Macrophage-derived inflammatory factor inhibitory lead compound and application thereof

Abstract

The invention discloses a macrophage-derived inflammatory factor inhibitory lead compound and application thereof, and relates to biological medicine. The lead compound is obtained by screening from a ChenDiv lead compound library by taking a menin / SETD2 interaction structural domain as a target spot, and is used for specifically inhibiting the interaction between menin and SETD2, preferably MSI-1-MSI-8. The SET functional structure domain of the SETD2 can be specifically combined with the SET functional structure domain of the SETD2. The invention also discloses an application of the lead compound in preparation of macrophage over-activation related inflammation regulation drugs, the inflammation comprises cytokine storm related inflammation, acute lung injury related inflammation and the like, and the drugs can play a role by inhibiting H3K36me3 expression and macrophage M1 type polarization. The lead compound is high in targeting property and wide in anti-inflammatory spectrum, and a new effective active component is provided for treatment of diseases such as cytokine storm and acute lung injury.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a class of macrophage-derived inhibitory lead compounds that target the menin / SETD2 interaction, and the application of such compounds in the preparation of drugs for regulating inflammation related to macrophage overactivation. Background Technology

[0002] Cytokine storm (CS) is a severe abnormal immune response triggered by pathogen infection or cell therapy, characterized by the abnormal secretion of large amounts of cytokines / inflammatory factors / chemokines such as GM-CSF, IL-1α, and IL-6 by immune cells. CS clinically presents with persistent fever, splenomegaly, hepatomegaly with liver failure, acute respiratory distress syndrome, and multiple organ failure, with an extremely high mortality rate. CS-related diseases can be divided into cytokine storm syndrome (CSS) and cytokine release syndrome (CRS). CSS is caused by pathogen infection leading to abnormal activation of the body's immune system, promoting a sharp increase in inflammatory cytokines; CRS is related to increased release of IL-6, IL-10, TNF-α, GM-CSF, and IFN-γ caused by CAR-T cell therapy.

[0003] Currently, there are significant limitations to clinical anti-inflammatory drugs for CS: glucocorticoids, as broad-spectrum immunosuppressants, can inhibit inflammatory responses but weaken the body's ability to clear pathogens, increasing patient mortality and secondary infection rates, and are accompanied by side effects such as hyperglycemia and hypertension. Tocilizumab, an antagonist of the cytokine IL-6 receptor, is a newly developed potential drug for the clinical treatment of CS. However, tocilizumab only antagonizes the single cytokine IL-6 and has no inhibitory effect on multiple other inflammatory factors, so its efficacy is limited, and its efficacy needs further clinical trials to verify (Cytokine Storm Caused by Novel Coronavirus and Its Drug Treatment. Chinese Pharmaceutical Journal, 55(5), pages 333–336 (2020)). Antagonists targeting the SARS-CoV-2 S protein receptor angiotensin-converting enzyme 2 (ACE2) are in clinical trials. These inhibitors block viral infection of target cells by antagonizing the binding of SARS-CoV-2 to ACE2, thus suppressing cytokine storms (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, in addition to the ACE2 receptor, SARS-CoV-2 can also infect monocytes / macrophages via the cell surface Fcγ receptor with antibody assistance, releasing large amounts of cytokines and inducing excessive inflammatory responses, leading to CS (FcγR-mediated SARS-CoV-2 infection of monocytesactivates inflammation. Nature, 606, pages 576–584 (2022)). In summary, current CS treatment lacks specific drugs with clear efficacy based on pathogenesis, and novel targeted drugs are urgently needed.

[0004] Alveolar macrophages (AMs) are the first-line defensive immune cells of the alveoli, playing a crucial physiological role in maintaining alveolar surfactant homeostasis and clearing pathogens. However, overactivated AMs release large amounts of pro-inflammatory cytokines, such as tumor cytokine-α (TNF-α) and interleukin-6 (IL-6), triggering a cytokine storm (CS) that ultimately leads to acute respiratory distress syndrome (ARDS). Therefore, elucidating the molecular mechanisms of AM overactivation and identifying key targets regulating this process is of significant theoretical and clinical value for the prevention and treatment of related diseases.

