Acute pneumonia inhibitory ginsenoside Rb1 monomer and application thereof

Abstract

The invention discloses an acute pneumonia inhibitory ginsenoside Rb1 monomer and application thereof, and relates to the technical field of biological medicines. The ginsenoside Rb1 is screened from a natural product library through a high-throughput screening method, and the ginsenoside Rb1 plays a role in inhibiting expression of inflammatory factors mediated by a menin / SETD2 compound. The monomer can be applied to preparation of anti-inflammatory drugs, anti-tumor drugs, radiotherapy and chemotherapy adjuvant therapy drugs, anti-cytokine storm syndrome (CSS) drugs, lung protection drugs and the like. The compound is applied to drugs for treating acute pneumonia related to inflammatory factor over-activation induced by microbial infection and physical and chemical factors, including inhibition of inflammatory factor expression, reduction of lung tissue inflammatory cell infiltration and pulmonary alveolar protein deposition and the like. The monomer can also effectively inhibit release of lung epithelial cell inflammatory factors induced by ionizing radiation and chemotherapeutic drug exposure. The compound has the advantages of definite action target, high efficiency, specificity, remarkable curative effect, low toxicity and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an acute pneumonia inhibitory ginsenoside Rb1 monomer and application thereof. Background Art

[0002] Abnormal activation of alveolar macrophages (AM) is a key factor leading to a series of severe pulmonary clinical manifestations. For example, when infected by pathogens such as the new coronavirus SARS-CoV-2, influenza virus, bacteria and mycoplasma, macrophages are activated to abnormally promote the expression of a series of cytokines, inflammatory factors and chemokines such as IL-1α and IL-6, thereby causing severe acute respiratory syndrome, which has a very high mortality rate. In addition, radio- and chemotherapy-induced pneumonia is also caused by the fact that when cancer patients receive radio- and chemotherapy treatment, normal lung tissue is damaged by ionizing radiation, chemotherapy drugs and other physical and chemical factors, resulting in a sharp increase in the expression of cytokines, inflammatory factors and chemokines. In severe cases, the lungs will undergo acute inflammatory reactions and extensive fibrosis, which will damage respiratory function and even lead to respiratory failure.

[0003] Cytokine Storm (CS) is an extremely serious immune response characterized by the activation of the body's immune cells by pathogen infection, which leads to the abnormal secretion of a large number of cytokines, inflammatory factors and chemokines, such as GM-CSF, IL-1α, IL-6, etc. The clinical manifestations of CS include persistent fever, splenomegaly, hepatomegaly with liver dysfunction, acute respiratory distress syndrome and multiple organ failure, and the mortality rate is also extremely high. CS-related diseases can be divided into Cytokine Storm Syndrome (CSS) and Cytokine Release Syndrome (CRS), and their causes are related to the increased release of multiple cytokines caused by pathogen infection and cell therapy, respectively.

[0004] At present, there is still a lack of targeted drugs with clear efficacy for the treatment of severe acute respiratory syndrome and cytokine storm-related diseases caused by abnormal activation of inflammatory factors. For example, although Tocilizumab, a monoclonal antibody to the IL-6 receptor, is a potential clinical therapeutic drug, it can only antagonize IL-6, a single cytokine, and has limited antagonistic effects on multiple other inflammatory factors, and its efficacy needs to be further verified in clinical applications. Although antagonists targeting the new coronavirus / SARS-CoV-2 S protein receptor-angiotensin converting enzyme 2 (ACE2) are in the clinical trial stage, SARS-CoV-2 can not only enter lung macrophages through ACE2, but also infect monocytes / macrophages through other pathways such as cell surface Fcγ receptors, release a large number of cytokines, and induce excessive inflammatory responses. Glucocorticoids are currently the most commonly used drugs for the clinical treatment of infectious acute pneumonia and CS. As a broad-spectrum immunosuppressant, they not only inhibit immune responses, increase the mortality rate and secondary infection rate of patients, but also have large side effects and limited efficacy.

[0005] The inventors' previous studies have found that the interaction between menin (encoding gene MEN1) and histone methyltransferase SETD2 remodels chromatin H3K36me3 (trimethylation of lysine 36 at position 3 of histone 3) to maintain the expression of multiple cytokines and inflammatory factors such as IL-1α and IL-6. Pathogens and other factors activate the expression of these cytokines and inflammatory factors by enhancing the menin / SETD2 interaction, which is a key node of the cytokine storm. Therefore, based on this pathogenesis, it is urgent to develop new target drugs to more effectively treat CS and related diseases. Summary of the invention

[0006] The first object of the present invention is to provide an acute pneumonia inhibitory ginsenoside Rb1 monomer. The ginsenoside Rb1 (Rb1) is an active substance extracted from the dried root of the Araliaceae plant Panax ginseng, which can target the menin / SETD2 protein interaction.

[0007] The second object of the present invention is to provide an application of an acute pneumonia inhibitory ginsenoside Rb1 monomer.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0009] The present invention provides an acute pneumonia inhibitory ginsenoside Rb1 monomer, the structural formula of which is as follows:

[0010]

[0011] The ginsenoside Rb1 monomer is screened from the natural product library TargetMol-NaturalCompound Library by a high-throughput screening method. It effectively blocks the interaction between menin and SETD2 by specifically binding to a specific domain of menin, thereby blocking the chromatin H3K36me3 histone modification mediated by the menin / SETD2 complex, and then inhibiting the abnormal expression of cytokines / inflammatory factors / chemokines mediated by the menin / SETD2 complex, including but not limited to IL-1β, IL-6, IL-8, IL-1α, etc.

[0012] The present invention provides the use of the acute pneumonia inhibitory ginsenoside Rb1 monomer in the preparation of a drug for treating or preventing diseases associated with abnormal expression of cytokines / inflammatory factors / chemokines such as IL-1α and IL-6, including but not limited to autoimmune diseases, inflammatory diseases, infectious diseases and tumors.

[0013] The ginsenoside Rb1 monomer can be used in the preparation of anti-inflammatory drugs. The ginsenoside Rb1 monomer can inhibit the expression of inflammatory factors activated by the menin / SETD2 complex and can be used as the main component of anti-inflammatory drugs. The inflammatory factors include but are not limited to IL-1α, IL-1β, IL-6, and IL-8. The ginsenoside Rb1 monomer can specifically inhibit the expression of macrophage inflammatory factors, inhibit LPS-induced acute lung injury, reduce lung inflammation and macrophage infiltration, and inhibit the expression of alveolar macrophage inflammatory factors, so it can be used as a lung anti-inflammatory drug, especially for diseases such as acute pneumonia caused by pathogen infection such as influenza and COVID-19.

