and use it as a molecular target to exert anti-atherosclerosis effects. Summary of the invention

[0004] The purpose of the present invention is to treat atherosclerosis by inhibiting pyroptotic inflammation and to develop the medical therapeutic effect of SFII.

[0005] The small molecule compound SFII is a flavonoid compound extracted from the root of the traditional Chinese medicine Scutellaria baicalensis. It shows excellent activity in inhibiting cell pyroptotic inflammation and resisting atherosclerosis. It can directly bind to the GSDMD protein to inhibit its pore-forming activity, thereby inhibiting the release of pyroptotic effectors and downstream inflammatory factors. At present, there is a lack of clinical drugs for treating atherosclerosis by inhibiting pyroptotic inflammation. On the one hand, the present invention reveals the target and mechanism of SFII in inhibiting cell pyroptosis, and on the other hand, it provides new drugs and theoretical basis for the treatment of atherosclerosis.

[0006] The present invention is achieved by providing a drug that resists cell pyroptosis and exerts anti-inflammatory effects to prevent or treat atherosclerosis, wherein the drug for treating or preventing atherosclerosis is a small molecule compound SFII. The small molecule compound SFII can be used for injection or oral preparation.

[0007] Another object of the present invention is to provide an injection (10-20 mg / kg / day) made of the drug that resists cell pyroptosis and exerts anti-inflammatory effects to prevent or treat atherosclerosis.

[0008] Another object of the present invention is to provide an oral preparation (25-50 mg / kg / day) made by utilizing the drug for resisting cell pyroptosis to exert anti-inflammatory effects to prevent or treat atherosclerosis.

[0009] Another object of the present invention is to provide a GSDMD inhibitor (25-50 mg / kg / day) prepared by using the drug to resist cell pyroptosis to exert anti-inflammatory effects to prevent or treat atherosclerosis. The GSDMD inhibitor with anti-pyroptosis activity is prepared from SFII and pharmaceutically acceptable excipients.

[0010] Further, acceptable excipients include but are not limited to acceptable carriers or fillers or binders or wetting agents or disintegrants or absorption promoters or surfactants or adsorption carriers or lubricants or 0.9% sodium chloride ("excipients" refer to conventional drug carriers in the pharmaceutical field, for example: diluents, excipients such as water, fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginates, gelatin and polyvinyl pyrrolidone; wetting agents such as glycerol; disintegrants such as agar, calcium carbonate and sodium bicarbonate; absorption promoters such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as kaolin and soap clay; lubricants such as talc, calcium / magnesium stearate, polyethylene glycol, etc.).

[0011] Another object of the present invention is to provide a tablet made of a GSDMD inhibitor made by using the drug for resisting cell pyroptosis to exert anti-inflammatory effects to prevent or treat atherosclerosis.

[0012] Another object of the present invention is to provide a pill made of a GSDMD inhibitor made by using the drug for resisting cell pyroptosis to exert anti-inflammatory effects to prevent or treat atherosclerosis.

[0013] Another object of the present invention is to provide a suspension prepared from a GSDMD inhibitor prepared by utilizing the drug for resisting cell pyroptosis to exert anti-inflammatory effects to prevent or treat atherosclerosis.

[0014] Another object of the present invention is to provide a granule prepared by using the GSDMD inhibitor prepared by the drug for resisting cell pyroptosis to exert anti-inflammatory effect to prevent or treat atherosclerosis.

[0015] Another object of the present invention is to provide an emulsion prepared by using the GSDMD inhibitor prepared by the drug for preventing or treating atherosclerosis by resisting cell pyroptosis and exerting anti-inflammatory effect.

[0016] The present invention obtains a potential inhibitor of GSDMD, SFII, by virtual screening of the flavonoid drug library. On THP-1 cells induced by PMA, a cell pyroptosis model was constructed by lipopolysaccharide (LPS) combined with nigericin stimulation, and it was found that SFII can significantly reduce the release of inflammatory factors and effectively improve the cell damage caused by pyroptosis. Subsequently, we used the surface plasmon resonance principle (SPR) to perform biomolecular interaction analysis and protein pulldown technology to confirm that SFII can directly bind to the GSDMD protein. In the mouse atherosclerosis animal model, SFII can effectively reduce cell pyroptosis, improve atherosclerosis, and reduce the area of ​​vascular intimal plaques. Through fluorescence localization, it was found that GSDMD was mainly expressed in macrophages, and SFII effectively reduced the content of GSDMD in macrophages. At the same time, SFII effectively inhibited the increase in lipids and inflammatory cell pyroptosis caused by ox-LDL stimulation in the mouse macrophage cell line RAW264.7. The above results indicate that SFII has the ability to specifically target GSDMD to resist the inflammatory effects caused by cell pyroptosis, thereby alleviating atherosclerosis.