[0005] The MEN1 gene encodes the nuclear protein menin, which participates in various biological processes, including gene transcription regulation, DNA damage repair, and cell proliferation and differentiation. Menin is a key scaffold protein for the directed recruitment of MLL fusion proteins to chromatin. Menin binds directly to the N-terminus of MLL and is crucial for the recruitment of MLL fusion proteins to chromatin targets (such as the HOX locus), an indispensable step in the malignant transformation of leukemia. SETD2 catalyzes the trimethylation of histone H3 at position 36 (H3K36me3), and is currently the only known histone H3K36-specific trimethyltransferase. SETD2 plays an important role in DNA replication, damage repair, and gene expression regulation by mediating H3K36me3 modification.

[0006] Previous research in this invention revealed that menin is a key scaffold protein mediating H3K36me3 covalent modification mediated by the histone methyltransferase SETD2. Menin directly interacts with SETD2, maintaining GM-CSF transcription and regulating macrophage maturation, differentiation, and activation through H3K36me3. Furthermore, it was confirmed that pathogen model receptors such as the SARS-CoV-2 spike protein and bacterial lipopolysaccharide (LPS) activate the expression of various cytokines / inflammatory factors, including IL-1α and IL-6, by promoting menin / SETD2 interaction and its recruitment at specific chromatin sites, as well as H3K36me3 histone remodeling, thus acting as a key node in cytokine / inflammatory storms. Based on this core mechanism, this invention screened and identified lead compounds that specifically inhibit menin / SETD2 interaction, providing a new strategy for the regulation of CS-related inflammation. Summary of the Invention

[0007] The purpose of this invention is to address the problems of unclear targets, poor specificity, narrow anti-inflammatory spectrum, or significant side effects of existing cytokine storm (CS) treatment drugs, and to provide a class of macrophage-derived inhibitory lead compounds that target the menin / SETD2 interaction and their applications.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0009] A first aspect of the present invention provides screening for inhibitory lead compounds of macrophage-derived inflammatory factors:

[0010] Using the minimal domain of the menin / SETD2 interaction as the target, 100 potential lead compounds that inhibit the menin / SETD2 interaction were screened in the ChemDiv library of 50,000 lead compounds through high-throughput screening. Based on ADME druggability analysis, lead compounds with potential side effects were excluded, and finally, 8 lead compounds that specifically inhibit the menin / SETD2 interaction and have low potential side effects were screened and named MSI-1, MSI-2, MSI-3, MSI-4, MSI-5, MSI-6, MSI-7, and MSI-8, respectively.

[0011]

[0012] The lead compound specifically binds to the SET functional domain (amino acids 1400-1800) of the SETD2 protein and does not bind directly to the menin protein.

[0013] The binding site of the lead compound to the SETD2 protein includes at least one amino acid residue from PHE-1635, GLN-1632, TYR-1561, TYR-1535, and ILE-1620.

[0014] The binding free energy of the complex formed by the lead compound and the SETD2 protein is ≤-24 kcal / mol, and the dissociation constant (Kd value) is ≤50 μM.

[0015] A second aspect of the present invention provides the use of the lead compound in the preparation of drugs for regulating inflammation related to macrophage overactivation.

[0016] The inflammation is either cytokine storm-related inflammation or acute lung injury-related inflammation.

[0017] The drug can inhibit the expression of one or more inflammatory factors among IL-1β, IL-6, TNF-α, IL-8, IL-18, and CCL2.

[0018] The drug exerts its inflammatory regulatory effect by inhibiting H3K36me3 protein expression and chromatin recruitment, and inhibiting macrophage M1 polarization.

[0019] The inflammation is induced by lipopolysaccharide, Streptococcus pneumoniae, or viral infection.

[0020] A third aspect of the present invention provides a pharmaceutical composition comprising the aforementioned lead compound, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0021] The dosage form of the pharmaceutical composition is an injection, aerosol, inhaler, or oral preparation.