[0014] The use of the acute pneumonia inhibitory ginsenoside Rb1 monomer in the preparation of drugs for resisting cytokine storm syndrome-related diseases, wherein the cytokine storm syndrome-related diseases include severe acute respiratory syndrome, influenza, and COVID-19.

[0015] The ginsenoside Rb1 monomer can be used in the preparation of lung protection drugs. Rb1 can improve the symptoms of acute lung injury induced by LPS and reduce alveolar protein deposition, indicating that it may have application potential in lung protection.

[0016] The ginsenoside Rb1 monomer can be used in the preparation of adjuvant therapeutic drugs for radiotherapy and chemotherapy. MEN1 / SETD2 knockdown can inhibit the expression of inflammatory factors induced by physical and chemical factors (such as cisplatin and ionizing radiation), indicating that the menin / SETD2 complex may be involved in the inflammatory response caused by radiotherapy and chemotherapy. Both pep242 and Rb1 can inhibit ionizing radiation-induced cell senescence and inflammatory factor expression, indicating that Rb1 can be used as an adjuvant therapeutic drug for radiotherapy and chemotherapy to reduce the side effects caused by radiotherapy and chemotherapy.

[0017] The ginsenoside Rb1 monomer can be used in the preparation of anti-tumor drugs. The ginsenoside Rb1 monomer can specifically inhibit the menin / SETD2 interaction and has indirect anti-tumor activity, especially in tumors with abnormal menin / SETD2 expression.

[0018] The ginsenoside Rb1 monomer can be used in the preparation of drugs for other diseases related to the menin / SETD2 complex.

[0019] The present invention provides a pharmaceutical composition, which comprises ginsenoside Rb1 and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition is used for treating acute pneumonia associated with cytokine storm or pneumonia associated with radiotherapy and chemotherapy caused by pathogen infection.

[0020] The pharmaceutical composition also contains other active ingredients for treating acute pneumonia or related diseases.

[0021] The pharmaceutical composition is in the form of tablets, capsules, injections, inhalants or any other pharmaceutical preparations suitable for clinical use, and the appropriate administration route and dosage can be selected according to the type of disease and the patient's condition.

[0022] Experiments have shown that the ginsenoside Rb1 monomer of the present invention inhibits the overexpression of macrophages and inflammatory factors (such as IL-1β, IL-6, etc.) activated by microorganisms (such as the S-protein of the new coronavirus or bacterial lipopolysaccharide LPS); in animal models, it inhibits LPS-induced acute lung injury, reduces inflammatory cell infiltration and alveolar protein deposition in lung tissue; and inhibits the release of inflammatory factors (such as IL-1β, IL-6, IL-8, etc.) in lung epithelial cells induced by ionizing radiation and chemotherapeutic drug exposure.

[0023] Compared with the prior art, the advantages and technical effects of the present invention are:

[0024] The theoretical basis of the present invention is that pathogens promote a series of CSS-related inflammatory factors / cytokines / chemokines through chromatin H3K36m3 reprogramming by activating the formation of menin / SETD2 complexes and chromatin-directed recruitment. Epigenetic molecular mechanism. Therefore, the natural product Rb1 that competitively inhibits the interaction of menin / SETD2 can directly inhibit the expression of a series of CSS-related inflammatory factors / cytokines / chemokines at the transcriptional level by specifically antagonizing the interaction of menin / SETD2. The present invention is designed based on the pathogenesis of acute pneumonia, with clear targets, strong targeting, and obvious effects. Ginsenosides have been proven to be low-toxic, and have potential application prospects for the treatment of diseases such as acute pneumonia caused by pathogen infection and physical and chemical factors. The ginsenoside Rb1 provided by the present invention shows good application prospects in the treatment of acute pneumonia related to cytokine storm caused by pathogen infection, as well as potential application prospects in the treatment of pneumonia related to radiotherapy and chemotherapy, with the advantages of clear mechanism and target, specific action, and simultaneous extensive inhibition of multiple inflammatory factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the identification diagram of the minimum domain of menin that interacts with SETD2. A is the verification of the interaction between SETD2 SET region and menin; B is the identification of the interaction between menin Palm domain and SETD2; C is the confirmation of menin 231-248 fragment as the minimum interaction domain; D is the verification of direct interaction between menin and SETD2 in vitro; E is the species conservation analysis of menin 231-248 fragment; F is the inhibition of menin and SETD2 interaction by Pep242.

[0026] Figure 2 This is a diagram of high-throughput screening of small molecule inhibitors in natural product libraries. A is a high-throughput screening flow chart; B is the determination of FITC-menin peptide concentration; C is the determination of SET protein concentration; D is the Z factor stability test; E is the chemical structure diagram of Rb1; and F is the specific inhibition verification of Rb1.

[0027] Figure 3 This is a diagram of biochemical evaluation of ginsenoside Rb1 inhibiting menin / SETD2 interaction. A is GST-pull down verification; B. Immunoprecipitation experiment verifies Rb1 inhibition; C. Raw264.7 cell experiment verifies Rb1 inhibition; D. menin protein thermal stability experiment; E. Dose-dependent menin protein thermal stability experiment; F. SETD2 protein thermal stability experiment.

[0028] Figure 4This is a bioinformatics analysis of the interaction between ginsenoside Rb1 and menin protein. A is the molecular docking of Rb1 and menin target protein using Schrodinger Maestro software; B is the visualization of the binding mode of Rb1 and menin protein using Pymol2.1 software; and C is the molecular docking score table showing the binding energy.

[0029] Figure 5 This is a graph showing that ginsenoside Rb1 inhibits the expression of inflammatory factors in macrophages. A shows the changes in the expression of inflammatory factors in alveolar macrophages after LPS and Rb1 treatment; B shows the changes in the expression of inflammatory factors in THP-1 cells after S-Pro and Rb1 treatment.

[0030] Figure 6 This is a diagram of ginsenoside Rb1 inhibiting LPS-induced acute lung injury. A shows that Rb1 improves the symptoms of LPS-induced acute lung injury in mice; B shows that Rb1 inhibits LPS-induced lung inflammation and macrophage infiltration; C shows that Rb1 reduces alveolar protein deposition in mice with LPS-induced acute lung injury; and D shows that Rb1 inhibits the expression of inflammatory factors in alveolar macrophages in mice induced by LPS.

[0031] Figure 7 This is a graph showing that MEN1 / SETD2 knockdown inhibits the expression of inflammatory factors induced by physical and chemical factors. A shows that MEN1 knockdown inhibits the expression of inflammatory factors in A549 cells induced by cisplatin; B shows that MEN1 knockdown inhibits the expression of inflammatory factors in A549 cells induced by ionizing radiation; C shows that SETD2 knockdown inhibits the expression of inflammatory factors in A549 cells induced by cisplatin; and D shows that SETD2 knockdown inhibits the expression of inflammatory factors in A549 cells induced by ionizing radiation.

