Application of tripterine in preparation of medicine for treating STING-related inflammation

Abstract

The invention belongs to the field of new application of medicines, and particularly relates to application of tripterine in preparation of a medicine for treating STING related inflammation. Experiments prove that tripterine can directly target STING protein and effectively inhibit excessive activation of a downstream signal channel of the STING protein for the first time, and the tripterine can be used as a potential STING inhibitor, a brand-new treatment candidate drug is provided for STING related inflammatory diseases, and a new thought is provided for treatment of immune and inflammatory diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of new use of drugs, and more particularly relates to an application of celastrol in preparation of a drug for treating STING-related inflammation. BACKGROUND

[0002] The cGAS-STING signaling pathway is an important innate immune pathway in cells. As a pattern recognition receptor, cGAS can recognize abnormal DNA in the cytoplasm and synthesize cyclic guanosine monophosphate-adenylic acid (cGAMP) from ATP and GTP, which can bind and activate the STING protein on the endoplasmic reticulum. After activation, the STING protein translocates to the Golgi and activates TANK-binding kinase 1 (TBK1). TBK1 then undergoes autophosphorylation and activates the transcription factor interferon regulatory factor 3 (IRF3). The cGAS-STING signaling pathway has been recognized as an important therapeutic target for immune and inflammatory-related diseases. In the case of infection, cell stress and tissue damage, this signaling pathway is a key mediator of inflammatory response. By targeting the inhibition of the STING signaling pathway, immune and inflammatory activation can be reduced, and STING-related immune and inflammatory diseases can be treated. Various STING inhibitors have been found, including H-151 and the like.

[0003] Celastrol is a natural active ingredient extracted from traditional Chinese medicine Tripterygium wilfordii. It has become a research hotspot due to its strong anti-tumor and weight loss effects. In order to further expand the application range of celastrol, further research on other pharmaceutical uses of celastrol is needed. SUMMARY

[0004] The application aims to provide an application of celastrol in preparation of a drug for treating STING-related inflammation.

[0005] The application provides an application of celastrol in preparation of a drug for treating STING-related inflammation.

[0006] Celastrol can induce cancer cell apoptosis by inhibiting proteasome activity, has anti-tumor, lipid metabolism regulation and anti-rheumatoid effects, and the present application first discovers that celastrol can directly target STING and inhibit downstream signal activation, reveals an important mechanism of the anti-inflammatory effect of celastrol, and provides a new candidate drug for further development of STING inhibitors based on celastrol to treat immune and inflammatory diseases.

[0007] Further, the celastrol is the only effective component in the drug.

[0008] Further, the drug further comprises a pharmaceutically acceptable excipient.

[0009] Further, the adjuvant comprises any one or more of non-toxic fillers, stabilizers, diluents, adjuvants.

[0010] Further, the diluent is any one of water and normal saline.

[0011] The application also provides a medicine for treating STING-related inflammation, which is prepared by mixing emladin and an adjuvant, and the content of emladin in the medicine is 0.1wt%-99wt%.

[0012] Further, the solid dosage form comprises granules, tablets, capsules, pills, dripping pills, and the solution dosage form comprises oral liquid preparation, intragastric administration, and injection administration.

[0013] Further, the solution dosage form is a solution of water and emladin, or a solution of normal saline and emladin.

[0014] The application has the following beneficial effects: A brand-new action target and mechanism are disclosed: the application first proves by experiments that emladin can directly target STING protein and effectively inhibit the overactivation of the downstream signal pathway.

[0015] A brand-new therapeutic candidate drug for STING-related diseases is provided: the abnormal activation of the STING signal pathway is a common pathological link of many autoimmune diseases (such as systemic lupus erythematosus and Aicardi-Goutières syndrome) and inflammatory diseases. The application clarifies that emladin is a natural STING direct inhibitor, which provides a brand-new drug development direction and lead compound for the treatment of the above-mentioned refractory diseases, and has important clinical conversion value.