[0022] Compared with the prior art, the advantages and technical effects of the present invention are as follows:

[0023] The theoretical basis of this invention is the epigenetic molecular mechanism by which pathogens promote the expression of a series of inflammatory factors / cytokines / chemokines through H3K36m3 reprogramming by activating the formation of the menin / SETD2 complex and chromatin-directed recruitment. Therefore, lead compounds that competitively inhibit the menin / SETD2 interaction directly inhibit the expression of CS-related inflammatory factors / cytokines / chemokines at the transcriptional level by specifically antagonizing the menin / SETD2 interaction. This invention screened and obtained eight lead compounds (MSI-1~MSI-8) with high specificity and low potential side effects, targeting the key structural domain of the interaction between menin and SETD2, thus solving the problems of poor targeting and narrow anti-inflammatory spectrum of existing drugs. This invention specifically binds to the SET functional domain of SETD2, blocking the formation of the menin / SETD2 complex, thereby inhibiting the transcription of inflammatory factors mediated by H3K36me3 chromatin reprogramming, and regulating the inflammatory response from the source. Experiments have confirmed that the lead compounds can effectively inhibit the expression of various inflammatory factors and alleviate tissue damage caused by excessive activation of macrophages, providing new active ingredients for the preparation of regulatory drugs for diseases such as CS and acute lung injury. Attached Figure Description

[0024] Figure 1 This presents the results of high-throughput screening of the lead compound library. A shows the high-throughput screening flowchart; B shows the fluorescence polarization assay used to detect the inhibitory effect of eight lead compounds on the binding of menin to SETD2.

[0025] Figure 2 The results show the validation of the lead compound's inhibition of the menin / SETD2 interaction. A: GST-pulldown experimental results; B~C, H~M: Co-IP / Western Blot experimental results; D: Endogenous Co-IP experimental results; E~G: CETSA experimental results.

[0026] Figure 3 The results show the specific binding verification of MSI-1 and SETD2. A: Schematic diagram of molecular docking; B: Enlarged view of the binding mode; C: Table of molecular docking parameters; D-E: MST experimental results; F: DSF experimental results.

[0027] Figure 4 The results are from molecular dynamics simulations of the MSI-1 / SETD2 complex. A: RMSD curve; B: SASA curve; C: Hydrogen bond number variation curve; D: Rg curve; E: RMSF curve; F: Binding free energy analysis results.

[0028] Figure 5 The results show the effects of the lead compound on the inhibition of LPS-induced inflammatory cytokine expression in AM cells. Specifically, A~D: the effects of MSI-1~MSI-4 on the relative expression levels of IL-6, TNF-α, and IL-1β.

[0029] Figure 6 The results show the effects of MSI-1 in alleviating LPS-induced acute lung injury in mice. A: Schematic diagram of the experimental procedure; B: Gross morphology of lung tissue; C: Quantitative analysis of protein concentration in bronchoalveolar lavage fluid; D: Wet-to-dry weight ratio of lung tissue; E-F: Relative expression levels of inflammatory factors; G: Results of HE, IHC, and PAS staining.

[0030] Figure 7 The results show the effects of MSI-1 in alleviating SP-induced acute lung injury in mice. A: Schematic diagram of the experimental procedure; B: Gross morphology of lung tissue; C: Quantitative analysis of protein concentration in bronchoalveolar lavage fluid; D: Wet-to-dry weight ratio of lung tissue; E-F: Relative expression levels of inflammatory factors; G: Results of HE, IHC, and PAS staining.

[0031] Figure 8 Results are for verifying the mechanism of action of the lead compound. A: Western blotting detection of H3K36me3 expression; B: ChIP results; C: Flow cytometry detection of macrophage polarization. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the following embodiments will be used in conjunction with the accompanying drawings to further illustrate the invention. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0033] Example 1: High-throughput screening of lead compounds and ADME druggability analysis