[0032] Figure 8 This is a graph showing that pep242 and ginsenoside Rb1 inhibit ionizing radiation / physicochemical factors-induced cell senescence / inflammatory factor expression. A shows that pep242 inhibits ionizing radiation-induced A549 cell senescence; B shows that pep242 inhibits ionizing radiation-induced A549 cell inflammatory factor expression; C shows that ginsenoside Rb1 inhibits DDP-induced A549 cell inflammatory factor expression; and D shows that ginsenoside Rb1 inhibits ionizing radiation-induced A549 cell inflammatory factor expression. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the following embodiments will further illustrate the present invention in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. On the contrary, the present invention covers any substitution, modification, equivalent method and scheme made on the essence and scope of the present invention as defined by the claims.

[0034] The first aspect of the present invention: identification of the minimal domain of menin / SETD2 interaction. (1) The SET region of SETD2 protein was divided into three fragments (SET-1, -2, -3, 1460-1550, 1551-1673, 1674-1720 amino acids, respectively), FLAG-tagged SET-1, -2, -3 fragments were co-transfected with HA-MEN1 in 293T cells, HA tag was used to immunoprecipitate menin protein, and Western blot was used to detect FLAG, which proved that SET-1, -2, -3 all interacted with menin. In contrast, the Palm domain of menin protein was segmented into 231-380, 243-380, 249-380, 281-380, 231-280, and HA-tagged MEN1 fragments were co-transfected with FLAG-tagged SET into 293T cells, and the menin protein was immunoprecipitated by HA tag. FLAG was detected by Western blot, demonstrating that menin interacted with SET through the 231-248 fragment ( Figure 1 ).

[0035] The second aspect of the present invention is to use the TargetMol-Natural Compound Library to screen for specific inhibitors of menin / SETD2 interaction. The natural product ginsenoside Rb1 (Rb1) was screened out by fluorescence polarization (FP) experiment to specifically inhibit the interaction between menin and SETD2 protein ( Figure 2 ); Co-Immunoprecipitation (Co-IP), Cellular Thermal Shift Assay (CETSA), GST-Pull down and Western Blotting demonstrated that Rb1 specifically inhibited the menin / SETD2 interaction ( Figure 3); Schrodinger Maestro software was used to perform molecular docking between Rb1 and the SETD2 target protein. The results showed that Rb1 and the target protein had a good binding effect and a high degree of match. The complex formed by Rb1 and the protein after docking was visualized using Pymol2.1 software to obtain the binding mode of Rb1 and the protein. According to the binding mode, the amino acid residues that bind to the protein pocket can be clearly seen. Rb1 forms strong hydrogen bond interactions with LYS-206, ASP-136, SER-159, ASP-185, TYR-324, and GLU-368 in the active site of menin protein, which makes an important contribution to anchoring small molecules in the protein pocket; in addition, since the natural product can also form van der Waals interactions with the surrounding residues, it makes an important contribution to the stabilization of natural products. These interactions can effectively promote the formation of stable complexes between natural products and proteins ( Figure 4 ). Rb1 natural product was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0036] The third aspect of the present invention: Rb1 significantly inhibits the expression of inflammatory factors induced by pathogens. In order to explore the potential therapeutic effect of Rb1 in cytokine storm syndrome / CSS caused by new coronavirus / SARS-CoV-2 infection in the lungs, macrophages THP-1 (PMA-induced differentiated M0 human monocytic leukemia cells THP-1) were treated with SARS-CoV-2 S protein (S-Pro), and mouse alveolar macrophages were treated with LPS. RT-qPCR results showed that S-Pro and LPS can significantly activate the expression of cytokine storm-related cytokines such as IL-1β, and Rb1 dose-dependently effectively inhibits the expression of inflammatory factors activated by pathogens ( Figure 5 ). LPS-induced acute lung injury model in mice, hematoxylin-eosin (HE) staining and F4 / 80 specific marker immunohistochemical staining (IHC) histopathological observation, the results showed that the LPS group had obvious acute inflammation, alveolar destruction, and hemorrhage in the lungs. Rb1 treatment significantly improved LPS-induced lung injury and effectively reduced inflammatory cell infiltration in lung tissue ( Figure 6 The results of BCA assay for alveolar lavage fluid protein concentration showed that Rb1 treatment significantly reduced LPS-induced alveolar protein deposition ( Figure 6 ); RT-qPCR results showed that Rb1 significantly inhibited the expression of cytokines / inflammatory factors in alveolar macrophages induced by LPS ( Figure 6 In addition, Rb1 effectively inhibits the excessive release of inflammatory factors from lung epithelial cells caused by ionizing radiation / chemotherapeutic drugs ( Figure 7 and 8 ).

[0037] Example 1: Identification of the minimal domain of menin that interacts with SETD2

[0038] Human embryonic kidney cell line 293T was selected for culture, and plasmid transfection was performed when the cells grew to the logarithmic phase. Using Lipofectamine 2000 reagent, according to the manufacturer's instructions, the plasmid containing the SET region of SETD2 protein divided into three fragments (SET-1, SET-2, SET-3, 1460-1550, 1551-1673, 1674-1720 amino acids, respectively) or the menin protein Palm domain fragment fragment, and the plasmid containing the HA-tagged menin protein or SET protein were co-transfected into 293T cells. After transfection for 48 hours, the cells were collected and lysed, and the menin protein was immunoprecipitated using HA tag antibody to capture the protein complex bound to the menin protein. The immunoprecipitated complex was subjected to Western blot analysis, and the presence of SETD2 protein or its fragment was detected using FLAG tag antibody. Prokaryotic expression vectors pET28a-menin and pET32a-SET containing the coding sequences of menin protein and SETD2 protein were constructed. The constructed vector was transfected into Escherichia coli BL21 and cultured in LB medium until the logarithmic growth phase. IPTG was used to induce protein expression, and different induction temperatures and induction times were set according to the properties of the protein. The obtained menin and SETD2 proteins were purified using a His tag affinity chromatography column. The purified menin protein was subjected to a GST-pull down experiment with a GST-fused SETD2 protein or a GST empty vector. The binding of menin protein was analyzed using Western blot to verify the direct interaction between menin and SETD2 protein. Pep242 peptide (231-248 fragment of menin protein) was chemically synthesized. In the GST-pull down experiment, different concentrations of Pep242 peptide were added to observe its inhibitory effect on the interaction between menin and SETD2 protein. Bioinformatics tools were used to perform species conservation analysis on the 231-248 fragment of menin protein to evaluate the degree of conservation of this fragment during species evolution.