[0016] The clinical application range and value of emladin are expanded: based on the newly discovered STING targeting and inhibiting ability, the potential application of emladin is no longer limited to the traditional anti-tumor and anti-rheumatism fields, but can be expanded to a series of new immune inflammatory diseases driven by the cGAS-STING signal axis, which greatly improves the development potential and application prospect of emladin as a drug mother nucleus. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1To investigate the inhibition of STING signaling activation by triptolide in stable cell lines, the following diagrams are presented: A shows Western blot images of the inhibition of STING, TBK1, and IRF3 phosphorylation by different doses of triptolide; B shows the Western blot results for TBK1; C shows the Western blot results for IRF3; and D shows the Western blot results for STING. * indicates P < 0.05 compared to the Control group, ** indicates P < 0.01 compared to the Control group, *** indicates P < 0.001 compared to the Control group, # indicates P < 0.05 compared to the STING treatment group, ## indicates P < 0.01 compared to the STING treatment group, and ### indicates P < 0.001 compared to the STING treatment group.

[0018] Figure 2 This diagram illustrates the inhibition of STING-mediated interferon pathway activation by triptolide. In the diagram, A represents the relative mRNA expression level of Ifn-β, B represents the relative mRNA expression level of Ifit1, C represents the relative mRNA expression level of Ifit2, and D represents the relative mRNA expression level of Ifit3. * indicates P < 0.05 compared to the Control group, ** indicates P < 0.01 compared to the Control group, *** indicates P < 0.001 compared to the Control group, # indicates P < 0.05 compared to the STING treatment group, ## indicates P < 0.01 compared to the STING treatment group, and ### indicates P < 0.001 compared to the STING treatment group.

[0019] Figure 3 The graph shows the inhibition of endogenous STING signal activation by triptolide. A represents the immunoblotting results of cGAMP-induced STING and TBK1 phosphorylation in THP1 cells by different doses of triptolide; B represents the Western blot quantitative statistical results of STING; and C represents the Western blot quantitative statistical results of TBK1. * indicates P < 0.05 compared to the Control group, ** indicates P < 0.01 compared to the Control group, *** indicates P < 0.001 compared to the Control group, # indicates P < 0.05 compared to the STING treatment group, ## indicates P < 0.01 compared to the STING treatment group, and ### indicates P < 0.001 compared to the STING treatment group.

[0020] Figure 4 The diagram shows the interaction results between STING protein and triptolide. A is the SPR spectrum of triptolide binding to human STING-CTD, B is the steady-state affinity fitting curve of SPR data, C is the thermogram obtained from ITC measurement, and D is the binding isotherm of ITC data.

[0021] Figure 5 The graph shows the inhibition of inflammatory factor expression in RAW264.7 macrophages by triptolide. In the graph, A represents the relative mRNA expression level of Ifn-β, B represents the relative mRNA expression level of the inflammation-related factor CCL5, and C represents the relative mRNA expression level of CXCL10. * indicates P < 0.05 compared to the Control group, ** indicates P < 0.01 compared to the Control group, *** indicates P < 0.001 compared to the Control group, # indicates P < 0.05 compared to the STING treatment group, ## indicates P < 0.01 compared to the STING treatment group, and ### indicates P < 0.001 compared to the STING treatment group.

[0022] Figure 6 Immunohistochemical images showing the effect of triptolide on MCD diet-induced non-alcoholic steatohepatitis (NASH). In the image, A shows representative liver immunohistochemical images for each group (Control group: normal control group; Model group: MCD diet-induced model group; Low group: 0.3 mg / kg triptolide intervention group; High group: 0.9 mg / kg triptolide intervention group). B shows the quantitative analysis results of STING protein expression in each group. *** indicates P < 0.001 compared to the Control group, and ### indicates P < 0.001 compared to the Model group. Detailed Implementation

[0023] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0024] Example 1 I. Based on the screening of stable cell lines, it was found that triptolide inhibits the activation of downstream signaling pathways of STING.