[0034] This embodiment involves high-throughput screening of small molecule inhibitors targeting the menin / SETD2 interaction from the ChemDiv lead compound library, followed by ADME druggability analysis. The specific procedure is as follows: Figure 1 As shown in A: Previous studies identified the 231-248 fragment as the minimal menin domain interacting with SETD2, and verified the high conservation of this fragment in species evolution (Patent Application No.: 2024119120475). Based on this key interacting fragment, high-throughput screening was performed using the ChemDiv lead compound library. The purified SET protein and the FITC-labeled menin 231-248 peptide were added to 384-well plates, and the ChemDiv lead compound library (containing 50,000 compounds) was used to screen for potential targeting inhibitors. Figure 1(A) After screening, the fluorescence polarization values ​​of FITC-labeled menin 231-248 peptide, SET protein, and eight lead compounds were detected by fluorescence polarization assay. The chemical structures of the eight lead compounds are as follows:

[0035]

[0036] Eight lead compounds with high specificity and low potential side effects were selected through high-throughput screening using ADME druggability analysis. These compounds were named MSI-1, MSI-2, MSI-3, MSI-4, MSI-5, MSI-6, MSI-7, and MSI-8, respectively. ADME druggability analysis was then performed on these eight lead compounds to evaluate their high specificity and low potential side effects. The results are summarized in Table 1. This example demonstrates the successful screening of multiple lead compounds (MSI-1 to MSI-8) from the compound library.

[0037] Table 1

[0038]

[0039] Example 2: Verification of the lead compound's inhibition of the menin / SETD2 interaction

[0040] The inhibitory effect of the lead compound obtained in Example 1 on the menin / SETD2 interaction was verified by various molecular biology experiments. The specific results are as follows:

[0041] MSI-1 inhibitory effect verification: GST-pull down confirmed that MSI-1 specifically disrupts the direct interaction between menin and SET protein ( Figure 2 (A) FLAG-SET and HA-MEN1 plasmids were co-transfected into 293T cells. HA-tag immunoprecipitation of menin protein was performed. Western blot analysis confirmed that MSI-1 (0, 7.5, 15, 30, 60 μM) dose-dependently inhibited the interaction between menin and SET proteins. Figure 2 (B in the text); FLAG-SET and HA-MEN1 plasmids were co-transfected into 293T cells, and SET protein was co-precipitated by FLAG-tag immunoprecipitation. Western blot analysis confirmed that MSI-1 dose-dependently inhibited the interaction between menin and SET protein. Figure 2 In vitro culture of C57BL6 / J mouse BMDM cells, followed by induction into macrophages with M-CSF, and then addition of MSI-1 (0, 6.25, 12.5, 25 μM), showed that MSI-1 gradient inhibition of the interaction between menin and SETD2 (C). Figure 2 (D in the middle)

[0042] MSI-1 protein binding specificity verification: 293T cells were transfected with SETD2 plasmid, and the thermostability of MSI-1 binding to SETD2 protein was detected by cell thermal displacement assay. The results showed that (50 μM) MSI-1 inhibited the degradation of SETD2 protein induced by increasing temperature, suggesting a direct interaction between MSI-1 and SETD2 protein. Figure 2 (E in the text); SETD2 plasmid transfected into 393T cells, and cell thermal displacement assay was used to detect the dose-dependent thermostability of MSI-1 binding to SETD2 protein at different concentrations. The results showed that the thermostability of SETD2 protein gradually increased with increasing MSI-1 dose, suggesting a direct interaction between MSI-1 and SETD2 protein. Figure 2 The MEN1 plasmid was transfected into 293T cells, and the thermostability of MSI-1 binding to menin protein was detected by cell thermal displacement assay. The results showed that MSI-1 (50 μM) had no significant effect on the thermostability of menin protein, suggesting that MSI-1 and menin protein do not interact directly. Figure 2 (G in the middle)

[0043] Verification of the inhibitory effects of MSI-2, MSI-3, and MSI-4: 293T cells were co-transfected with FLAG-SET and HA-MEN1 plasmids. Menin protein was co-precipitated using HA-tag immunoprecipitation. Western blot analysis confirmed that MSI-2, MSI-3, and MSI-4 (0, 1, 6.25, 12.5, 25, 50 μM) dose-dependently inhibited the interaction between menin and SET proteins. Figure 2 The inhibitory effects of HM in MSI-5 to MSI-8 were similarly verified.

[0044] The results above demonstrate that the selected lead compounds directly inhibit the menin / SETD2 interaction.

