Novel use of drug for intervention in IPMK target

Abstract

A novel use of a drug for intervention in an IPMK target. It is found for the first time that HIF2α and GATA3 jointly regulate IPMK and a PI3K-AKT signaling pathway to mediate pathogenic Th2 cell differentiation, and it is verified for the first time that intervention in the IPMK target enables prevention and treatment of type II immune diseases and diseases caused by dysregulated Th2 / ILC2 differentiation and acts against tumors, aging and aging related diseases, thereby providing a new potential target for clinical treatment of type II immune diseases, tumors, diseases caused by dysregulated Th2 / ILC2 differentiation, and aging and aging related diseases, and achieving good prospects for application.

Description

A novel use of drugs targeting IPMK Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a novel use of a drug that intervenes in the IPMK target. Background Technology

[0002] Type II immune diseases are inflammations primarily mediated by Th2 cells, ILC2 cells, and related cytokines. Currently, type II inflammation is considered an immunopathological mechanism for a variety of diseases involving multiple systems such as the skin, respiratory, and digestive systems. This type of reaction is characterized by sensitivity to a variety of allergens, rapid response, and distribution in various barrier tissues.

[0003] Asthma is a common type II allergic immune disease affecting approximately 300 million people worldwide. It is primarily characterized by narrowing of the airways, excessive mucus production, tracheal wall remodeling, and dysregulation of both the innate and adaptive immune systems, leading to a hyperresponsiveness of the bronchi to harmless antigens such as pollen or cold air. Asthma patients often experience symptoms such as shortness of breath, wheezing, and chest tightness. Currently, conventional clinical treatments mainly utilize glucocorticoids and short- or long-acting β2-adrenergic agonists. Corticosteroids primarily suppress inflammatory responses in the bronchi, while β2-adrenergic agonists primarily open constricted bronchial smooth muscle. However, these treatments only control symptoms to a certain extent and do not cure asthma. Therefore, a deeper understanding of the pathogenesis of asthma and the identification of new therapeutic targets are important research topics in this field.

[0004] Th2 cells play a central role in allergic asthma. They are the main source of type 2 cytokines such as interleukin (IL)-4 and IL-13, which drive the immune and physiological characteristics of asthma, such as IgE-mediated inflammation, eosinophilia, airway hyperresponsiveness, and goblet cell proliferation. Th2 cells also interact with other T cell subsets, such as Th17 and Th1 cells, jointly participating in the pathogenesis of asthma. Furthermore, the role of Th2 cells in asthma is not limited to the immune response; they can also act as a bridge between inflammation and neuronal regulatory behavioral responses, and play an important role in epithelial differentiation and damage repair. Therefore, Th2 cells are a key factor in the pathogenesis of allergic asthma, and understanding their regulatory mechanisms and biological characteristics is crucial for developing new therapeutic strategies.

[0005] IPMK (inositol polyphosphokinase) is a multifunctional enzyme that plays a crucial role in the cell nucleus and is involved in various cellular signaling and metabolic regulatory processes. IPMK plays a key role in the differentiation and function of Th1 and Th17 cells. For example, IPMK regulates the differentiation and effector function of Th1 and Th17 cells by controlling the Akt-mTOR signaling pathway. Furthermore, IPMK deficiency leads to a significant reduction in the immune response of Th1 and Th17 cells, thereby affecting the host's resistance to certain pathogens.

[0006] Currently, there are no literature reports on the relationship between IPMK and Th2 cell differentiation and function, nor are there any literature reports on the relationship between IPMK and type II immune diseases. Summary of the Invention

[0007] To address the problems of existing technologies, this invention provides a novel use for drugs that intervene in IPMK targets.

[0008] The use of drugs that target IPMK in the preparation of drugs for the prevention and / or treatment of type II immune diseases and their complications, anti-tumor, anti-aging or anti-aging-related diseases, wherein the drugs that target IPMK are selected from IPMK inhibitors, drugs that inhibit IPMK gene expression, drugs that knock out or knock down the IPMK gene, and drugs that degrade IPMK protein.

[0009] Preferably, the IPMK inhibitor is selected from small molecule compounds, peptides, and antibodies, and more preferably vilazone, LI-2242, UNC7437, UNC9750, quercetin, and chlorogenic acid.

[0010] Preferably, the drug that inhibits IPMK gene expression is selected from circular RNA, antisense nucleic acid, small interfering nucleic acid, nucleic acid aptamer, small activating nucleic acid, micronucleic acid, mRNA drug, and ribozyme.

[0011] Preferably, the drug for knocking out or knocking down the IPMK gene can be a CRISPR / Cas9 gene editing system.

[0012] Preferably, the drug for degrading IPMK protein includes a protein degrading agent, and the protein degrading agent is preferably selected from PROTAC and molecular gels.

[0013] Preferably, the drug intervening in the IPMK target is an antibody-drug conjugate, which is formed by linking an antibody targeting the target cell with an active ingredient intervening in the IPMK target through a linker; preferably, the target cell is a Th2 cell and / or an ILC2 cell.

