Acute pneumonia inhibitory oleanolic acid OA monomer and application thereof

Abstract

The invention discloses an acute pneumonia inhibitory oleanolic acid OA monomer and application thereof, and relates to the field of biological medicines. An oleanolic acid OA monomer is screened out from a natural product library, and the interaction between menin and SETD2 protein can be specifically inhibited. The OA obviously improves the LPS-induced acute lung injury of mice and inhibits the expression of IR / DDP-induced cell senescence inflammatory factors. OA is extracted from plants and is low in toxicity, and the extraction process is simple and economical. And hydrogen bonds and hydrophobic interaction can be formed with SETD2 protein active sites, so that a stable compound is formed. It is proved that OA is specifically combined with a specific structural domain of SETD2 to antagonize the interaction of menin. Meanwhile, inflammatory factor overexpression induced by LPS, S-Pro and the like can be effectively inhibited, and LPS-induced inflammatory acute lung injury can be remarkably inhibited. The compound has the advantages of high targeting property, definite action mechanism, remarkable treatment effect and the like, and can be further prepared into various dosage forms of pharmaceutical compositions containing OA for clinical application.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an acute pneumonia-inhibiting oleanolic acid OA monomer and its application, especially for acute pneumonia associated with cytokine storm caused by pathogen infection, radiotherapy and chemotherapy. Background Art

[0002] Abnormal activation of alveolar macrophages (AM) causes a series of clinical manifestations in the lungs. For example, infections such as the novel coronavirus SARS-CoV-2, influenza virus, bacteria, and mycoplasma activate macrophages to abnormally promote the expression of a series of cytokines / inflammatory factors / chemokines such as IL-1α and IL-6, causing severe acute respiratory syndrome with a very high mortality rate. In addition, radiotherapy and chemotherapy pneumonia is caused by a sharp increase in the expression of a series of cytokines / inflammatory factors / chemokines when normal lung tissues are damaged by ionizing radiation, chemotherapy drugs and other physical and chemical factors when cancer patients receive radiotherapy and chemotherapy. In severe cases, the lungs undergo acute inflammatory reactions and extensive fibrosis, leading to respiratory function damage and even respiratory failure.

[0003] Pathogen infection activates the body's immune cells, abnormally secreting a large number of cytokines / inflammatory factors / chemokines such as GM-CSF, IL-1α, IL-6, etc., causing cytokine storm (CS). The clinical manifestations of CS are persistent fever, splenomegaly, hepatomegaly with liver dysfunction, acute respiratory distress syndrome and multiple organ failure, with a very high mortality rate. CS-related diseases can be divided into cytokine storm syndrome (CSS) and cytokine release syndrome (CRS). The cause of CSS is that pathogen infection causes abnormal activation of the body's immune system, promoting a sharp increase in inflammatory cytokines; the cause of CRS is related to the increased release of multiple cytokines such as IL-6, IL-10, TNF-α, GM-CSF and IFN-γ caused by cell therapy such as CAR-T. In addition, radiotherapy and chemotherapy pneumonia is an inflammatory response caused by excessive activation of inflammatory factors / cytokines / chemokines expression in normal lung tissue after ionizing radiation or chemotherapy for lung cancer, breast cancer, esophageal cancer, malignant lymphoma or other malignant tumors in the chest. Pathological changes are manifested as exudative inflammatory response in the acute phase and extensive lung tissue fibrosis in the chronic phase. In severe cases, extensive lung fibrosis occurs, leading to respiratory function damage and even respiratory failure. At present, there is a lack of targeted drugs with clear efficacy for the treatment of the above acute pneumonia. Tocilizumab, a monoclonal antibody to the receptor of cytokine IL-6, is a recently developed potential drug for the clinical treatment of CS. However, tocilizumab only antagonizes IL-6, a cytokine, and cannot effectively antagonize other multiple inflammatory factors, so its effect is limited and its efficacy needs to be further verified in clinical applications (Cytokine storm caused by new coronavirus and its drug treatment. Chinese Journal of Pharmaceutical Sciences, 55(5), pages333–336 (2020)). Antagonists targeting the novel coronavirus / SARS-CoV-2 S protein receptor, angiotensin-converting enzyme 2 (ACE2), are in clinical trials. These inhibitors block the infection of target cells by antagonizing the binding of SARS-CoV-2 to ACE2, thereby inhibiting the cytokine storm (Angiotensin-converting enzyme 2—at the heart of the COVID-19 pandemic. Cell, 186 (5), pages 906–922 (2023)).However, recent studies have found that SARS-CoV-2 can not only enter lung macrophages through a limited number of ACE2, but also infect monocytes / macrophages through cell surface Fcγ receptors with the assistance of antibodies, and release a large amount of cytokines, inducing excessive inflammatory response and leading to CS (FcγR-mediated SARS-CoV-2 infection of monocytes activates inflammation. Nature, 606, pages576–584 (2022)).

[0004] Glucocorticoids are currently the most commonly used drugs for the clinical treatment of CS. However, as a broad-spectrum immunosuppressant, glucocorticoids inhibit the immune response and the clearance of pathogens such as the new coronavirus / SARS-CoV-2 by the human immune system, increase the mortality rate and secondary infection rate of patients, have significant side effects, and have limited efficacy. In summary, there is currently a lack of clinically effective treatments for CS, and there is an urgent need to develop new target drugs based on the pathogenesis.