[0025] HEK-293T cells stably expressing mouse STING protein were cultured in vitro in six-well plates. The results of this study were published in "Nan Y, Cui D, Guo J, Ma X, Wang J, Guo L, Li T, Yang M, Huang G, Xu A, Ma W. STINGCOPII ER Export Trafficking and Signaling Primed by PhosphorylationSwitches. Adv Sci (Weinh). 2025 Sep;12(36):e03660.doi:10.1002 / advs.202503660.Epub 2025 Jul 1. PMID:40598830; PMCID:PMC12463132. In the article, cells were treated with 0.1 μM, 1 μM, and 10 μM triptolide and 10 μM H-151, respectively, followed by incubation with 25 μM DMXAA for 1 hour. The control group was a blank group without treatment. Cells were then lysed using RIPI (Beyotime, P0038) lysis buffer containing 1% PMSF and phosphatase cocktail inhibitor. After centrifugation at 12000g for 15 minutes at 4°C, the supernatant was collected. Protein concentration was determined using Beyotime's BCA protein quantification kit (catalog number P0012) with a standard curve plotted using BSA standards. The supernatant was diluted to the same concentration according to the BSA results. The diluted supernatant was mixed with preheated 5× loading buffer at a volume ratio of 4:1, denatured by boiling in a water bath, aliquoted, and stored or analyzed at -80℃. For analysis, 20 μg of protein was loaded onto each well and separated by electrophoresis on a 10% SDS-polyacrylamide gel. The stacking gel voltage was set to 80V, and the separating gel voltage to 120V. The protein was then transferred to a polyvinylidene fluoride (PVDF) membrane activated with methanol for 30 seconds at a constant current of 200mA for 2 hours. After transfer, the membrane was blocked with 5% BSA (diluted with TBST) solution at room temperature for 1 hour, followed by incubation with primary antibody at 4℃ overnight. The next day, the membrane was washed three times with TBST for 10 minutes each time, and then incubated with secondary antibody at room temperature with shaking for 1 hour. The membrane was washed three more times with TBST for 10 minutes each time. Finally, ECL chemiluminescence buffer was used for development, and the signal was captured using a chemiluminescence imaging system with automatic exposure time for optimal results.The primary antibodies include: anti-STING (CST, 13647), anti-pSTING (CST, 50907), anti-TBK1 (CST, 3013S), anti-pTBK1 (CST, 5483), anti-IRF3 (Proteintech, 11312), anti-pIRF3 (CST, 4947), and anti-GAPDH (Proteintech, 60004-1-Ig). The secondary antibodies include horseradish peroxidase-labeled goat anti-rabbit secondary antibody (Proteintech, SA00001-2) and goat anti-mouse secondary antibody (Proteintech, RGAM001).

[0026] like Figure 1 As shown, in HEK-293T cells stably expressing STING protein, treatment with 0.1 μM, 1 μM, and 10 μM triptolide for 1 h significantly inhibited DMXAA-induced STING protein phosphorylation and significantly suppressed the phosphorylation activation of downstream STING proteins TBK1 and IRF3, exhibiting a significant dose-dependent effect. Since HEK-293T cells do not express the upstream protein cGAS of STING, these results suggest that triptolide may directly act on STING protein to inhibit downstream signaling activation.

[0027] II. Study on the inhibition of STING downstream interferon and interferon-stimulated gene (ISG) expression by triptolide.

[0028] HEK-293T cells stably expressing mouse STING protein were cultured in six-well plates in vitro. After treatment with 0.1 μM, 1 μM, and 10 μM triptolide and 10 μM H-151, respectively, they were incubated with 25 μM DMXAA for 1 hour. Total RNA was then extracted from the cells using a column extraction kit. 100 ng of total RNA was used to synthesize the first strand of cDNA via reverse transcription. Amplification was performed using SYBR Green real-time quantitative PCR premixed reagent (Vazyme Biotech, China) on a real-time quantitative PCR instrument. The PCR program was set as follows: 95℃ pre-denaturation for 2.5 min; followed by 44 cycles of 95℃ denaturation for 15 seconds and 60℃ annealing / extension for 30 seconds; finally, dissociation curve analysis was performed. The expression levels of the target gene were normalized using GAPDH or β-actin as internal control genes.