[0045] Example 3: Specific interaction analysis between MSI-1 and SETD2 proteins

[0046] This embodiment verifies the specific interaction between MSI-1 and the SETD2 protein through molecular docking and biophysical experiments. MSI-1 was molecularly docked with the SETD2 target protein using Schrodinger Maestro software. The results show that MSI-1 and SETD2 protein have a good binding affinity and a high degree of matching. Figure 3(A) The complex formed by MSI-1 and SETD2 protein after docking was visualized using Pymol 2.1 software to obtain the binding mode of MSI-1 and SETD2 protein. Figure 3 (B in the text); combined with pattern analysis, it was found that MSI-1 forms strong hydrogen bond interactions with PHE-1635 and GLN-1632 of the SETD2 protein active site, which makes an important contribution to anchoring small molecules in the protein pocket; since MSI-1 can form van der Waals interactions with surrounding residues, it makes an important contribution to stabilizing the natural product. These interactions can effectively promote the formation of a stable complex between MSI-1 and the protein. Molecular docking results showed that the binding energy was -9.53 kcal / mol (< -7 kcal / mol) ( Figure 3 (C in the text); EGFP-SET plasmid transfected 293T cells, cells were lysed with RIPA, and the protein solution was subjected to microscale thermophoresis (MST) with MSI-1 (1.25 mM). The results showed that MSI-1 specifically bound to SET protein with good affinity, and the Kd value was 41.5 μM (C in the text). Figure 3 (D in the text); EGFP-MEN1-Palm plasmid transfected 293T cells, cells were lysed with RIPA, and the protein solution was subjected to microscale thermophoresis (MST) with MSI-1 (1.25 mM). The results showed that MSI-1 did not bind to menin-Palm protein. Figure 3 The purified SET protein was incubated with FITC-HNS-Fluorescein at room temperature using differential scanning fluorometry (DSF) with fluorescent labeling. The temperature melting curve was detected by quantitative real-time PCR. The results showed that the MSI-1 small molecule inhibitor specifically bound to the SET protein and stabilized the SET protein in a dose-dependent manner. Figure 3 (F in the text). The above results indicate that MSI-1 specifically interacts with the SETD2 protein.

[0047] Example 4: Molecular dynamics simulation analysis of the interaction between MSI-1 and SETD2 proteins

[0048] In this embodiment, Gromacs 2020 software was used to perform 100 ns molecular dynamics simulations on MSI-1 and SETD2 to analyze the interaction stability. The RMSD plots show that the average RMSD of the complexes is less than 3.5 Å, and the complexes reach dynamic equilibrium around 10 ns. Figure 4As shown in Figure A), the SASA plot reveals a significant decrease in the accessible surface area of ​​the complex. The binding of MSI-1 to SETD2 protein did not impair the protein's stability; rather, it improved it. This indirectly indicates that MSI-1 and SETD2 bind well. Figure 4 (B in the text); Statistical analysis of the changes in the number of hydrogen bonds between the protein and MSI-1 throughout the simulation revealed that MSI-1 can form one or more hydrogen bonds with protein pocket amino acids, and these hydrogen bonds play an important role in stabilizing the MSI-1 and SETD2 proteins. Figure 4 (C in the text); Analysis of the relative compactness and stability induced by the binding of the complex at major and minor sites, and measurement of the target protein's radius of gyration (Rg), revealed a slight decrease in the Rg of the SETD2 protein, suggesting that the binding of the SETD2 protein to MSI-1 promotes the maintenance of more hydrophobic contacts and the formation of more effective interactions within the protein (C in the text). Figure 4 (D in the diagram); As can be seen from the RMSF plot, a small number of amino acids in the complex formed by the interaction of SETD2 protein and MSI-1 undergo significant conformational changes (around 1450). This is mainly because these amino acids are located in the hinge region of the protein, which is inherently more flexible, and their conformation changes somewhat during the simulation. However, the conformational changes of most amino acids are within an acceptable range. Figure 4 The binding free energy of MSI-1 to SETD2 protein is -24.5 + / -1.35 kcal / mol, with van der Waals forces playing a major role (-45.43 + / -0.9 kcal / mol). This indicates that MSI-1 can stably remain in the protein site pocket and interact strongly with surrounding residues through van der Waals forces. Due to the effective hydrogen bonding between MSI-1 and the protein pocket, electrostatic interactions also contribute significantly to the stability of the complex (-10.18 + / -0.04 kcal / mol). In summary, MSI-1 has a strong affinity for SETD2 protein, which promotes the formation of a stable complex, thereby exerting its active effect. Figure 4 (F in the text).