[0017] In specific applications, the drug is various preparations prepared from SFII and pharmaceutically acceptable excipients. Among them, injections for injection and oral capsules are preferred. The preparation of the present invention can be in unit dosage form, such as injections, capsules (including sustained release or delayed release forms), tablets, pills, suspensions, granules, tinctures, syrups, emulsions, suspensions and various sustained-release forms, so as to be suitable for various modes of administration, such as oral, parenteral injection, mucosal, intramuscular, intravenous, subcutaneous, intraocular, intradermal or transdermal administration (among which injections for injection and oral capsules are preferred).

[0018] The characteristics of the present invention are that the active ingredients in the drug are easy to obtain, can specifically target gasdermin D (GSDMD) to inhibit cell pyroptosis, have excellent anti-inflammatory activity, and can prevent or treat atherosclerosis. It can be used as a drug for treating atherosclerotic diseases with significant effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the molecular structural formula of the small molecule compound SFII described in the present invention.

[0020] Figure 2 This is a diagram showing the effect of the small molecule compound SFII described in the present invention on GSDMD-mediated cell pyroptosis in the macrophage cell line THP-1 model.

[0021] Figure 3 This is a diagram showing the structure of the binding of the small molecule compound SFII described in the present invention to the GSDMD protein and the related molecular mechanism results.

[0022] Figure 4 , Figure 5 , Figure 6 This is a graph showing the effect of the small molecule compound SFII described in the present invention on the degree of atherosclerosis in mice.

[0023] Figure 7 This is the effect of the small molecule compound SFII described in the present invention on cell pyroptosis in a mouse atherosclerosis model.

[0024] Figure 8 , Fig. 9 , Fig.10 This is a diagram showing the effect of the small molecule compound SFII described in the present invention on cell pyroptosis in the mouse macrophage cell line RAW264.7 stimulated by ox-LDL. DETAILED DESCRIPTION

[0025] The specific implementation modes of the present invention are described in detail below with reference to the embodiments.

[0026] The structural formula of small molecule compound SFII is as follows Figure 1 , is a flavonoid compound extracted from the root of the traditional Chinese medicine Scutellaria baicalensis, which shows excellent activity in inhibiting cell pyroptotic inflammation and resisting atherosclerosis. It can directly bind to the GSDMD protein to inhibit its pore-forming activity, thereby inhibiting the release of pyroptotic effectors and downstream inflammatory factors. At present, there is a lack of drugs for treating atherosclerosis by inhibiting pyroptotic inflammation. The present invention, on the one hand, reveals the targets and mechanisms of SFII in inhibiting cell pyroptosis, and on the other hand, provides new drugs and theoretical basis for the treatment of atherosclerosis.

[0027] Experimental animals: All animal care and experimental procedures used in the present invention were approved by the Animal Policy and Welfare Committee of Wenzhou Medical University (approval ID: WYYY-AEC-2022-038). APOE knockout mice of the C57BL / 6 strain were obtained from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. - / -After being fed a high-fat diet, mice were given SFII (10 mg / kg / d, dissolved in 1% CMC-Na, 0.25% tween-80) and SFII (20 mg / kg / d, dissolved in 1% CMC-Na, 0.25% tween-80) by oral gavage for 42 days.

[0028] Experimental cells: The human THP-1 cell line and mouse RAW264.7 cell line used in the present invention were purchased from the American Type Culture Collection (ATCC). The culture media used were RPMI 1640 culture medium and DMEM culture medium, respectively. 10% fetal bovine serum (FBS, Thermo Fisher Scientific) and 1% penicillin-streptomycin (ThermoFisher Scientific) were added during use. The cell culture environment was 5% CO2, 37°C constant temperature incubator. Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich, oxidized low-density lipoprotein (ox-LDL) was purchased from Guangzhou Yiyuan Biotechnology Co., Ltd., and Nigericin (Nig) was purchased from aladdin. The small molecule compounds used in the present invention were purchased from Wuhan Tianzhi Co., Ltd., and the remaining reagents were also purchased commercially and used according to the instructions or published literature.

[0029] Example 1: This example explores the effect of SFII on the resistance of pyroptosis damage in the classic pyroptosis model of macrophages; The present invention inoculated the THP-1 cells used in the six-well plate, added PMA to stimulate the cells for 24 hours, and then treated them with LPS (10 μg / ml) for 4 hours, and then stimulated with Nigericin (Nig) (10 μM) for 30 minutes to construct a classic cell pyroptosis model. SFII was added to pre-treat THP-1 cells for 1 hour according to the group. We found that SFII can inhibit the secretion of IL-1β inflammatory factor in the classic pyroptosis model ( Figure 2 Middle A), effectively improve cell activity ( Figure 2 Middle B).