[0005] At present, there is still a lack of targeted drugs with clear efficacy for acute pneumonia such as severe acute respiratory syndrome caused by abnormal activation of inflammatory factors. Tocilizumab, a monoclonal antibody to the IL-6 receptor, only antagonizes IL-6, a cytokine, and cannot effectively antagonize other inflammatory factors. Its efficacy needs to be further verified in clinical applications. Antagonists targeting the new coronavirus / SARS-CoV-2 S protein receptor, angiotensin-converting enzyme 2 (ACE2), are in the clinical trial stage. However, SARS-CoV-2 can not only enter lung macrophages through ACE2, but also infect monocytes / macrophages through cell surface Fcγ receptors with the help of antibodies to release a large number of cytokines, which can still induce excessive inflammatory responses. Glucocorticoids are currently the most commonly used drugs for the clinical treatment of infectious and chemoradiotherapy-induced acute pneumonia. However, as a broad-spectrum immunosuppressant, glucocorticoids inhibit immune responses, have large side effects, and have limited efficacy. Summary of the invention

[0006] The purpose of the present invention is to provide a natural product library (TargetMol-Natural Compound Library) for high-throughput screening of acute pneumonia inhibitory oleanolic acid OA monomers that specifically inhibit menin / SETD2 interaction.

[0007] Another object of the present invention is to provide the application of the acute pneumonia inhibitory oleanolic acid OA monomer.

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

[0009] The acute pneumonia inhibitory oleanolic acid OA monomer is separated and extracted from the whole herb of Swertia serrata of the Gentianaceae family or the fruit of Ligustrum lucidum, and its structural formula is as follows:

[0010]

[0011] The present invention uses the high-throughput screening technology of the natural product library (TargetMol - Natural Compound Library) to identify natural product monomers that can specifically inhibit the menin / SETD2 interaction. Based on the previous study that identified the smallest and highly conserved 231-248 fragment of menin that interacts with SETD2, the purified SET protein and the FITC-labeled menin polypeptide were placed in a 384-well plate and screened from 1,500 natural products, and finally oleanolic acid OA was obtained.

[0012] The oleanolic acid OA monomer can specifically inhibit the interaction between menin and SETD2 protein, and significantly inhibit the expression of pathogen-induced inflammatory factors through this mechanism.

[0013] Fluorescence polarization (FP) experiments confirmed that OA could specifically inhibit the interaction between menin and SETD2 proteins. Biochemical experiments such as co-immunoprecipitation (Co-IP), cell thermal shift assay (CETSA), GST-Pull down and protein blotting (Western Blotting) further demonstrated the inhibitory effect of OA on the interaction between menin and SETD2. Among them, GST-pull down showed that OA specifically destroyed the direct interaction between the two; co-immunoprecipitation combined with Western blot showed that OA dose-dependently inhibited the interaction between the two; cell thermal shift assay revealed that OA directly interacted with SETD2 and had no obvious effect on the thermal stability of menin protein.

[0014] Schrodinger Maestro software was used to perform molecular docking of OA and SETD2 target protein, and it was found that OA bound well to the target protein and had a high degree of match. The docking complex was visualized by Pymol2.1 software, which confirmed that OA formed strong hydrogen bond interactions with GLN-1623, ALA-1564, and GLU-1592 in the active site of SETD2 protein, and had hydrophobic interactions with PHE-1620, ALA-1564, MET-1563, and TYR-1622, which effectively promoted the stable formation of the complex.

[0015] The present invention provides the use of the oleanolic acid OA monomer in the preparation of drugs related to the treatment of cytokine storm syndrome caused by pathogen infection. The pathogens include but are not limited to the new coronavirus SARS-CoV-2, influenza virus, bacteria and mycoplasma, and the oleanolic acid OA monomer can dose-dependently inhibit the abnormal expression of cytokines such as IL-1α and IL-6 in macrophages activated by the S protein of SARS-CoV-2 and bacterial lipopolysaccharide LPS.

[0016] The present invention provides the use of the oleanolic acid OA monomer in the preparation of a drug for treating radio- and chemotherapy-induced pneumonia. The oleanolic acid OA monomer can dose-dependently inhibit the expression of cell senescence inflammatory factors induced by ionizing radiation IR or cisplatin DDP, and improve the acute inflammatory response caused by normal lung tissue damage caused by radio- and chemotherapy.

[0017] The present invention provides application of oleanolic acid OA in preparing a drug for inhibiting menin / SETD2 protein interaction.

[0018] The present invention provides application of oleanolic acid OA in preparing a drug for inhibiting the expression of inflammatory factors.

[0019] The present invention provides application of oleanolic acid OA in preparing a medicine for improving acute lung injury.

[0020] The oleanolic acid OA antagonizes the interaction between menin and SETD2 by specifically binding to a specific domain of SETD2, effectively inhibits the overexpression of inflammatory factors induced by LPS, S-Pro, IR, DDP, etc., and significantly improves LPS-induced inflammatory acute lung injury.

[0021] The present invention provides a pharmaceutical composition for treating acute pneumonia, comprising the oleanolic acid OA monomer as an active ingredient, and a pharmaceutically acceptable carrier, excipient or diluent.

[0022] The dosage form of the pharmaceutical composition is tablet, capsule, injection, oral liquid preparation or aerosol.

[0023] The present invention provides the use of the oleanolic acid OA monomer in preparing a drug for inhibiting the expression of pathogen-induced inflammatory factors, in particular, in preparing a drug for treating or preventing acute pneumonia activated by LPS or S-Pro.

[0024] Compared with the prior art, the technical effects and advantages of the present invention are:

[0025] The present invention is based on the molecular mechanism of gene transcriptional regulation of key factors related to the pathogenesis of CS, such as IL-1α and IL-6, and aims to develop new targeted drugs. Studies have found that menin (encoding gene MEN1) interacts with histone methyltransferase SETD2, and remodels chromatin H3K36me3 (trimethylation of lysine 36 of histone 3) to maintain the expression of multiple cytokines / inflammatory factors such as IL-1α and IL-6; pathogen infection or ionizing radiation / chemotherapeutic drugs will enhance the menin / SETD2 interaction, thereby activating the expression of a series of cytokines / inflammatory factors such as IL-1α and IL-6, which is the key node of the cytokine storm.