[0049] Example 5: Lead compound inhibits LPS-induced expression of macrophage inflammatory factors.

[0050] This embodiment verifies the inhibitory effect of the lead compound on the expression of inflammatory factors through cell experiments. Mouse alveolar macrophages were treated with LPS (100 ng / ml) and MSI-1 (0, 1, 6.25, 12.5, 25, 50 μM) for 24 hours, and the cells were harvested. 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 MSI-1 (0, 1, 6.25, 12.5, 25, 50 μM) dose-dependently inhibited the LPS-induced upregulation of these inflammatory factors. Figure 5 AM cells were treated with LPS (100 ng / ml) and MSI-2 (0, 1, 6.25, 12.5, 25, 50 μM) for 24 hours, 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 MSI-2 (0, 1, 6.25, 12.5, 25, 50 μM) dose-dependently inhibited the LPS-induced upregulation of these inflammatory factors. Figure 5 Mouse alveolar macrophages were treated with LPS (100 ng / ml) and MSI-3 (0, 1, 6.25, 12.5, 25, 50 μM) for 24 hours, 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 MSI-3 (0, 1, 6.25, 12.5, 25, 50 μM) dose-dependently inhibited the LPS-induced upregulation of these inflammatory factors. Figure 5 Mouse alveolar macrophages were treated with LPS (100 ng / ml) and MSI-4 (0, 1, 6.25, 12.5, 25, 50 μM) for 24 hours, 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 MSI-4 (0, 1, 6.25, 12.5, 25, 50 μM) dose-dependently inhibited the LPS-induced upregulation of these inflammatory factors. Figure 5 (D in the text). Specifically, MSI-1, MSI-2, MSI-3, and MSI-4 all showed significant inhibitory effects, and experiments confirmed that these lead compounds effectively alleviated the inflammatory response at the cellular level.

[0051] Example 6: MSI-1 alleviates LPS-induced acute lung injury

[0052] This embodiment evaluates the therapeutic effect of MSI-1 on LPS-induced acute lung injury through animal experiments. An acute lung inflammation and injury model was induced in C57BL / 6J mice by intratracheal instillation of LPS (5 mg / kg). Figure 6(A) ; After 24 hours, the mice were sacrificed and their lungs were dissected. The lung tissue showed extensive hemorrhage and edema. Intratracheal instillation of 1 mg / kg and 10 mg / kg MSI-1 significantly improved the above symptoms. Figure 6 (B in the text); BCA method was used to determine protein concentration in bronchoalveolar lavage fluid. The results showed that LPS-induced alveolar protein deposition was significant in mice with acute lung injury, while MSI-1 treatment significantly improved LPS-induced alveolar protein deposition (B in the text). Figure 6 C); In mice with acute lung inflammation, the wet / dry weight ratio of lung tissue was significantly increased, while that in the treatment group was significantly decreased. Figure 6 (D in the text); primary isolated alveolar macrophages, RNA extracted using the Trizol method, and RT-qPCR detection of cytokine expression in mouse alveolar macrophages. The results showed that LPS significantly activated the expression of inflammatory factors such as IL-1β, TNF-α, IL-6, and IL-18, while MSI-1 significantly inhibited LPS-induced inflammatory factor expression in alveolar macrophages. Figure 6 (E in the text); RNA was extracted from lung tissue using the Trizol method, and cytokine expression in mouse alveolar macrophages was detected by RT-qPCR. The results showed that LPS significantly activated the expression of inflammatory factors / chemokines such as IL-1β, TNF-α, IL-6, and IL-18, while MSI-1 significantly inhibited LPS-induced expression of inflammatory factors / chemokines in lung tissue parenchymal cells (…). Figure 6 The results of hematoxylin-eosin (HE) staining of mouse lung tissue showed that the LPS group had obvious acute inflammation, diffuse inflammatory cell infiltration in the alveolar cavity and pulmonary interstitium, widening of lung septa, rupture of alveolar septa in some areas, alveolar atrophy, and even structural loss, while the MSI-1 treatment group significantly inhibited the lung damage induced by LPS; the results of immunohistochemical staining (IHC) for F4 / 80, CD4, and CD11b specific antibodies showed that the LPS group had increased infiltration of macrophages, CD4-positive T cells, and neutrophils, while the MSI-1 treatment group significantly improved macrophage infiltration; the results of glycogen (PAS) staining showed that the LPS group had increased glycogen accumulation, while the MSI-1 treatment group had decreased glycogen accumulation. Figure 6 (G in the text). Experimental results show that MSI-1 can effectively alleviate LPS-induced acute lung injury.