[0014] Preferably, the drug for treating type II immune diseases is a drug for treating Th2 cell-mediated immune diseases and / or ILC2 cell-mediated immune diseases.

[0015] Preferably, the drug is a drug that inhibits stem cell differentiation into Th2 cells, inhibits the differentiation of stem cell-like Th2 cell subsets into pathogenic Th2 cell subsets, and inhibits Th2 cells from secreting effector cytokines.

[0016] Preferably, the type II immune diseases include atopic dermatitis, chronic spontaneous urticaria, nodular prurigo, bullous pemphigoid, chronic sinusitis with or without nasal polyps, allergic rhinitis, asthma, allergic bronchopulmonary aspergillosis, chronic obstructive pulmonary disease, eosinophilic granulomatous polyangiitis, food allergy, eosinophilic esophagitis, allergic conjunctivitis, ulcerative colitis, lichen planus, and lymphedema.

[0017] Preferably, the complications of the type II immune disease include tissue fibrosis and organ fibrosis.

[0018] Preferably, the tumor includes prostate cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, gastric cancer, endometrial cancer, uterine cancer, brain cancer, liver cancer, bladder cancer, ovarian cancer, testicular cancer, head cancer, neck cancer, skin cancer, mesothelial carcinoma, leukocyte carcinoma, esophageal cancer, breast cancer, muscle cancer, connective tissue cancer, intestinal cancer, lung cancer, adrenal cancer, thyroid cancer, kidney cancer, or bone cancer; or glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma, choriocarcinoma, basal cell carcinoma of the skin, or seminoma of the testis.

[0019] Preferably, the aging includes nervous system aging, immune aging, tissue aging, cardiovascular system aging, and skin aging.

[0020] Preferably, the age-related diseases include sarcopenia, chronic low-grade inflammation, Alzheimer's disease, Parkinson's disease, neuromyelitis optica, chronic infection, age-related obesity, and cardiac fibrosis.

[0021] Use of IPMK agonists or drugs that overexpress IPMK in the preparation of drugs for the prevention and / or treatment of diseases caused by Th2 / ILC2 differentiation disorders.

[0022] Preferably, the IPMK agonist is a small molecule compound, peptide, or antibody capable of activating IPMK.

[0023] Preferably, the drugs for overexpressing IPMK include drugs for T-cell directed overexpression, drugs for adeno-associated virus-mediated overexpression, drugs for lentivirus-mediated overexpression, drugs for adenovirus-mediated overexpression, drugs for retrovirus-mediated overexpression, drugs for regulator-enhancer-induced overexpression, drugs for regulation-transcriptional-induced overexpression, drugs for regulation-transcriptional-element-induced overexpression, small molecule drug response systems for overexpressing genes, Cre-loxp systems, Flp-frt systems, and Dre-rox systems.

[0024] Preferably, the IPMK agonist or IPMK overexpressing drug is an antibody-drug conjugate, which is formed by linking an antibody targeting the target cell with an active ingredient via a linker; the active ingredient has IPMK agonist activity or IPMK overexpressing activity; preferably, the target cell is a Th2 cell and / or an ILC2 cell.

[0025] Preferably, the diseases caused by Th2 / ILC2 differentiation disorder include worm infection, obesity, obesity-related complications, primary immune thrombocytopenic purpura, rheumatoid arthritis, systemic lupus erythematosus, rheumatoid arthritis, type I diabetes, multiple sclerosis, chronic thyroiditis, and tumors.

[0026] Preferably, the obesity-related complications include diseases caused by abnormal accumulation of lipid droplets.

[0027] Preferably, the diseases caused by abnormal lipid droplet accumulation include fatty liver, insulin resistance, diabetes, cardiovascular and cerebrovascular diseases, hyperlipidemia, chronic kidney disease, atherosclerosis, coronary heart disease, heart failure, myocardial lipotoxicity, and metabolic syndrome.

[0028] Preferably, the tumor includes prostate cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, gastric cancer, endometrial cancer, uterine cancer, brain cancer, liver cancer, bladder cancer, ovarian cancer, testicular cancer, head cancer, neck cancer, skin cancer, mesothelial carcinoma, leukocyte carcinoma, esophageal cancer, breast cancer, muscle cancer, connective tissue cancer, intestinal cancer, lung cancer, adrenal cancer, thyroid cancer, kidney cancer, or bone cancer; or glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma, choriocarcinoma, basal cell carcinoma of the skin, or seminoma of the testis.

[0029] This invention is the first to discover that HIF2α and GATA3 co-regulate the IPMK and PI3K-AKT signaling pathways, mediating pathogenic Th2 cell differentiation. IPMK plays a crucial role in type II immune diseases, tumors, diseases caused by Th2 / ILC2 differentiation dysregulation, and aging and related diseases. This invention is the first to demonstrate that intervention on the IPMK target can be used for the prevention and treatment of type II immune diseases and diseases caused by Th2 / ILC2 differentiation dysregulation, as well as for anti-tumor, anti-aging, and anti-aging-related diseases. This invention provides a new potential target for the clinical prevention and treatment of type II immune diseases, anti-tumor, anti-aging and related diseases, and the treatment of diseases caused by Th2 / ILC2 differentiation dysregulation. This invention also provides new uses for existing drugs such as vilazorone for the prevention and treatment of type II immune diseases, particularly for asthma and allergic rhinitis.