[0039] Figure 1 A in the figure shows the interaction between the SET region of SETD2 protein and menin protein after it is divided into three fragments: SET-1, SET-2, and SET-3. The results show that the three fragments SET-1, SET-2, and SET-3 can all interact with menin protein, suggesting that there is no minimum domain in the SETD domain that interacts with menin.

[0040] Figure 1B in the figure is the identification of the interaction between menin Palm domain and SETD2. The Palm domain of HA-tagged menin protein was divided into different fragments 231-380, 243-380, 249-380, 281-380, and 231-280. The HA-tagged MEN1 fragments were co-transfected with FLAG-tagged SET in 293T cells, and the menin protein was immunoprecipitated using HA tag. FLAG was detected by Western blot to determine the minimum fragment for the interaction between menin and SET. The results showed that menin interacted with SET through the 231-248 fragment, indicating that this fragment is the key region for the interaction between menin and SETD2.

[0041] Figure 1 C in the figure is the confirmation that the menin 231-248 fragment is the minimum interaction domain. The HA-tagged menin protein 231-248 fragment was further divided into two segments, 231-242 and 243-248, and the interaction with the SETD2 protein was analyzed. HA-GFP-tagged 231-242, 243-248, and 231-248 fragments were co-transfected into 293T cells with FLAG-tagged SET. The menin protein was immunoprecipitated using the HA tag, and the FLAG tag was detected by Western blot to verify that the 231-248 fragment was the minimum domain for the interaction between menin and SET. The results showed that the 231-242, 243-248, and complete 231-248 fragments could interact with the SETD2 protein, confirming that the 231-248 fragment of the menin protein is the minimum domain that interacts with SETD2.

[0042] Figure 1 D in the figure is the direct interaction verification between menin and SETD2 in vitro. By constructing a prokaryotic expression vector and purifying menin and SETD2 proteins, an in vitro GST-pull down experiment was performed to detect the direct interaction between menin and SET proteins to confirm the direct interaction between menin and SET proteins in vitro. The results showed that menin and SET proteins can directly interact in vitro, verifying the direct binding ability between the two.

[0043] Figure 1 E in the figure is a species conservation analysis of the 231-248 fragment of the menin protein. A schematic diagram of the sequence structure of the Pep242 polypeptide (231-248) was drawn, and a species conservation analysis was performed to confirm the conservation of the 231-248 fragment of the menin protein during species evolution. The results showed that the sequence of the menin 231-248 fragment was highly conserved during species evolution.

[0044] Figure 1 Figure F shows that Pep242 inhibits the interaction between menin and SETD2. GST-pull down experiments were performed using menin and SET proteins purified in vitro. Different concentrations of Pep242 peptide were added to observe its inhibitory effect on the interaction between menin and SET. It was found that menin interacted directly with SET protein, and Pep242 could inhibit the interaction between menin and SET in a dose-dependent manner (7.5, 15, 30, 60 μM), suggesting that specific blocking of the interaction domain can antagonize protein interaction, and Pep242 can inhibit the interaction between menin and SETD2 protein in a dose-dependent manner.

[0045] This example successfully identified the minimum domain of menin that interacts with SETD2 as fragment 231-248 through a series of cell experiments and in vitro experiments, and verified the high conservation of this fragment during species evolution. At the same time, it was also found that the Pep242 polypeptide can specifically inhibit the interaction between menin and SETD2.

[0046] Example 2: High-throughput screening of small molecule inhibitors of menin-SETD2 interaction using natural product libraries

[0047] SET protein was obtained by affinity chromatography purification to ensure that the purity and activity of the protein met the experimental requirements. FITC-labeled menin 231-248 peptide was used as a fluorescent probe to detect the interaction with SET protein. TargetMol-Natural Compound Library (natural product library), containing 1500 natural products, was used for high-throughput screening of potential inhibitors of menin-SETD2 interaction.

[0048] SET protein and FITC-labeled menin 231-248 peptide were added to a 384-well plate in a certain ratio, and equal amounts of protein and peptide were added to each well to ensure the consistency of the reaction system. Compounds in the TargetMol-Natural Compound Library (1500) were used to screen potential targeted inhibitors one by one, and multiple concentration gradients were set for each compound (such as 0.1 μM, 0.5 μM, 1 μM, 2 μM, 5 μM). The binding of peptides to proteins was detected using a fluorescence polarization instrument, and the inhibitory effect of each compound was recorded to determine the optimal reaction concentration of FITC-menin 231-248 peptide, that is, the concentration corresponding to the maximum fluorescence polarization signal. Under the condition of fixed SET protein concentration, the peptide concentration was adjusted, the changes in fluorescence polarization signal were observed, and the optimal reaction concentration was determined to be 1 μM. The optimal reaction concentration of SET protein was determined using a fluorescence polarization experiment. Under the condition of fixed peptide concentration, the concentration of SET protein was changed, the changes in fluorescence polarization signal were observed, and the optimal reaction concentration was determined to be 500 nM. High-throughput screening of stability Z factor detection was performed. The sensitivity and stability of the screening system can be evaluated by calculating the Z factor. The closer the Z factor is to 1, the better the performance of the screening system.

[0049] Each compound in the TargetMol-Natural Compound Library was added to a 384-well plate containing SET protein and FITC-menin 231-248 peptide, with one replicate well set for each compound. The fluorescence signal of each well was detected using a fluorescence polarization experiment, and the changes in the fluorescence signal before and after the addition of the compound were compared to screen out compounds that can significantly inhibit the menin-SETD2 interaction.

[0050] The specificity of the potential inhibitors screened out was verified. Fluorescence polarization experiments were used to detect the effects of the inhibitors on the menin-SETD2 interaction and the menin-other protein (such as non-specific protein) interaction to confirm the specificity of the inhibitors. Chemical structure analysis was performed on the screened specific inhibitors to understand their chemical properties and possible mechanisms of action.

[0051] Figure 2 A in Figure 1 shows a flow chart of high-throughput screening of small molecule inhibitors of menin-SETD2 interaction from natural product libraries. High-throughput screening was performed by adding SET protein and FITC-labeled menin 231-248 peptides and compounds in the TargetMol-Natural Compound Library to a 384-well plate.

[0052] Figure 2B in the figure gives the results of the fluorescence polarization experiment, which shows that when the concentration of FITC-menin 231-248 peptide is 1 μM, the fluorescence polarization signal reaches the optimal state, so this concentration is determined to be the optimal reaction concentration.

[0053] Figure 2 C in the figure gives the results of the fluorescence polarization experiment, which shows that when the concentration of SET protein is 500 nM, the interaction with FITC-menin231-248 polypeptide is most significant, so this concentration is determined to be the optimal reaction concentration.

[0054] Figure 2 D in the figure is the Z factor stability test. The result of the high-throughput screening stability Z factor test is Z=0.95, indicating that the screening system has good sensitivity and stability and can meet the requirements of high-throughput screening.