[0030] in, Figure 2 The CCK8 (Cell Counting Kit-8) experiment indicated in A in the middle detects the cell activity after LPS+Nig modeling; Figure 2 ELISA in medium B detects the inflammatory factor IL-1β released into the extracellular space by macrophages.

[0031] Example 2: This example explores that SFII can directly bind to the GSDMD protein and is kinetically stable; The present invention uses AlphaFold to model the structure of human GSDMD (hGSDMD). After verifying the reliability of the hGSDMD model, the receptor tunnel and channel analysis web server Caver web is used to predict the binding site of hGSDMD. Then, AutoDock 4.2 software is used to predict the binding mode between hGSDMD and SFII. A cubic grid box with a size of 18.75 A × 18.75 A × 18.75 A is used to cover the binding site of hGSDMD ( Figure 3 SPR (Surface Plasmon Resonance, SPR) refers to the use of Surface Plasmon Resonance to analyze biomolecular interactions using Biacore TM The molecular interaction analysis system is Cytiva's fully automatic testing platform to evaluate the binding relationship between drugs and proteins. The present invention uses SPR to analyze the binding of SFII and GSDMD protein. We diluted the purified GSDMD protein with PBS at pH 7.4 to 100 μg / mL, coupled the GSDMD protein to a blank CM5 chip in 10 mM sodium acetate buffer; diluted SFII with 2‰ DMSO+PBS to 7 concentrations, with the highest concentration of 40 μM and the lowest concentration of 0.625 μM for subsequent detection; added the above samples to the sample plate in sequence and ran the program, using Biacore TM The software performs data fitting for kinetic analysis and steady-state analysis. Finally, we perform fitting analysis on the obtained data using the fitting model 1:1 ( Figure 3 Middle B).

[0032] In addition, the present invention uses BeaverbeadsTM streptavidin-agarose beads and biotinylated SFII to perform protein pulldown experiments to verify the binding of GSDMD and SFII. We added biotinylated SFII to streptavidin-agarose beads at a ratio of 10:1, and used non-biotinylated SFII, biotin alone, and untreated beads as control groups. Then, the purified GSDMD protein was added to the streptavidin-agarose beads with Bio-SFII. The mixture was incubated on a shaker at 25°C for 3 hours and then centrifuged. The precipitate was washed 3 times, and finally the protein was eluted by boiling the precipitate with 5x loading to obtain a GSDMD protein sample bound to SFII. The samples were grouped for Western Blot analysis, loaded on polyacrylamide gel for electrophoresis, detected with GSDMD primary antibody and secondary antibody of the corresponding species, and developed with ultrasensitive exposure solution ( Figure 3Based on the results of BLI and protein pulldown experiments, we found that SFII can directly bind to purified GSDMD protein.

[0033] in, Figure 3 A in the middle shows the schematic diagram of the virtual docking of the structurally modeled SFII and the GSDMD molecule, and the enlarged image (right) marks the predicted binding amino acid sites; Figure 3 Middle B shows that SPR experiments show that SFII binds to GSDMD; Figure 3 Center C shows a pulldown experiment in which GSDMD binds to SFII.

[0034] Example 3: This example explores the effect of SFII on targeted inhibition of GSDMD protein to alleviate cell pyroptosis and reduce atherosclerosis; Animal level: APOE in healthy age-matched individuals - / - The mice were divided into five groups, with 10 mice in each group. Two groups were fed with a normal low-fat diet (LFD), and the other three groups were fed with an atherosclerotic high-fat diet (HFD). They were divided into a low-fat control group (LFD), a high-fat modeling group (HFD), a high-fat low-dose medication group (HFD-10 mg / Kg / d), and a high-fat high-dose medication group (HFD-20 mg / Kg / d). High-fat feeding was continued for 12 weeks, and different doses of SFII were continuously gavaged from the 7th week until the end of 12 weeks of high-fat feeding. The entire length of the mouse aorta root to the iliac artery was stripped, and the lipid plaques on the intimal surface were stained with oil red ( Figure 4 We found that SFII can significantly reduce atherosclerotic plaques in mice and alleviate the increase in lipids and vascular fibrosis caused by atherosclerosis ( Figure 5 C, E, and Figure 6 (D, F).

[0035] Since macrophages are the main cells that play a role in atherogenesis and are also the classic cell type of cell pyroptosis pathway, we used macrophage labeling antibody F4 / 80 and GSDMD to perform immunofluorescence double staining experiments on mouse aortic root sections. The results showed that GSDMD expression increased in macrophages at the vascular root of atherosclerotic mice, and SFII could significantly reduce the expression of GSDMD on macrophages ( Figure 7 In order to verify the effect of SFII on cell pyroptosis in animals, we tested the effect of SFII on serum IL-1β. We found that SFII can effectively reduce the content of IL-1β in mouse serum ( Figure 7 Middle B).