[0026] The research basis of the present invention is the epigenetic molecular mechanism of menin / SETD2 complex regulating macrophage maturation, differentiation, activation and phagocytic function through H3K36m3 modification. Therefore, the design molecule of the natural product OA that competitively inhibits the interaction between menin and SETD2 is highly targeted, has a clear mechanism of action, and has a significant effect. OA has a significant potential therapeutic effect on cytokine storm-related diseases CSS and CRS caused by pathogens by specifically antagonizing the interaction between menin and SETD2; it has the advantages of obvious effect, high efficiency, specificity, and low toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a preliminary verification of the high-throughput screening of the natural product library and the interaction between OA and proteins. A is a process display of the high-throughput screening of small molecule inhibitors in the natural product library, showing the steps from preparing materials to screening potential targeted inhibitors; B is the screening results of the natural product library and the chemical structure of OA, indicating that OA was screened and its chemical structure was displayed; C is the verification of the inhibitory effect of OA on the interaction between menin and SET proteins through fluorescence polarization experiments.

[0028] Figure 2The biochemical method verifies the inhibitory properties of OA on the interaction between menin and SETD2. A is the GST-pull down experiment to confirm the destruction of the direct interaction between menin and SET protein by OA; B is the dose-dependent inhibition of the interaction between menin and SET protein detected by co-transfected cells and immunoprecipitation combined with Western blot; C is the dose-dependent inhibition of OA on the interaction between menin / SETD2 verified in the mouse mononuclear macrophage cell line; D is the effect of 50μM OA on the thermal stability of SETD2 protein detected by cell thermal shift experiment to verify the interaction between the two; E is the cell thermal shift experiment to detect the dose-dependent effect of different concentrations of OA on the thermal stability of SETD2 protein; F is the cell thermal shift experiment to detect the effect of OA on the thermal stability of menin protein to determine whether there is a direct interaction between OA and menin protein.

[0029] Figure 3 The interaction mechanism between OA and SETD2 protein was analyzed by bioinformatics. A is the molecular docking using SchrodingerMaestro software to show the binding between OA and SETD2 protein; B is the visualization of the docking complex using Pymol2.1 software to show the binding mode between OA and SETD2 protein; C is the molecular docking score table showing key data such as binding energy.

[0030] Figure 4 The molecular dynamics characteristics of OA and SETD2 proteins were analyzed by bioinformatics. A is the RMSD diagram obtained after 100ns simulation by Gromacs2020 software, showing the dynamic equilibrium of the complex; B is the SASA diagram, which reflects the effect of OA and SETD2 protein binding on protein stability and the binding effect of the two; C is the change in the number of hydrogen bonds between SETD2 protein and OA during the statistical simulation process, indicating the stabilizing effect of hydrogen bonds on the interaction between the two; D is the measurement of the gyration radius (Rg) of the target protein, analyzing the relative density and stability of the complex after binding; E is the RMSF diagram, showing the changes in amino acid conformation in the complex; F is the analysis of the binding free energy and various forces of OA and SETD2 protein, indicating the affinity of the two and the stability of the complex.

[0031] Figure 5 The results show that OA can inhibit the expression of inflammatory factors in macrophages. A shows the inhibitory effect of OA on the expression of inflammatory factors induced by LPS in the LPS-treated alveolar macrophage experiment; B shows the inhibitory effect of OA on the expression of inflammatory factors induced by S-Pro in the S-Pro-treated THP-1 cell experiment.

[0032] Figure 6The figure shows the inhibitory effect of OA on LPS-induced acute lung injury. A shows the improvement of OA on the gross symptoms of lung tissue in the mouse acute lung injury model; B shows the improvement of OA on LPS-induced lung injury and macrophage infiltration through HE and IHC staining; C shows the improvement of OA on LPS-induced alveolar protein deposition through BCA assay; D shows the inhibition of OA on the expression of inflammatory factors in alveolar macrophages induced by LPS through RT-qPCR detection.

[0033] Figure 7 This is a verification of the inhibitory effect of OA on IR / DDP-induced cell senescence / inflammatory factor expression. A shows the inhibition of OA on DDP-induced inflammatory factor expression in the DDP-treated A549 cell experiment; B shows the inhibition of OA on IR-induced inflammatory factor expression in the IR-treated A549 cell experiment. DETAILED DESCRIPTION

[0034] 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.

[0035] Studies have found that menin (encoding gene MEN1) interacts with histone methyltransferase SETD2, and remodels chromatin H3K36me3 (trimethylation of lysine 36 at position 3 of histone 3) to maintain the expression of multiple cytokines / inflammatory factors such as IL-1α and IL-6; pathogen infection or ionizing radiation / chemotherapeutic drugs activate the expression of a series of cytokines / inflammatory factors such as IL-1α and IL-6 by enhancing the menin / SETD2 interaction, which is the key node of cytokine storm. Based on the key target of menin / SETD2 interaction, the present invention provides a natural product that specifically inhibits the interaction between the two, named oleanolic acid (OA). Oleanolic acid OA is a pentacyclic triterpenoid compound isolated and extracted from the whole grass of the genus Swertia or the fruit of Ligustrum lucidum of the Gentianaceae plant Swertia, and exists in a variety of plants as free bodies and glycosides. Oleanolic acid is widely present in plants, and the extraction process is simple and economical. It can be made into monomer preparations on a large scale, and it has been proven to be low toxic. The OA provided by the present invention specifically inhibits the menin / SETD2 interaction; OA inhibits the abnormal expression of cytokines such as IL-1α and IL-6 in macrophages activated by the new coronavirus S-protein, bacterial lipopolysaccharide (LPS), etc.; in animal experiments, OA inhibits the expression of multiple inflammatory factors induced by LPS, and significantly alleviates LPS-induced acute lung injury in mice; OA dose-dependently inhibits the expression of cell aging inflammatory factors induced by ionizing radiation (IR) / cisplatin (DDP).

[0036] Example 1: Screening for menin / SETD2 interaction inhibitors based on natural product libraries

[0037] 1. Preparation of experimental materials

[0038] The TargetMol-Natural Compound Library was selected, which contains 1,500 natural products. SET protein was expressed and purified by genetic engineering technology. Preliminary studies determined that the minimum domain of menin that interacts with SETD2 is the 231-248 fragment, and verified that the fragment is highly conserved during species evolution (patent application number, 2024119120475). 293T cells and mouse mononuclear macrophage cell line Raw264.7 were prepared. Plasmids such as FLAG-SET, HA-MEN1, MEN1, and SETD2 were constructed and extracted.