[0055] Figure 2 Figure E shows the chemical structure of ginsenoside Rb1, a specific inhibitor screened from a natural product library. Ginsenoside Rb1 can significantly inhibit the menin-SETD2 interaction.

[0056] Figure 2 F in the figure gives the results of fluorescence polarization experiments, showing that ginsenoside Rb1 can specifically inhibit the interaction between menin and SET protein, further verifying its potential as an inhibitor of menin-SETD2 interaction.

[0057] In this example, a small molecule inhibitor, ginsenoside Rb1, which can specifically inhibit the interaction between menin and SETD2 (SET protein) was successfully screened out from the natural product library TargetMol-Natural Compound Library by a high-throughput screening method.

[0058] Example 3: Biochemical evaluation of the inhibitory effect of ginsenoside Rb1 on the interaction between menin and SETD2

[0059] This example aims to evaluate the effect of ginsenoside Rb1 on the interaction between menin and SETD2 protein by biochemical methods and explore its potential mechanism of action.

[0060] GST-fused menin and SET proteins, as well as unfused control proteins, were prepared. GST-fused proteins were bound to glutathione beads to form GST-bead complexes. Cell lysate (containing menin and SET proteins) was mixed with the GST-bead complexes, and different concentrations of Rb1 (0, 25, 50 μM) were added and incubated to allow protein-protein interaction. After incubation, the beads were washed to remove unbound proteins. Proteins bound to GST-beads were detected by Western blot to evaluate the effect of Rb1 on the direct interaction between menin and SET proteins.

[0061] FLAG-SET and HA-MEN1 plasmids were co-transfected into 293T cells, or FLAG-MEN1 plasmid was transfected alone into Raw264.7 cells. After the cells were cultured to an appropriate density, the cells were collected and lysed. The corresponding HA tag antibodies (such as HA or FLAG antibodies) were used for co-immunoprecipitation to collect the menin protein complex and capture the target protein and its interacting proteins. Western blot was used to detect menin and SET proteins in the co-immunoprecipitation products. At the same time, different concentrations of Rb1 (0, 25, 50 μM) treatment groups were set up to evaluate the inhibitory effect of Rb1 on the interaction between menin and SET proteins.

[0062] MEN1 or SETD2 plasmids were transfected into 293T cells. Cells were treated at different temperatures and with different concentrations of Rb1. Cell lysates were collected and the expression levels of menin or SETD2 proteins were detected by Western blot. The effect of Rb1 on the thermal stability of menin or SETD2 proteins and the direct interaction between Rb1 and menin were evaluated.

[0063] Figure 3 A in the figure shows the results of GST-pull down validation, which shows that Rb1 can specifically disrupt the direct interaction between menin and SET protein. In the absence of Rb1, menin and SET protein can bind; after the addition of Rb1, this binding is significantly inhibited. As the concentration of Rb1 increases, the binding of menin and SET protein gradually weakens, indicating that Rb1 specifically disrupts the direct interaction between menin and SET protein.

[0064] Figure 3B in the figure is an immunoprecipitation experiment to verify Rb1 inhibition. In 293T cells, FLAG-SET and HA-MEN1 plasmids were co-transfected and then immunoprecipitated using the HA tag. Western blot results showed that as the concentration of Rb1 increased (0, 25, 50 μM), the interaction between menin and SET protein gradually weakened, and the interaction between menin and SET protein was gradually inhibited in a dose-dependent manner, confirming that Rb1 inhibited the interaction between menin and SET protein in a dose-dependent manner.

[0065] Figure 3 C in the figure is the Raw264.7 cell experiment to verify Rb1 inhibition. In the mouse mononuclear macrophage cell line Raw264.7 cells, after FLAG-MEN1 plasmid transfection, the cells were collected and lysed, and immunoprecipitation was performed using the FLAG tag. Western blot was used to detect SETD2 protein, and different concentrations of Rb1 treatment groups were set to evaluate the effect of Rb1 on the interaction between menin and SETD2 proteins. Western blot results showed that Rb1 also inhibited the interaction between menin and SETD2 proteins in a dose-dependent manner. This is consistent with the results observed in 293T cells.

[0066] Figure 3 D in the figure is the result of the menin protein thermal stability experiment. The MEN1 plasmid was transfected into 293T cells. After the cells were cultured to an appropriate density, a cell thermal shift experiment was performed. Different concentrations of Rb1 treatment groups (0, 25, 50 μM) were set up to detect the thermal stability of Rb1 binding to menin protein. In the presence of 50 μM Rb1, the degradation of menin protein was significantly inhibited as the temperature increased. This shows that Rb1 can directly interact with menin and increase its thermal stability.

[0067] Figure 3 E in the figure is the result of the dose-dependent menin protein thermal stability experiment. Further cell thermal shift experiments showed that with the increase of Rb1 concentration, the thermal stability of menin protein gradually increased. This further confirmed the direct interaction between Rb1 and menin and revealed its dose-dependent effect in stabilizing menin protein.

[0068] Figure 3F in the figure is the SETD2 protein thermal stability experiment. The SETD2 plasmid was transfected into 293T cells. A cell thermal shift experiment was performed, and a 50 μM Rb1 treatment group was set up. The cell thermal shift experiment detected the thermal stability of Rb1 binding to SETD2 protein, evaluated the effect of Rb1 on the thermal stability of SETD2 protein, and the direct interaction between Rb1 and SETD2 protein. The results of the cell thermal shift experiment (SETD2 protein stability) showed that Rb1 (50 μM) had no obvious effect on the thermal stability of SETD2 protein, indicating that there was no direct interaction between Rb1 and SETD2 protein.

[0069] In this example, various biochemical methods such as GST-Pull Down, cell co-transfection and immunoprecipitation, and cell thermal shift experiments were used to comprehensively evaluate the inhibitory effect of ginsenoside Rb1 on the interaction between menin and SETD2. The experimental results showed that Rb1 could specifically destroy the direct interaction between menin and SET protein and increase the thermal stability of menin protein, but had no direct interaction with SETD2 protein.

[0070] Example 4: Bioinformatics analysis of the interaction between ginsenoside Rb1 and menin protein

[0071] Using bioinformatics methods and molecular docking technology, the interaction between ginsenoside Rb1 and menin protein was analyzed to determine their binding mode, key interaction residues and binding energy.

[0072] Import the molecular structure files of ginsenoside Rb1 and menin protein into Schrödinger Maestro software. Configure docking parameters, including the selection of docking region (active site of menin protein), the selection of docking algorithm, the setting of docking accuracy, etc. Run the molecular docking program to allow the Rb1 molecule to dock at the active site of menin protein. Analyze the docking results and evaluate the binding effect and matching degree between Rb1 and menin protein.