[0029] like Figure 2As shown, RT-qPCR detection revealed that the expression of downstream IFN-β and ISGs (including IFIT1, IFIT2, and IFIT3) was significantly upregulated in cells treated with the agonist DMXAA alone. However, the administration of triptolide significantly inhibited the upregulation of the expression of these genes, indicating that triptolide can inhibit the activation of the STING-mediated interferon pathway.

[0030] III. Study on the inhibition of endogenous STING signaling activation by triptolide.

[0031] To further evaluate the inhibitory effect of triptolide on STING signaling activation, THP1 cells endogenously expressing STING protein were used for detection. Cells were pretreated with 0.3 μM, 0.9 μM, and 1.5 μM triptolide for 1 hour, followed by stimulation with 5 μM cGAMP for 1 hour. Proteins were then collected, and STING and TBK1 phosphorylation was detected by Western blot to evaluate the activation of the STING signaling pathway. Figure 3 As shown, STING and TBK1 were significantly phosphorylated in cells stimulated with 5 µM cGAMP for 1 hour, while in cells co-administered with triptolide, the phosphorylation of STING and TBK1 was significantly inhibited in a dose-dependent manner. Figure 1 The test results in the stable cell lines were consistent, further demonstrating that triptolide can dose-dependently inhibit STING signaling activation.

[0032] IV. Research on the targeted binding of triptolide to STING protein.

[0033] To further demonstrate the direct binding of triptolide and STING, surface plasmon resonance (SPR) was used to detect the binding of the two.

[0034] The experiment was conducted on a Biacore 1K system. STING protein was immobilized using a CM5 chip (Cytiva, catalog number BR-1005-30) via amino-coupling (the reference channel was used to subtract background interference). Tripterygium wilfordii was serially diluted (0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM) before detection. The chip was regenerated with 10 mM glycine-HCl (pH 2.0) after each round of detection. The kinetic parameters of triptolide-STING protein were measured by fitting a Langmuir binding model using Biacore Insight software.

[0035] like Figure 4 As shown in Figure A, the response value of the binding between the two gradually increases with the increase of triptolide dosage. Through steady-state affinity fitting, the dissociation constant KD value was obtained as 4.27 × 10⁻⁶. -6μM such as Figure 4 The B result indicates that the STING protein has a strong direct interaction with triptolide.

[0036] To further verify the binding effect and analyze its thermodynamic characteristics, isothermal titration (ITC) was used for detection. First, each sample was placed in a vacuum chamber for 10 minutes to remove any gases. Then, using a TA Nano ITC isothermal calorimeter, 300 μL of 1 μM STING (as the titrant) was drawn into the sample cell using the instrument's built-in sampling needle, and 60 μL of 10 μM triptolide (as the titrant) was drawn into the titration needle. The experimental conditions were then set to 25°C, and the injection was repeated 20 times at this temperature, with 200-second intervals between each injection. Data was collected and analyzed using nanoAnalyzer software during the experiment, and an independent fitting model was used to obtain the equilibrium dissociation constant (K0). D ), stoichiometry (n), enthalpy change (ΔH), and entropy change (ΔS).

[0037] like Figure 4 As shown in Figure C, titration of the GST-tagged C-terminal domain of the STING protein (STING-CTD) with triptolide yielded a significantly exothermic binding isotherm. The molar heat release (kcal / mol) was fitted to the change in molar ratio to obtain the dissociation constant (K). D The concentration was 1.7 μM, which is consistent with the SPR data. These results indicate that triptolide can specifically and directly bind to the STING protein, and its binding site is located in the 152-C region (cytoplasmic domain) of the STING protein.