[0039] 2. High-throughput screening experiments

[0040] Purified SET protein and FITC-labeled menin 231-248 peptide were added to a 384-well plate, followed by various compounds from the natural product library for high-throughput screening to identify potential targeted inhibitors that can specifically inhibit the menin / SETD2 interaction ( Figure 1 A in the figure). After screening, it was determined that oleanolic acid OA can specifically inhibit the menin-SETD2 interaction, and the chemical structure of oleanolic acid OA was obtained ( Figure 1 B in the figure).

[0041] 3. Fluorescence polarization experiment

[0042] According to the standard procedure of fluorescence polarization experiment, OA with different concentration gradients was fully mixed with the reaction system containing SET protein and FITC-labeled menin 231-248 peptide, and incubated under specific temperature and time conditions. The fluorescence polarization value of the reaction system was accurately measured using a fluorescence polarization detector. By analyzing the changes in the fluorescence polarization value, it was strongly confirmed that oleanolic acid OA specifically inhibited the interaction between menin and SET protein ( Figure 1 C in.

[0043] 4. Biochemical experimental verification

[0044] GST-pull down experiment: construct GST-menin and His-SET expression vectors, induce expression in E. coli, and purify GST-menin and His-SET fusion proteins using affinity chromatography. After incubating GST-menin and OA in a suitable buffer for a period of time, add His-SET protein and continue incubation. After the incubation, add GST beads for precipitation. After multiple rigorous washings, the precipitate is subjected to SDS-PAGE electrophoresis and then transferred to a membrane. Western blot detection using anti-His antibody confirms that oleanolic acid OA specifically destroys the direct interaction between menin and SET protein ( Figure 2 A in the figure).

[0045] Co-transfection and immunoprecipitation experiments: FLAG-SET and HA-MEN1 plasmids were co-transfected into 293T cells using liposome transfection reagents. After transfection, the cells were cultured under appropriate cell culture conditions (such as 37°C, 5% CO2) for 24-48 hours. The cells were collected and lysed with a cell lysis buffer containing protease inhibitors. The supernatant of the lysed cells was incubated with ProteinA / G beads bound to anti-HA antibodies at 4°C overnight for immunoprecipitation. The next day, the beads were washed several times with washing buffer, and then SDS loading buffer was added. After boiling and denaturation, SDS-PAGE electrophoresis was performed. After transfer to the membrane, Western blot detection was performed with anti-FLAG antibody, which confirmed that oleanolic acid OA dose-dependently (0, 6.25, 12.5, 25, 50 μM) inhibited the interaction between menin and SET protein ( Figure 2 B in the figure).

[0046] Mouse mononuclear macrophage cell line experiment: The FLAG-MEN1 plasmid was efficiently transfected into the mouse mononuclear macrophage cell line Raw264.7 by electroporation transfection, and cultured in a culture medium containing appropriate antibiotics after transfection. After the cells were cultured to the logarithmic growth phase, the cells were collected and lysed with cell lysis buffer. The cell lysate supernatant was incubated with Protein A / G beads bound to anti-FLAG antibodies at 4°C overnight for immunoprecipitation. The subsequent washing, denaturation, electrophoresis and transfer steps were the same as those in the co-transfection and immunoprecipitation experiments. Finally, Western blot detection was performed with anti-SETD2 antibodies. The results confirmed that oleanolic acid OA dose-dependently inhibited the interaction between menin / SETD2 proteins ( Figure 2 C in.

[0047] Cell thermal shift experiment: Effect of 50 μM OA on the thermal stability of SETD2 protein: MEN1 plasmid was transfected into 293T cells and cultured for a certain period of time after transfection. The cells were collected and lysed with cell lysis buffer. The cell lysate was divided into several portions, and OA with a final concentration of 50 μM was added to each portion. Then, these samples were incubated at different temperature gradients. After the incubation, the cells were quickly cooled in an ice bath, followed by centrifugation. The supernatant was taken for SDS-PAGE electrophoresis. After transfer to the membrane, Western blot detection was performed with anti-SETD2 antibody. By observing the degradation of SETD2 protein as the temperature increased, it was suggested that oleanolic acid OA directly interacted with SETD2 ( Figure 2 D in.

[0048] Dose-dependent effect of different concentrations of OA on the thermal stability of SETD2 protein: Similar to the above experiment, 293T cells were transfected with MEN1 plasmid and then lysed. The cell lysate was divided into several portions, and different concentrations of OA (0, 6.25, 12.5, 25, 50 μM) were added respectively. Subsequently, different temperature gradient incubation, ice bath cooling, centrifugation, SDS-PAGE electrophoresis, membrane transfer and Western blot detection with anti-SETD2 antibody were performed to observe the changes in SETD2 protein stability with increasing OA doses. With the increase of oleanolic acid OA dose, the stability of SETD2 protein gradually increased, suggesting that oleanolic acid OA directly interacts with SETD2 ( Figure 2 E in.

[0049] Effect of OA on the thermal stability of menin protein: SETD2 plasmid was transfected into 293T cells, and the cells were cultured and lysed after transfection. The cell lysate was divided into several portions, and OA with a final concentration of 50 μM was added. The operation steps such as incubation under different temperature gradients were the same as the above-mentioned cell thermal shift experiment. Finally, Western blot detection was performed with anti-menin antibody to determine the effect of OA on the thermal stability of menin protein, and then to indicate whether there is a direct interaction between oleanolic acid OA and menin protein; the results showed that oleanolic acid OA (50 μM) had no obvious effect on the thermal stability of menin protein, indicating that oleanolic acid OA and menin protein do not have a direct interaction ( Figure 2 The above results are biochemical evaluations of the inhibitory effect of oleanolic acid OA on the interaction between menin and SETD2.