[0073] Import the complex structure file of Rb1 and menin protein after docking into Pymol 2.1 software. Adjust the view angle and color scheme to clearly observe the binding mode of Rb1 molecule and menin protein, especially the interaction with the amino acid residues in the active site. Record and analyze the hydrogen bond interactions between Rb1 and the active site of menin protein LYS-206, ASP-136, SER-159, ASP-185, TYR-324, GLU-368, as well as the van der Waals interactions with other surrounding residues, and export the analysis results in the form of images.

[0074] Extract the molecular docking score from the docking results, and calculate the binding energy between Rb1 and menin protein based on the molecular docking results. Evaluate the size of the binding energy and determine the binding strength and stability of Rb1 and menin protein.

[0075] Molecular docking results ( Figure 4 A in Figure 1): Through docking analysis, it was found that Rb1 and menin protein have a good binding effect and a high degree of match. This indicates that Rb1 may become an effective inhibitor or regulator of menin protein.

[0076] Complex visualization and binding mode ( Figure 4 B in the figure): The visualization analysis results of Pymol 2.1 software show that the binding mode of Rb1 and menin protein is clearly observed. Ginsenoside Rb1 forms strong hydrogen bond interactions with LYS-206, ASP-136, SER-159, ASP-185, TYR-324, and GLU-368 at the active site of menin protein, and these hydrogen bonds play an important role in anchoring small molecules (i.e., Rb1) in the protein pocket. In addition, Rb1 also forms van der Waals interactions with surrounding residues, and these interactions jointly promote the formation of a stable complex between Rb1 and menin protein. The amino acid residues and Rb1 molecules involved in the interaction are marked with different colors in the figure to clearly show their binding mode.

[0077] Molecular docking score evaluation ( Figure 4 C in the figure): According to the molecular docking results, the calculated binding energy is less than -6 kcal / mol. This indicates that the binding between Rb1 and menin protein is stable and has a strong interaction force; this result further supports the results of molecular docking and visualization analysis.

[0078] This example successfully analyzed the interaction between ginsenoside Rb1 and menin protein by bioinformatics methods. The results showed that Rb1 and menin protein had a good binding effect and a high degree of match. The binding mode and key interaction residues of Rb1 and menin protein were determined by visual analysis. In addition, the calculation results of binding energy showed that the binding between Rb1 and menin protein was stable. These results provide valuable reference and basis for further experimental research.

[0079] Example 5: Experimental study on the inhibition of ginsenoside Rb1 on the expression of inflammatory factors in macrophages

[0080] In this example, in vitro cell experiments were conducted to study the inhibitory effect of ginsenoside Rb1 on the expression of inflammatory factors in alveolar macrophages and THP-1 cells (PMA-induced differentiated M0 human monocytic leukemia cells) to verify its anti-inflammatory activity, especially its inhibitory effect on the upregulation of inflammatory factors induced by LPS (lipopolysaccharide) and S-Pro (a known inflammatory inducer).

[0081] Alveolar macrophages were inoculated in a culture dish containing RPMI 1640 medium and 10% fetal bovine serum and cultured in a cell culture incubator until the cells adhered to the wall and grew well. The cells were divided into three groups: control group (untreated), LPS group (LPS 100 ng / mL added), and ginsenoside Rb1 treatment group (LPS 100 ng / mL and ginsenoside Rb1 25 μM or 50 μM added, respectively). After treating alveolar macrophages for 24 hours, the cells were collected. Total cell RNA was extracted using Trizol reagent. The extracted RNA was reverse transcribed into cDNA, and real-time fluorescence quantitative PCR analysis was performed to detect the expression levels of inflammatory factors such as IL-6, TNF-α, and IL-1β. RT-qPCR results showed that in the alveolar macrophage experiment, the LPS treatment group significantly promoted the expression of inflammatory factors such as IL-6, TNF-α, and IL-1β compared with the control group. The ginsenoside Rb1 treatment group (25 μM and 50 μM) effectively inhibited the upregulation of the above inflammatory factors induced by LPS, indicating that ginsenoside Rb1 has the effect of inhibiting the expression of inflammatory factors in alveolar macrophages ( Figure 5 A in ).

[0082] THP-1 cells were inoculated in a culture dish containing RPMI 1640 medium and 10% fetal bovine serum, and PMA (the concentration was adjusted according to the experimental needs) was added to induce differentiation into M0 phase cells, and the cells were cultured in a cell culture incubator until the cells were completely differentiated. The differentiated cells were divided into three groups: control group (untreated), S-Pro group (addition of S-Pro 100 nM), and Rb1 treatment group (addition of S-Pro 100 nM and ginsenoside Rb1 25 μM or 50 μM, respectively). After 48 hours of treatment, the cells were collected. Total cell RNA was extracted using Trizol reagent. The extracted RNA was reverse transcribed into cDNA, and real-time fluorescence quantitative PCR analysis was performed to detect the expression levels of inflammatory factors such as IL-6, TNF-α, IL-1β, and IL-8. Figure 5Figure B shows that in the THP-1 cell experiment, the S-Pro treatment group significantly promoted the expression of inflammatory factors such as IL-6, TNF-α, IL-1β, and IL-8 compared with the control group. Similarly, the Rb1 treatment group (25 μM and 50 μM) effectively inhibited the upregulation of the above inflammatory factors induced by S-Pro, further confirming the inhibitory effect of ginsenoside Rb1 on the expression of inflammatory factors in macrophages.

[0083] This example verifies the significant effect of ginsenoside Rb1 in inhibiting the expression of inflammatory factors through alveolar macrophage and THP-1 cell experiments. Whether in LPS or S-Pro induced inflammation models, Rb1 can effectively inhibit the upregulation of inflammatory factors, indicating that it has strong anti-inflammatory activity.

[0084] Example 6: Study on the protective effect of ginsenoside Rb1 on LPS-induced acute lung injury

[0085] Healthy male C57BL / 6J mice were selected, with body weight controlled within the range of 20-25g, and randomly divided into three groups: normal control group, LPS model group and Rb1 treatment group, with at least 10 mice in each group. The normal control group did not receive any treatment and served as the baseline data. The LPS model group was given LPS (5 mg / kg) by tracheal instillation to induce an acute lung injury model. The Rb1 treatment group was given ginsenoside Rb1 (8 mg / kg) by tracheal instillation before or at the same time as LPS instillation to observe its protective effect on lung injury. 24 hours after LPS instillation, mice in each group were killed, lung tissues were dissected and removed, and the morphological changes of lung tissues, including hemorrhage, edema, etc., were observed with the naked eye, and photographed and recorded. The removed lung tissues were stained with hematoxylin-eosin (HE) to observe the pathological changes of lung tissues, including inflammatory cell infiltration, widening of alveolar septa, and alveolar atrophy. Immunohistochemical staining (IHC) was performed using F4 / 80-specific antibodies to detect macrophage infiltration. After the mice were killed, alveolar lavage was performed and the lavage fluid was collected. The protein concentration in the lavage fluid was determined by the BCA method to evaluate the alveolar protein deposition. Primary alveolar macrophages were isolated, and the expression levels of inflammatory cytokines such as IL-1β, TNF-α, IL-6, and IL-18 in the cells were detected by RT-qPCR technology.