[0053] Example 7: MSI-1 alleviates pneumococcal (SP)-induced acute lung injury

[0054] This embodiment evaluates the effect of MSI-1 on SP-induced acute lung injury. C57BL / 6J mice were administered SP via intratracheal instillation (10... 6 CFU / ml) induced acute lung inflammation injury model ( Figure 7(A) After 48 hours, mice were sacrificed and their lungs were dissected. The examination revealed extensive hemorrhage and edema in the lung tissue. Intratracheal infusion of 1 mg / kg and 10 mg / kg MSI-1 significantly improved these symptoms. Figure 7 (B in the text); BCA method was used to determine protein concentration in bronchoalveolar lavage fluid. The results showed that SP acute lung injury mice had significant alveolar protein deposition, while MSI-1 treatment significantly improved SP-induced alveolar protein deposition (B in the text). Figure 7 (C); In mice with acute lung inflammation, the wet / dry weight ratio of lung tissue was significantly increased, while that in the treatment group was significantly decreased. Figure 7 (D in the text); Primary alveolar macrophages were isolated, RNA was extracted using the Trizol method, and cytokine expression in mouse alveolar macrophages was detected by RT-qPCR. The results showed that SP significantly activated the expression of inflammatory factors such as IL-1β, TNF-α, IL-6, and CCL2, while MSI-1 significantly inhibited the expression of inflammatory factors in alveolar macrophages induced by SP. Figure 7 (E in the text); RNA was extracted from lung tissue using the Trizol method, and cytokine expression in mouse alveolar macrophages was detected by RT-qPCR. The results showed that SP significantly activated the expression of inflammatory factors / chemokines such as IL-1β, TNF-α, IL-6, and CCL2, while MSI-1 significantly inhibited SP-induced expression of inflammatory factors / chemokines in lung tissue parenchymal cells (…). Figure 7 The results of hematoxylin-eosin (HE) staining of mouse lung tissue showed that the SP group had obvious acute inflammation, diffuse inflammatory cell infiltration in the alveolar cavity and pulmonary interstitium, widening of lung septa, rupture of alveolar septa in some areas, alveolar atrophy, and even structural loss, while the MSI-1 treatment group significantly inhibited the lung damage induced by SP; the results of immunohistochemical staining (IHC) of F4 / 80, CD4, and CD11b specific antibodies showed that the SP group had increased infiltration of macrophages, CD4-positive T cells, and neutrophils, while the MSI-1 treatment group significantly improved macrophage infiltration; the results of glycogen (PAS) staining showed that the SP group had increased glycogen accumulation, while the MSI-1 treatment group had decreased glycogen accumulation. Figure 7 (G in the text). Experimental results confirmed that MSI-1 has a protective effect against SP-induced acute lung injury.