[0036] in, Figure 4A and B show schematic diagrams of Oil Red O staining of plaques in the aorta. Figure 5 Middle C and Figure 6 Middle D shows a schematic diagram of Oil Red O staining of frozen sections of the aortic root. Figure 5 ZhongE and Figure 6 F in the middle shows Masson staining of frozen sections of vascular roots. ; n = 6, * p < 0.05, **p < 0.005, ****p < 0.0001.

[0037] Figure 7 A in the middle shows the co-localization diagram of F4 / 80 and GSDMD immunofluorescence double staining in frozen sections of vascular roots taken using a confocal microscope; Figure 7 B in the middle represents the content of IL-1β in mouse serum detected by ELISA. n = 6, * p<0.05, ****p < 0.0001.

[0038] Example 4: This example explores the effect of SFII on alleviating GSDMD-mediated macrophage pyroptosis under ox-LDL stimulation; Mouse macrophage cell line RAW264.7 was inoculated on a six-well plate. After the cells adhered to the plate, they were pretreated with SFII for 2 hours and stimulated with ox-LDL to establish a model. The model was divided into five groups: control group (Control), model group (ox-LDL), low-dose drug model group (ox-LDL + SFII-2.5 μM), and high-dose drug model group (ox-LDL + SFII-5 μM). Oil red O staining experiments showed that the lipid content of macrophages stained with oil red was significantly reduced under the action of SFII ( Figure 8 Next, we detected the IL-1β content in the cell supernatant. SFII can still reduce the secretion of inflammatory factors at the cellular level ( Fig. 9 Middle C), Western Blot experiments detected the same trend in the supernatant proteins. Extracellular IL-1β and IL-18 decreased with the action of SFII, and the expression of GSDMD-N protein also decreased significantly ( Fig. 9 (D, E). GSDMD immunofluorescence showed that SFII can reduce the expression of GSDMD on the macrophage membrane ( Fig. 9 The above animal and cell results indicate that SFII can target and inhibit GSDMD protein to alleviate cell pyroptosis and thus reduce atherosclerosis.

[0039] Figure 8 A and B in the middle are photos of RAW264.7 cells stained with Oil Red O under an optical microscope; Fig. 9 Middle C indicates the content of IL-1β inflammatory factor in cell supernatant detected by ELISA; Fig. 9D and E show Western Blot detection of the protein levels of IL-1β, IL-18 and N-GSDMD in the cell supernatant of RAW264.7 cells and the bar chart of the analysis; Fig.10 F and G in the middle represent GSDMD immunofluorescence staining photos of RAW264.7 cells under confocal microscopy. n = 6, * p < 0.05, ** p < 0.005, *** p < 0.001, **** p < 0.0001.

[0040] The preparation method of the drug for treating or preventing atherosclerosis provided in the embodiment of the present invention includes: various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional production methods in the pharmaceutical field. For example, the active ingredient SFII is mixed with one or more carrier excipients, and then made into the desired dosage form, such as injection, capsule and tablet.

[0041] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the design concept of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of the small molecule compound SFII in the preparation of drugs for resisting cell pyroptosis, characterized in that: The small molecule compound SFII is used in the preparation of a drug for resisting cell pyroptosis and exerting an anti-inflammatory effect to prevent or treat atherosclerosis. The molecular structure of the small molecule compound SFII is shown in the following formula: 。 2. The use according to claim 1, characterized in that: The drug prepared from the small molecule compound SFII is an injection for exerting an anti-inflammatory effect in resisting cell pyroptosis to prevent or treat atherosclerosis.

3. The use according to claim 1, characterized in that: The drug prepared from the small molecule compound SFII and used for preventing or treating atherosclerosis by exerting anti-inflammatory effects in resisting cell pyroptosis is an oral preparation.

4. A GSDMD inhibitor with anti-pyroptosis activity prepared by comprising the small molecule compound SFII according to claim 1, characterized in that: The GSDMD inhibitor with anti-cell pyroptosis activity is prepared from a small molecule compound SFII and pharmaceutically acceptable excipients.

5. The anti-pyroptosis activity GSDMD inhibitor according to claim 4, characterized in that: The pharmaceutically acceptable excipients include but are not limited to acceptable carriers, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, and 0.9% sodium chloride.

6. The anti-pyroptosis GSDMD inhibitor according to claim 4, characterized in that: The anti-pyroptosis activity GSDMD inhibitor can be prepared into any one of tablets, pills, suspensions, granules and emulsions.

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