[0086] Figure 6 A in the figure shows the general view of lung tissue. The surface of lung tissue in the normal control group was smooth and the color was normal; the lung tissue in the LPS model group had extensive bleeding and obvious edema; while these symptoms were significantly improved in the Rb1 treatment group, with a smoother surface of lung tissue and reduced bleeding and edema.

[0087] Figure 6B in the figure is the result of hematoxylin-eosin (HE) staining of mouse lung tissue. The LPS model group had obvious acute inflammation in the lungs, diffuse inflammatory cell infiltration in the alveolar cavity and lung interstitium, widened alveolar septa, ruptured alveolar septa in some areas, alveolar atrophy, and even structural disappearance. The Rb1 treatment group significantly inhibited these pathological changes caused by LPS, the lung tissue structure was relatively intact, and the infiltration of inflammatory cells was reduced. The results of F4 / 80 specific antibody immunohistochemistry (IHC) staining showed that the infiltration of macrophages increased in the LPS model group, while the Rb1 treatment group significantly improved the infiltration of macrophages.

[0088] Figure 6 Figure C shows that Rb1 reduces alveolar protein deposition in mice with LPS acute lung injury. The protein concentration of alveolar lavage fluid was measured by BCA method. The results showed that the alveolar protein deposition in the LPS model group was significantly increased, while the Rb1 treatment group significantly improved the LPS-induced alveolar protein deposition and the protein concentration was significantly reduced.

[0089] Figure 6 D in the figure indicates that Rb1 inhibits the expression of inflammatory factors in mouse alveolar macrophages induced by LPS. The results of RT-qPCR test showed that the expression of inflammatory cytokines such as IL-1β, TNF-α, IL-6, and IL-18 in alveolar macrophages in the LPS model group was significantly increased, while the Rb1 treatment group significantly inhibited the expression of inflammatory factors induced by LPS, indicating that ginsenoside Rb1 has anti-inflammatory effects and can alleviate LPS-induced acute lung injury.

[0090] This example verifies through animal experiments that ginsenoside Rb1 has a significant protective effect on LPS-induced acute lung injury, providing an experimental basis for further development of drugs for the treatment of acute lung injury.

[0091] Example 7: Study on the inhibitory effect of MEN1 and SETD2 knockdown on the expression of inflammatory factors induced by physical and chemical factors

[0092] Human lung adenocarcinoma cell line A549 was selected for culture, and when the cells grew to the logarithmic phase, they were grouped and treated. A549 cells were divided into three groups: shLuc group (control group, transfected with empty vector), shMEN1 group (divided into two subgroups, shMEN1-1 and shMEN1-2, transfected with different MEN1 shRNA plasmids), and shSETD2 group (divided into two subgroups, shSETD2-1 and shSETD2-2, transfected with different SETD2 shRNA plasmids). The grouped A549 cells were treated with two physical and chemical factors: one was treated with 5μM cisplatin (DDP) for 24 hours, and the other was irradiated with 5Gy ionizing radiation. After treatment, the cells were cultured for 72 hours to observe the effects of physical and chemical factors on the cells and the effects after gene knockdown. The RNA of each group of cells was extracted using the Trizol method, and reverse transcribed to generate cDNA. RT-qPCR technology was used to detect the expression levels of inflammatory factors such as IL-1β, IL-6, and IL-8 in each group of cells to evaluate the effects of physical and chemical factors and gene knockdown on the expression of inflammatory factors.

[0093] Figure 7 Figure A shows the expression of IL-1β, IL-6, and IL-8 inflammatory factors in A549 cells in the shLuc group, shMEN1-1 group, and shMEN1-2 group under 5μM DDP treatment. RT-qPCR results showed that DDP significantly promoted the expression of IL-1β, IL-6, and IL-8 inflammatory factors, and knocking down MEN1 effectively inhibited the upregulation of the above inflammatory factors induced by DDP.

[0094] Figure 7 B in the figure shows that MEN1 knockdown inhibits the expression of inflammatory factors in A549 cells induced by ionizing radiation. The expression of inflammatory factors in each group of cells was different from that in the control group after 5 Gy ionizing radiation. Figure 7 Similar to Figure A in Figure 1. Ionizing radiation significantly promoted the expression of IL-1β, IL-6, and IL-8, and knockdown of MEN1 effectively inhibited the upregulation of the above inflammatory factors induced by ionizing radiation.

[0095] Figure 7 C in the figure shows that SETD2 knockdown inhibits the expression of inflammatory factors in A549 cells induced by cisplatin, and shows the expression of inflammatory factors in A549 cells in the shLuc group, shSETD2-1 group, and shSETD2-2 group under 5 μM DDP treatment. Figure 7 Similar to Figure A in Figure , RT-qPCR results showed that DDP promoted the expression of IL-1β, IL-6, and IL-8 inflammatory factors, and knockdown of SETD2 effectively inhibited the DDP-induced upregulation of the above inflammatory factors.

[0096] Figure 7D in the figure indicates that SETD2 knockdown inhibited the expression of inflammatory factors in A549 cells induced by ionizing radiation. The expression of inflammatory factors in each group of cells was different from that in the control group after 5 Gy ionizing radiation. Figure 7 The results of RT-qPCR showed that ionizing radiation significantly promoted the expression of IL-1β, IL-6, and IL-8 inflammatory factors, and knockdown of SETD2 effectively inhibited the upregulation of the above inflammatory factors induced by ionizing radiation.

[0097] In summary, this example verifies the inhibitory effect of MEN1 and SETD2 knockdown on the expression of inflammatory factors induced by physical and chemical factors (such as DDP and ionizing radiation) through cell experiments.