[0050] Example 2: OA interacts with SETD2 protein

[0051] 1. Preparation of experimental materials

[0052] Prepare Schrodinger Maestro software, Pymol2.1 software, and Gromacs2020 software. Obtain a high-resolution three-dimensional structure file of SETD2 protein from the Protein Data Bank (PDB). If there are missing or incomplete parts in the structure, use relevant software to complete and optimize the structure. Draw the structure of OA using chemical structure drawing software (such as ChemDraw) and convert it into a format compatible with the docking software to ensure the accuracy of the structure.

[0053] 2. Molecular docking experiment

[0054] The docking module in Schrodinger Maestro software was used to perform molecular docking between the structure file of OA and the three-dimensional structure file of SETD2 protein. During the docking process, the active site was carefully defined, and the key area of ​​interaction with menin determined in previous studies was referred to. A suitable docking algorithm (such as the Glide algorithm) was selected, and a series of docking parameters were set. Multiple docking calculations were performed, and the binding of OA and SETD2 target protein was deeply analyzed from the docking results, including key information such as binding mode, binding site, and binding energy. The results showed that OA and the target protein had a good binding effect and a high degree of match ( Figure 3 A in the figure).

[0055] The complex formed by OA and SETD2 protein after docking was visualized using Pymol2.1 software to obtain the binding mode of OA and SETD2 protein. The binding mode diagram shows the amino acid residues that bind to the pocket of SETD2 protein, such as OA and GLN-1623, ALA-1564, and GLU-1592 in the active site of SETD2 protein, which form strong hydrogen bond interactions, making an important contribution to anchoring small molecules in the protein pocket; in addition, since the natural product can also have good hydrophobic interactions with PHE-1620, ALA-1564, MET-1563, and TYR-1622, it has an important contribution to stabilizing the natural product. These interactions can effectively promote the formation of a stable complex between the natural product and SETD2 protein ( Figure 3 B in the figure).

[0056] Record the data in the molecular docking score table, such as binding energy, number of hydrogen bonds, hydrophobic interactions, etc. The molecular docking score table results show that the binding energy is less than -6kcal / mol ( Figure 3 C). The above results simulate the molecular basis of the interaction between oleanolic acid OA and SETD2 protein.

[0057] 3. Molecular dynamics simulation experiment

[0058] Gromacs2020 software was used to perform 100ns molecular dynamics simulations of OA and SETD2. The SETD2 protein structure was preprocessed and appropriate force field parameters were added to ensure the rationality of the protein structure during the simulation. The OA and SETD2 protein complex was built, and preliminary simulation steps such as energy minimization and equilibration were performed to make the system reach a stable state. During the simulation, the structure file of the complex was output regularly, and the root mean square deviation (RMSD) was accurately calculated using the analysis tool of Gromacs2020 software, and the RMSD graph was drawn. It can be seen from the RMSD graph that the average RMSD of the complex is less than 3.5Å, and the complex basically reaches dynamic equilibrium in about 10ns, indicating that the complex maintains a relatively stable structure during the simulation ( Figure 4 A in the figure).

[0059] SASA analysis: The solvent-accessible surface area (SASA) was calculated using the analysis tool of Gromacs2020 software, and a SASA graph was drawn. The SASA graph showed that the accessible surface area of ​​the complex decreased significantly, indicating that the binding of OA to SETD2 protein did not damage the stability of the protein itself, but improved the stability of the protein. This also indirectly indicates that OA has a good binding effect with SETD2 protein ( Figure 4 B in the figure).

[0060] Hydrogen bond analysis: The hydrogen bond analysis tool provided by Gromacs2020 software was used to count the changes in the number of hydrogen bonds between the protein and OA during the entire simulation process. It was found that OA can form one or more hydrogen bond interactions with amino acids in the protein pocket, and these hydrogen bonds play an important role in stabilizing OA and SETD2 protein ( Figure 4 C in.

[0061] Gyration radius analysis: Analyze the relative compactness and stability of the complex induced by binding at the primary and secondary sites, measure the gyration radius (Rg) of the target protein, and draw an Rg graph. The Rg graph shows that the Rg of the SETD2 protein decreases slightly, suggesting that the binding of the SETD2 protein to OA causes the protein to maintain more hydrophobic contacts and form more effective interactions within the protein. On the other hand, it can also better match OA, thereby promoting the stability of the complex ( Figure 4 D in.

[0062] RMSF analysis: Calculate the root mean square fluctuation (RMSF) of the amino acids in the complex and draw the RMSF graph. According to the RMSF graph, a small number of amino acids in the complex formed by the interaction between SETD2 protein and OA have large conformational changes (around 1450). The main reason is that these amino acids in the complex are located in the hinge region of the protein, which is relatively flexible. Their conformation changes during the simulation process, but most of the amino acid conformational changes are within an acceptable range ( Figure 4 E in.

[0063] Binding free energy calculation: The free energy calculation module of Gromacs2020 software was used, and the MM / PBSA (Molecular Mechanics / Poisson-Boltzmann Surface Area) method was used to accurately calculate the binding free energy of OA and SETD2 protein. The results showed that the binding free energy of OA and SETD2 protein was -29.14+ / -1.43kcal / mol, of which van der Waals force played a major role (-37.36+ / -1.19kcal / mol), indicating that OA can be stably maintained in the protein site pocket and has strong van der Waals interactions with surrounding residues. Since OA forms effective hydrogen bond interactions with the protein pocket, electrostatic interactions also make an important contribution to stabilizing the complex (-8.35+ / -0.76kcal / mol). In summary, OA has a strong affinity for SETD2 protein, and these affinities can prompt OA to form a stable complex with SETD2 protein, thereby exerting an active effect ( Figure 4 F in.