[0038] 5. Tripterygium wilfordii inhibits the expression of interferon-β and inflammatory factors in macrophages.

[0039] To further evaluate the effects of triptolide on STING signaling and downstream interferons and inflammatory factors in macrophages, RAW264.7 (mouse macrophages) were used for detection. Cells were pretreated with 0.3 μM, 0.9 μM, and 1.5 μM triptolide for 1 hour, followed by stimulation with 25 μM DMXAA for 1 hour. Cell RNA was then extracted, and the mRNA expression of IFN-β, CCL5, and CXCL10 was detected by RT-qPCR (as described above).

[0040] like Figure 5 As shown, after intervention with triptolide, the mRNA expression levels of DMXAA-induced IFN-β and its downstream inflammatory factors CCL5 and CXCL10 were significantly reduced. These results demonstrate that Tripterygium wilfordii can inhibit the activation of the STING signaling pathway and the expression of downstream inflammatory factors in macrophages.

[0041] VI. Tripterygium wilfordii inhibits the expression of interferon-β and inflammatory factors in macrophages.

[0042] To further evaluate the application of triptolide-targeted inhibition of STING in disease treatment, a non-alcoholic steatohepatitis (NAH) model was induced in C57BL / 6 mice using a methionine- and choline-deficient diet. Animal grouping and specific implementation were as follows: Male C57BL / 6 mice were divided into a normal control group (Control) and a MCD diet (purchased from Chengdu Dashuo Experimental Animal Co., Ltd.) feeding group. After 2 weeks, the MCD feeding group was further divided into a model group, a low-dose (0.3 mg / kg) triptolide intervention group, and a high-dose (0.9 mg / kg) triptolide intervention group. The model group received intraperitoneal injection of saline, while the low-dose and high-dose groups received intraperitoneal injection of the corresponding doses of triptolide, respectively. This intervention was continued for 7 weeks. Immunohistochemical analysis was performed on the livers of mice in each group. The immunohistochemical results of the livers in each group are shown below. Figure 6 As shown, the liver tissue in the model group exhibited numerous vacuoles and disordered hepatocyte arrangement, with a significant increase in STING protein-positive cells. In contrast, the low-dose and high-dose triptolide intervention groups significantly improved hepatocyte vacuolation and significantly reduced the area of ​​STING protein-positive cells. This demonstrates that triptolide may alleviate non-alcoholic steatohepatitis in vivo by targeting and binding to STING and inhibiting STING protein expression, suggesting the potential application of triptolide in alleviating non-alcoholic steatohepatitis by targeting STING.

[0043] The above studies indicate that triptolide is a direct inhibitor of the STING protein, and the STING signaling pathway has been recognized as an important therapeutic target for immune and inflammatory diseases. Therefore, more potent STING inhibitors can be developed based on this drug.

[0044] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0045] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0046] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Application of triptolide in the preparation of drugs for treating STING-related inflammation.

2. The application according to claim 1, characterized in that, Tripterygium wilfordii is the only active ingredient in the drug.

3. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

4. The application according to claim 3, characterized in that, The excipients include any one or more of fillers, stabilizers, diluents, and adjuvants.

5. The application according to claim 4, characterized in that, The diluent is either water or physiological saline.

6. A drug for treating STING-related inflammation, characterized in that, The drug is a mixture of triptolide as described in claim 1 and excipients, wherein the content of triptolide in the drug is 0.1 wt% to 99 wt%.

7. The drug according to claim 6, characterized in that, The drug is available in either a solid dosage form or a solution dosage form. The solid dosage forms include granules, tablets, capsules, pills, and drop pills, while the solution dosage forms include oral liquid preparations, oral enemas, and injectable dosage forms.

8. The medicament according to claim 7, characterized in that, The solution formulation is a solution composed of water and triptolide, or a solution composed of physiological saline and triptolide.

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