[0055] Example 8: MSI-1 inhibits H3K36me3 protein expression and macrophage M1 polarization

[0056] This example explores the mechanism of action of MSI-1. BMDM cells were treated with LPS (100 ng / ml) and MSI-1 (25 μM) for 24 hours, and then the cells were harvested. Proteins were extracted using RIPA. Western blotting results showed that LPS significantly promoted the expression of menin, SETD2, and H3K36me3 proteins, while MSI-1 inhibited LPS-induced H3K36me3 protein expression. Figure 8 BMDM cells were treated with LPS (100 ng / ml) and MSI-1 (25 μM) for 24 hours, and the cells were harvested. Chromatin immunoprecipitation (ChIP) experiments showed that MSI-1 inhibited the binding of H3K36me3 to Csf2. Figure 8 BMDM cells were treated with LPS (100 ng / ml) and INF-γ (20 ng / ml) for 48 hours before cell harvesting. Flow cytometry experiments showed that MSI-1 significantly inhibited LPS-induced macrophage M1 polarization. Figure 8 (C in the text). Experimental results show that MSI-1 inhibits the inflammatory response by regulating H3K36me3-mediated histone reprogramming and macrophage polarization.

[0057] Experiments show that the various lead compounds provided in this invention effectively inhibit the menin / SETD2 interaction: at the cellular level, they effectively inhibit the expression of multiple cytokines such as IL-1α, IL-1β, IL-6, and IL-8 in LPS-induced acute lung injury (AM); at the animal level, they significantly inhibit the expression of multiple inflammatory factors induced by LPS and Streptococcus pneumoniae (SP), alleviating acute lung injury in mice. The small molecule targeted inhibitors provided in this invention demonstrate their application potential in the treatment of diseases related to cytokine storms caused by abnormal macrophage activation, possessing advantages such as clear target, specific action, and broad inhibition of multiple inflammatory factors.

[0058] This invention, based on the theoretical understanding that the menin / SETD2 complex promotes the expression of cytokines / inflammatory factors / chemokines such as IL-1α and IL-6 through chromatin H3K36me3 histone reprogramming, develops small molecule lead compounds that specifically inhibit the expression of key inflammatory factors such as IL-1α and IL-6 by blocking the interaction between menin / SETD2 proteins. This invention utilizes the ChemDiv lead compound library for high-throughput screening to identify small molecule lead compounds MSI-1, MSI-2, MSI-3, MSI-4, MSI-5, MSI-6, MSI-7, and MSI-8 that specifically block the menin / SETD2 interaction. A series of biochemical experiments and bioinformatics analyses confirmed that the lead compounds specifically antagonize the menin / SETD2 interaction. Cellular and animal experiments demonstrated that the lead compounds effectively inhibit the expression of inflammatory factors induced by LPS and Streptococcus pneumoniae, significantly alleviating their induced acute lung injury. The lead compounds provided by this invention have significant advantages such as clear target, high efficiency and specificity, and significant therapeutic effects.

[0059] In summary, this invention screened a variety of lead compounds targeting the menin / SETD2 interaction and verified their potential application in the treatment of cytokine storm syndrome caused by macrophage overactivation.

[0060] The above embodiments are merely preferred embodiments of the present invention and should not be considered as limiting the scope of the present invention. All equivalent variations and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. Use of macrophage-derived inhibitory lead compounds for the prevention and / or treatment of diseases mediated by cytokine storms; wherein, The lead compound is at least one of MSI-1, MSI-2, MSI-3, MSI-4, MSI-5, MSI-6, MSI-7, and MSI-8, with the following molecular formula: The lead compound was obtained through high-throughput screening of the ChemDiv lead compound library.

2. The use according to claim 1, characterized in that, The screening method includes the following steps: 1) The minimum menin domain interacting with SETD2 was identified as segment 231-248, and its evolutionary conservation was verified; 2) The purified SET protein and FITC-labeled menin 231-248 peptide were added to a 384-well plate and then added to the ChemDiv lead compound library for high-throughput screening. 3) The binding activity of the compound with menin / SETD2 was verified by fluorescence polarization experiments; 4) Perform ADME drug development analysis to screen for lead compounds with high specificity and low potential side effects.

3. The use according to claim 1, characterized in that, The diseases mediated by cytokine storms are acute respiratory distress syndrome caused by pathogen infection or bacterial-induced acute lung injury.

4. The use according to claim 3, characterized in that, The pathogen is a bacterium; preferably, the bacterium is lipopolysaccharide or Streptococcus pneumoniae.

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