[0064] Example 3: Study on the inhibitory effect of OA on the expression of pathogen-induced inflammatory factors

[0065] 1. Preparation of experimental materials

[0066] Cell line: Human monocytic leukemia THP-1 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum (FBS) and 1% double antibody (penicillin-streptomycin mixture) in a cell culture incubator at 37°C and 5% CO2. Mouse alveolar macrophages were obtained from healthy mice by bronchoalveolar lavage and cultured in DMEM medium containing 10% FBS and 1% double antibody.

[0067] Reagents: SARS-CoV-2 S protein (S-Pro), bacterial lipopolysaccharide (LPS), Trizol reagent, reverse transcription kit, SYBRGreen fluorescent dye, primers (designed for inflammatory factors such as IL-6, TNF-α, IL-1β, IL-8 and internal reference genes).

[0068] 2. Cell culture and treatment

[0069] THP-1 cell treatment: THP-1 cells were cultured in RPMI1640 medium containing 10% FBS, and PMA with a final concentration of 100 nM was added to the culture system to induce differentiation for 48 hours to make them into M0 macrophages. The differentiated cells were then divided into different groups, and different concentrations (0, 12.5, 25 μM) of OA and 100 nM S-Pro were added to each group, and the culture was continued for 48 hours.

[0070] Treatment of mouse alveolar macrophages: Mouse alveolar macrophages were obtained by alveolar lavage, cultured in DMEM medium containing 10% FBS, divided into different groups, and added with different concentrations (0, 12.5, 25 μM) of OA and 100 ng / mL of LPS, and cultured for 24 h.

[0071] 3. RNA extraction and RT-qPCR

[0072] RNA extraction: After the culture, strictly follow the instructions of Trizol reagent to extract total RNA from the above treated cells, and use a spectrophotometer to accurately measure the RNA concentration and purity to ensure A 260 / A 280 Between 1.8-2.0.

[0073] Reverse transcription: Take an appropriate amount of total RNA and reverse transcribe the RNA into cDNA according to the operating procedures of the reverse transcription kit.

[0074] qPCR: cDNA was used as a template and SYBR Green fluorescent dye was used for real-time fluorescence quantitative PCR reaction. The amplification reaction was performed on a PCR instrument under the following conditions: 95°C pre-denaturation for 30 s, then 95°C denaturation for 5 s, 60°C annealing for 30 s, for a total of 40 cycles. By analyzing the Ct value, the 2^(-ΔΔCt) method was used to accurately calculate the relative expression of inflammatory factors to evaluate the inhibitory effect of OA on the expression of inflammatory factors induced by S-Pro and LPS.

[0075] After treating alveolar macrophages with LPS (100 ng / mL) and oleanolic acid OA (12.5, 25 μM) for 24 h, the cells were harvested and RNA was extracted using the Trizol method. RT-qPCR results showed that LPS significantly promoted the expression of inflammatory factors such as IL-6, TNF-α, and IL-1β, while oleanolic acid OA (12.5, 25 μM) effectively inhibited the upregulation of the above inflammatory factors induced by LPS ( Figure 5A in the figure). THP-1 cells (PMA-induced differentiation of M0 human monocytic leukemia THP-1 cells) were treated with S-Pro (100 nM) and oleanolic acid OA (12.5, 25 μM) for 48 h, and the cells were harvested and RNA was extracted using the Trizol method. RT-qPCR results showed that S-Pro significantly promoted the expression of inflammatory factors such as IL-6, TNF-α, IL-1β, and IL-8, while oleanolic acid OA (12.5, 25 μM) effectively inhibited the upregulation of the above inflammatory factors induced by S-Pro ( Figure 5 B in the figure).

[0076] Example 4: Study on the inhibitory effect of OA on LPS-induced acute lung injury

[0077] 1. Preparation of experimental materials

[0078] Experimental animals: C57BL6 / J mice, 6-8 weeks old, male, purchased from a regular experimental animal center, raised in a specific pathogen-free (SPF) animal room, with free access to food and water.

[0079] Reagents: LPS, oleanolic acid OA, hematoxylin, eosin, F4 / 80 specific antibody, BCA protein concentration determination kit, 4% paraformaldehyde fixative.

[0080] 2. Animal model construction and processing

[0081] C57BL6 / J mice were randomly divided into a control group, an LPS model group, and an OA treatment group, with a certain number of mice in each group (determined according to the statistical requirements of the experimental design).

[0082] The mice in the LPS model group and OA treatment group were instilled with 5 mg / kg LPS solution through the trachea, and the mice in the control group were instilled with an equal amount of normal saline to induce acute lung injury model. 24 hours later, the mice in the OA treatment group were instilled with 25 mg / kg OA solution through the trachea, and the mice in the control group and LPS model group were instilled with an equal amount of normal saline.

[0083] 3. Observation and analysis of lung tissue

[0084] Gross observation: After another 24 hours, the mice were killed, and the lung tissue was quickly dissected and removed. The gross morphology of the lung tissue was carefully observed, and the presence of bleeding and edema in the lung tissue was recorded in detail to visually evaluate the improvement effect of OA on LPS-induced lung tissue damage. It was found that large-area bleeding and edema in the lung tissue could be significantly improved by intratracheal instillation of oleanolic acid OA (25 mg / kg) ( Figure 6 A) Figure 6 A in the figure).

[0085] Tissue staining: Part of the lung tissue was fixed with 4% paraformaldehyde, and after standard steps such as dehydration, transparency, wax immersion, and embedding, paraffin sections were made. The paraffin sections were stained with hematoxylin-eosin (HE), and the pathological changes of the lung tissue were carefully observed under a microscope, including the infiltration of inflammatory cells in the alveolar cavity and interstitial lung, the width of the alveolar septa, the integrity of the alveolar structure, etc., in order to accurately evaluate the inhibitory effect of OA on LPS-induced lung inflammation. The results of hematoxylin-eosin (HE) staining of mouse lung tissue showed that the LPS group had obvious acute inflammation in the lungs, diffuse inflammatory cell infiltration in the alveolar cavity and interstitial lung, widened alveolar septa, rupture of alveolar septa in some areas, atrophy of alveoli, and even disappearance of the structure. The oleanolic acid OA treatment group significantly inhibited the lung damage caused by LPS ( Figure 6 B in the figure).