[0098] Example 8: Study on the inhibitory effects of pep242 and ginsenoside Rb1 on cell senescence and inflammatory factor expression induced by ionizing radiation / physical and chemical factors

[0099] Human lung adenocarcinoma cell line A549 was selected for culture, and when the cells grew to the logarithmic phase, they were grouped and treated. For groups that required ionizing radiation treatment, A549 cells were exposed to 5 Gy of ionizing radiation. For groups that required drug treatment, different concentrations of pep242 (specific blocking menin / SETD2 interaction peptide, patent application number 202311371849.5) and ginsenoside Rb1 were given for pretreatment. The concentration of pep242 was 50 μM, and the concentration of ginsenoside Rb1 was set to 0, 6.25, 12.5, and 25 μM. After drug pretreatment, some groups also needed to be treated with 5 μM cisplatin (DDP). SA-βgal staining was used to detect cell senescence. After ionizing radiation or drug treatment, the cells were cultured for 72 hours, and then the instructions of the SA-βgal staining kit were followed to observe and record the proportion of cell senescence. After ionizing radiation or drug treatment, the cells were cultured for 72 hours, and the RNA of each group of cells was extracted using the Trizol method. After reverse transcription to generate cDNA, the expression levels of inflammatory factors such as IL-1β, IL-6, and IL-8 in each group of cells were detected using RT-qPCR technology.

[0100] Figure 8 Figure A shows the cell senescence of A549 cells after pep242 pretreatment under ionizing radiation. The results showed that ionizing radiation significantly promoted the senescence of A549 cells, while the proportion of cell senescence was significantly reduced after pretreatment with 50 μM pep242, indicating that pep242 treatment can inhibit cell senescence induced by ionizing radiation.

[0101] Figure 8B in the figure shows that pep242 inhibits the expression of inflammatory factors in A549 cells induced by ionizing radiation. Under the same treatment conditions, the expression levels of inflammatory factors in each group of cells were detected by RT-qPCR. The results of RT-qPCR showed that ionizing radiation significantly promoted the expression of inflammatory factors such as IL-1β, IL-6, and IL-8, while after pretreatment with pep242, the expression levels of these inflammatory factors were significantly reduced, indicating that pep242 effectively inhibited the upregulation of inflammatory factor expression induced by ionizing radiation.

[0102] Figure 8 C in the figure indicates that ginsenoside Rb1 inhibits the expression of inflammatory factors in A549 cells induced by DDP, and shows the expression of inflammatory factors in A549 cells after pretreatment with ginsenoside Rb1 at different concentrations under DDP treatment. RT-qPCR results showed that DDP significantly promoted the expression of IL-1β, IL-6, and IL-8 inflammatory factors, while after pretreatment with Rb1, the expression levels of these inflammatory factors gradually decreased with the increase of Rb1 concentration, indicating that Rb1 can inhibit the upregulation of inflammatory factor expression induced by DDP.

[0103] Figure 8 D in the figure shows that ginsenoside Rb1 inhibits the expression of inflammatory factors in A549 cells induced by ionizing radiation. Under ionizing radiation, the expression of inflammatory factors in A549 cells pretreated with different concentrations of ginsenoside Rb1 is similar to that in Figure C. Ionizing radiation promotes the expression of inflammatory factors, and after pretreatment with Rb1, the expression levels of these inflammatory factors are significantly reduced, and the reduction is more obvious with the increase of Rb1 concentration.

[0104] This example verifies the inhibitory effects of pep242 and ginsenoside Rb1 on cell senescence and inflammatory factor expression induced by ionizing radiation / physicochemical factors through cell experiments. It provides a new perspective for understanding the role of menin / SETD2 interaction in cell senescence and inflammation regulation, and also provides new ideas for developing treatment strategies for inflammation-related diseases and aging-related diseases. At the same time, this example also preliminarily verifies the potential of ginsenoside Rb1 as a natural product that targets the menin / SETD2 interaction, and provides experimental basis for its application prospects in the treatment of diseases such as pathogen infection and acute pneumonia induced by physical and chemical factors.

[0105] The present invention reveals the epigenetic molecular mechanism by which pathogens promote a series of CSS-related inflammatory factors / cytokines / chemokines through chromatin H3K36me3 reprogramming by activating the formation of menin / SETD2 complexes and chromatin-directed recruitment. This provides a new perspective for understanding the pathogenesis of diseases such as acute pneumonia. Based on the above-mentioned new mechanism of action, the present invention designs a natural product Rb1 (i.e., ginsenoside Rb1) that competitively inhibits the interaction between menin / SETD2 as a drug candidate. This drug design is highly targeted and can directly inhibit the expression of a series of CSS-related inflammatory factors / cytokines / chemokines, thereby being expected to achieve significant therapeutic effects in the treatment of diseases such as acute pneumonia. Ginsenosides, as a natural product, have been proven to have low toxicity. This makes Rb1 as a drug candidate have better safety and tolerability, providing more possibilities for clinical application. At the same time, due to the high incidence of diseases such as acute pneumonia worldwide and the lack of effective treatment methods, the potential application prospects of Rb1 are very broad. The present invention provides a natural product Rb1 that targets and destroys the menin / SETD2 interaction, and verifies the potential application prospect of Rb1 in the treatment of acute pneumonia induced by pathogen infection and physical and chemical factors.

[0106] The above embodiments are only preferred embodiments of the present invention and cannot be considered to limit the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. Acute pneumonia inhibitory ginsenoside Rb1 monomer, characterized in that It was screened from the natural product library TargetMol-Natural Compound Library by high-throughput screening method, and its structural formula is as follows: The ginsenoside Rb1 monomer can specifically target and inhibit the interaction between menin and SETD2 protein.

2. The use of the acute pneumonia inhibitory ginsenoside Rb1 monomer as claimed in claim 1 in the preparation of anti-inflammatory drugs, characterized in that The monomer can inhibit the expression of macrophage inflammatory factors, inhibit LPS-induced acute lung injury, reduce lung inflammation and macrophage infiltration, and inhibit the expression of alveolar macrophage inflammatory factors.

3. The use of the acute pneumonia suppressive ginsenoside Rb1 monomer as claimed in claim 1 in the preparation of anti-tumor drugs, characterized in that It is particularly useful in tumors with abnormal expression of menin / SETD2.

4. Use of the acute pneumonia inhibitory ginsenoside Rb1 monomer as claimed in claim 1 in the preparation of radiotherapy and chemotherapy adjuvant therapeutic drugs for alleviating the side effects caused by radiotherapy and chemotherapy.

5. The use of the acute pneumonia inhibitory ginsenoside Rb1 monomer as claimed in claim 1 in the preparation of drugs for treating diseases related to cytokine storm syndrome, characterized in that The cytokine storm syndrome-related diseases include severe acute respiratory syndrome, influenza, and COVID-19.

6. The use of the acute pneumonia suppressive ginsenoside Rb1 monomer as claimed in claim 1 in the preparation of a lung protection drug, characterized in that Used to improve the symptoms of LPS-induced acute lung injury and reduce alveolar protein deposition.

7. A pharmaceutical composition, characterized in that The composition comprises the acute pneumonia inhibitory ginsenoside Rb1 monomer as claimed in claim 1 and a pharmaceutically acceptable carrier or excipient, and is used for treating acute pneumonia associated with cytokine storm or pneumonia associated with radiotherapy and chemotherapy caused by pathogen infection.

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