[0086] Immunohistochemical staining: After the above paraffin sections were dewaxed and hydrated, they were incubated with 3% hydrogen peroxide solution to block endogenous peroxidase activity, and then nonspecific binding sites were blocked with goat serum. Then, F4 / 80-specific antibody was added and incubated overnight at 4°C. The next day, biotin-labeled secondary antibody was incubated, and finally DAB color developer was used for color development, and the cell nuclei were counterstained with hematoxylin. The infiltration of F4 / 80-positive cells (i.e., macrophages) was carefully observed under a microscope to evaluate the improvement effect of OA on macrophage infiltration. The results of F4 / 80-specific antibody immunohistochemical staining (IHC) showed that the infiltration of macrophages in the LPS group increased, while the oleanolic acid OA treatment group significantly improved the infiltration of macrophages ( Figure 6 B in the figure).

[0087] Determination of protein concentration in alveolar lavage fluid: alveolar lavage was performed on mice, and alveolar lavage fluid was collected. The protein concentration in the alveolar lavage fluid was accurately determined according to the operating procedures of the BCA protein concentration determination kit to evaluate the improvement effect of OA on LPS-induced alveolar protein deposition. The protein concentration of alveolar lavage fluid was determined by the BCA method. The results showed that alveolar protein deposition was obvious in mice with LPS acute lung injury, and oleanolic acid OA treatment significantly improved LPS-induced alveolar protein deposition ( Figure 6 C in.

[0088] Detection of inflammatory factors in alveolar macrophages: Alveolar macrophages were obtained by alveolar lavage, and the expression levels of inflammatory factors such as IL-1β, TNF-α, IL-6, and IL-18 were detected according to the RNA extraction and RT-qPCR methods in Example 3 to evaluate the inhibitory effect of OA on the expression of inflammatory factors in alveolar macrophages induced by LPS. The results showed that LPS significantly activated the expression of inflammatory cytokines such as IL-6, TNF-α, IL-1β, and IL-18, while oleanolic acid OA significantly inhibited the expression of inflammatory factors induced by LPS ( Figure 6 D).

[0089] Example 5: Study on the inhibitory effect of OA on IR / DDP-induced cell senescence / inflammatory factor expression

[0090] 1. Preparation of experimental materials

[0091] Cell line: Human lung epithelial cells A549 were cultured in high-glucose DMEM medium containing 10% fetal bovine serum (FBS) and 1% double antibody (penicillin-streptomycin mixture) in a cell culture incubator at 37°C and 5% CO2. The medium was changed regularly and the cell growth status was observed.

[0092] Reagents: Cisplatin (DDP), prepared into a 10 mM stock solution with physiological saline, stored at -20°C, and diluted with culture medium to the required concentration when used; ionizing radiation source (such as a γ-ray irradiator that can provide a stable dose of 5 Gy); Trizol reagent for extracting total cellular RNA; reverse transcription kit for reverse transcription of RNA into cDNA; SYBR Green fluorescent dye for real-time fluorescence quantitative PCR reaction; oleanolic acid OA, the purity of which was tested to be greater than 98%.

[0093] Instruments: High-precision cell culture incubator to ensure a stable cell growth environment; high-speed centrifuge for centrifugal separation of cells and liquid stratification during RNA extraction; real-time fluorescence quantitative PCR instrument to accurately detect the expression of inflammatory factors; UV spectrophotometer to measure the concentration and purity of RNA.

[0094] 2. Cell processing

[0095] A549 cells were plated at 5 × 10 5 The cells were inoculated in a 6-well plate at a density of 100 cells and cultured in an incubator for 24 hours until the cells adhered to the wall and grew well. After the cells adhered to the wall, they were divided into different groups and different concentrations of OA (0, 6.25, 12.5, 25 μM) were added respectively. Three replicate wells were set for each concentration to ensure the reliability of the experimental data.

[0096] DDP-treated group: 2 h after the addition of OA, DDP with a final concentration of 5 μM was added to the corresponding group, and the culture was continued for 72 h, during which the changes in cell morphology were regularly observed.

[0097] IR treatment group: After 2 hours of OA addition, the corresponding group cells were washed twice with serum-free medium, and then the 6-well plate was placed under an ionizing radiation source to receive 5 Gy of ionizing radiation. After the irradiation, the medium was replaced with DMEM containing 10% FBS and cultured for another 72 hours.

[0098] 3. RNA extraction and RT-qPCR

[0099] RNA extraction: After the culture is completed, aspirate the culture medium in the 6-well plate, add 1 mL of Trizol reagent to each well, and operate strictly according to the Trizol reagent instructions. Use a pipette to repeatedly blow the cells to fully lyse them, and then transfer the lysate to a 1.5 mL centrifuge tube. Add 200 μL of chloroform, shake vigorously for 15 seconds, let stand at room temperature for 3 minutes, and centrifuge at 4°C and 12000 rpm for 15 minutes. Transfer the upper aqueous phase to a new centrifuge tube, add 500 μL of isopropanol, invert to mix, let stand at room temperature for 10 minutes, and centrifuge at 4°C and 12000 rpm for 10 minutes. Discard the supernatant, wash the RNA precipitate twice with 75% ethanol, and centrifuge at 4°C and 7500 rpm for 5 minutes. Finally, dry the RNA precipitate, dissolve it with an appropriate amount of RNase-free water, and measure the RNA concentration and purity with a UV spectrophotometer to ensure A 260 / A 280 Between 1.8-2.0, ensuring that the RNA quality meets the requirements of subsequent experiments.

[0100] Reverse transcription: Take 1 μg of total RNA and perform reverse transcription according to the operating procedures of the reverse transcription kit. The reaction system includes 5× reverse transcription buffer, dNTPs, reverse transcriptase, random primers and RNA template, with a total volume of 20 μL. The reaction conditions are: incubate at 25℃ for 10 min, incubate at 42℃ for 60 min, and incubate at 70℃ for 15 min. The obtained cDNA product is stored at -20℃ for future use.

[0101] qPCR: Using cDNA as a template, SYBRGreen fluorescent dye was used for real-time fluorescence quantitative PCR reaction. Specific primers were designed for inflammatory factors such as IL-1β, IL-6, and IL-8. The primer sequences were strictly analyzed and verified by bioinformatics to ensure their specificity and amplification efficiency.

[0102] The PCR reaction system was 20 μL, including 10 μL 2×SYBR Green Master Mix, 0.5 μL upstream primer (10 μM), 0.5 μL downstream primer (10 μM), 2 μL cDNA template and 7 μL ddH2O. The reaction conditions were: 95°C pre-denaturation for 30 s, then 95°C denaturation for 5 s, 60°C annealing for 30 s, for a total of 40 cycles. Three replicate wells were set for each sample.

[0103] Data analysis: The Ct values ​​generated by real-time fluorescence quantitative PCR were analyzed, and the relative expression of inflammatory factors was calculated using the 2^(-ΔΔCt) method. The untreated group (cells with only culture medium added) was used as the control to compare the expression changes of inflammatory factors in different treatment groups and evaluate the inhibitory effect of OA on the expression of inflammatory factors induced by DDP and IR. GraphPad Prism 8 software was used for data analysis and drawing. One-way ANOVA was used for inter-group comparison. P<0.05 indicated that the difference was statistically significant.

[0104] The experimental results showed that in the DDP treatment group, 5 μM DDP significantly promoted the expression of IL-1β, IL-6, and IL-8 inflammatory factors, and with the increase of OA concentration (0, 6.25, 12.5, 25 μM), the expression of these inflammatory factors showed a significant dose-dependent downward trend, indicating that oleanolic acid OA effectively inhibited the upregulation of inflammatory factors induced by DDP ( Figure 7 In the IR treatment group, 5Gy of ionizing radiation also significantly promoted the expression of IL-1β, IL-6, and IL-8 inflammatory factors, while after adding different concentrations of OA, the upregulation of inflammatory factor expression was significantly inhibited, further proving that OA has an inhibitory effect on the expression of inflammatory factors induced by IR ( Figure 7 This result fully demonstrates the potential application value of OA in combating cell aging and inflammatory response caused by ionizing radiation or chemotherapy drugs, and provides a strong experimental basis for its research in the field of related disease treatment.

[0105] The present invention develops targeted drugs based on the molecular mechanism of gene transcription regulation of CS-related key factors such as IL-1α and IL-6, that is, the menin / SETD2 complex promotes the expression of the above cytokines / inflammatory factors / chemokines through chromatin H3K36me3 histone modification, so specific blocking of the interaction between menin / SETD2 proteins can antagonize the expression of IL-1α, IL-6, etc. The present invention uses the natural product library (TargetMol-Natural Compound Library) to high-throughput screen out the natural product monomer oleanolic acid OA that specifically blocks the menin / SETD2 interaction. Through a series of biochemical experiments and bioinformatics analysis, it is confirmed that OA antagonizes the interaction of menin by specifically binding to the specific domain of SETD2; cytological and animal experiments have confirmed that OA effectively inhibits the overexpression of inflammatory factors induced by LPS, S-Pro, etc., and significantly inhibits LPS-induced inflammatory acute lung injury.

[0106] In summary, the present invention provides a natural product OA that targets and destroys the interaction between menin and setd2, and verifies the potential application prospects of OA in the treatment of acute pneumonia associated with cytokine storm caused by over-activation of macrophages. The OA provided by the present invention shows good application prospects in acute pneumonia associated with cytokine storm caused by pathogen infection and radiotherapy and chemotherapy, and has the advantages of clear mechanism of action and target, specific action, and simultaneous broad inhibition of multiple inflammatory factors.

[0107] 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 oleanolic acid OA monomer, characterized in that: Its structural formula is as follows: The oleanolic acid OA monomer has the ability to specifically inhibit the interaction between menin and SETD2 protein, and significantly inhibits the expression of pathogen-induced inflammatory factors through this mechanism.

2. Use of the oleanolic acid OA monomer as claimed in claim 1 in the preparation of drugs for treating cytokine storm syndrome caused by pathogen infection.

3. Use of the oleanolic acid OA monomer as claimed in claim 1 in the preparation of a drug for inhibiting the expression of pathogen-induced inflammatory factors, in particular in the preparation of a drug for treating or preventing acute pneumonia activated by LPS or S-Pro.

4. The use according to claim 2 or 3, characterized in that The pathogens include but are not limited to the novel coronavirus SARS-CoV-2, influenza virus, bacteria and mycoplasma. The oleanolic acid OA monomer can dose-dependently inhibit the abnormal expression of IL-1α and IL-6 cytokines in macrophages activated by the S protein of SARS-CoV-2 and bacterial lipopolysaccharide LPS.

5. Use of the oleanolic acid OA monomer as claimed in claim 1 in the preparation of a drug for treating radio- and chemotherapy-induced pneumonia.

6. The use according to claim 5, characterized in that The oleanolic acid OA monomer can dose-dependently inhibit the expression of cell senescence inflammatory factors induced by ionizing radiation IR or cisplatin DDP, and improve the acute inflammatory response caused by normal lung tissue damage caused by radiotherapy and chemotherapy.

7. Use of the oleanolic acid OA monomer as claimed in claim 1 in the preparation of a drug for inhibiting menin / SETD2 protein interaction.

8. Use of the oleanolic acid OA monomer as claimed in claim 1 in the preparation of a drug for inhibiting the expression of inflammatory factors.

9. Use of the oleanolic acid OA monomer as claimed in claim 1 in the preparation of a medicament for improving acute lung injury.

10. A pharmaceutical composition for treating acute pneumonia, characterized in that: The invention comprises the oleanolic acid OA monomer according to claim 1 as an active ingredient, and a pharmaceutically acceptable carrier, excipient or diluent.

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