[0030] definition:

[0031] "Medications for the prevention and / or treatment of type II immune diseases and their complications" refers to medications that can prevent type II immune diseases alone, treat type II immune diseases alone, prevent type II immune disease complications alone, or treat type II immune disease complications alone, or prevent type II immune diseases and their complications simultaneously, or treat and prevent type II immune diseases or their complications simultaneously.

[0032] IPMK inhibitors refer to any substance that inhibits IPMK activity. These substances can be small molecule compounds, peptides, or antibodies, including molecules that have been reported in the art to inhibit IPMK activity, such as vilazorone, LI-2242, UNC7437, and UNC9750.

[0033] Drugs that inhibit IPMK gene expression: These are any substances that can inhibit IPMK gene expression, including antisense nucleic acids (ASO), small interfering nucleic acids (siRNA), nucleic acid aptamers, small activating nucleic acids, micronucleic acids, mRNA drugs, ribozymes, etc.

[0034] Drugs that knock out or knock down the IPMK gene: Any substance that has the effect of knocking out or knocking down the IPMK gene, such as the CRISPR / Cas9 gene editing system.

[0035] Drugs that degrade IPMK: These refer to any substance that can degrade IPMK proteins, such as protein degrading agents. Protein degrading agents include Protac, molecular gels, etc.

[0036] IPMK agonists: These are any substances that inhibit IPMK activity. These substances can be small molecule compounds, peptides, or antibodies.

[0037] Drugs that overexpress IPMK: These are drugs that increase the expression level of IPMK in cells or organisms to a level higher than normal. These substances can be small molecule compounds, peptides, enzymes, nucleic acid drugs, and systems composed of them that have gene editing or gene expression functions.

[0038] Type II immune diseases are immune diseases primarily mediated by Th2 cells, ILC2 cells, and related cytokines.

[0039] Th2 cells: refers to helper T cells 2.

[0040] ILC2 cells: type 2 intrinsic lymphoid cells.

[0041] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0042] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0043] Figure 1 shows the experimental results of high HIF2α expression in Th2 cells compared to other types of CD4-positive helper T cells.

[0044] Figure 2 shows the experimental results of positive regulation of Th2 cell differentiation efficiency after overexpression of HIF2α during in vitro differentiation, compared with the control group.

[0045] Figure 3 shows the clustering and visualization results of helper T cells in asthma and sinusitis patients.

[0046] Figure 4 shows the experimental results of heterogeneity of Th2 cells in patients with asthma and sinusitis.

[0047] Figure 5 shows the experimental results of the positive correlation between the expression of Epas1 and the expression of pathogenic Th2 genes.

[0048] Figure 6 shows the experimental results of HIF2α deficiency inhibiting Th2 cell differentiation in vitro.

[0049] Figure 7 shows the experimental results of HIF2α deficiency reducing lung infiltrating cells in asthmatic mice.

[0050] Figure 8 shows the experimental results of HIF2α deficiency causing a decrease in BALF and total lung cell count.

[0051] Figure 9 shows the experimental results of reduced immune cell infiltration in the BALF of asthmatic mice caused by HIF2α deficiency.

[0052] Figure 10 shows the experimental results of reduced immune cell infiltration in the lungs of asthmatic mice caused by HIF2α deficiency.

[0053] Figure 11 shows the experimental results of HIF2α deficiency inhibiting Th2 cell differentiation in BALF of asthmatic mice.

[0054] Figure 12 shows the experimental results of HIF2α deficiency inhibiting Th2 cell differentiation in the lungs of asthmatic mice.

[0055] Figure 13 shows the experimental results of HIF2α deficiency inhibiting the expression of Th2 cell-related effector molecules.

[0056] Figure 14 shows the experimental results of reduced eosinophil infiltration in BALF caused by HIF2α deficiency.

[0057] Figure 15 shows the experimental results of reduced eosinophil infiltration in the lungs caused by HIF2α deficiency.

[0058] Figure 16 shows the experimental results of GATA3 knockout mice, where HIF2α was no longer induced after IL-4 stimulation, compared to the control group.

[0059] Figure 17 shows the experimental results of the luciferin reporter system and EMSA experiment demonstrating that GATA3 can bind to the HIF2α promoter DNA sequence.

[0060] Figure 18 shows the experimental results of chromatin immunoprecipitation q-PCR and CUT&Tag experiments demonstrating that GATA3 protein can bind to the HIF2α promoter DNA sequence.

[0061] Figure 19 shows the experimental results that simultaneous overexpression of HIF2α and GATA3 in 293T cells increases the expression levels of both.

[0062] Figure 20 shows the results of the Western blot experiment, which demonstrated that, compared with the control group mice, the expression level of GATA3 in T cells was reduced by T cell-specific knockout of HIF2α.

[0063] Figure 21 shows the experimental results of the luciferin reporter system demonstrating that the HIF2α protein can bind to the GATA3 promoter DNA sequence.

[0064] Figure 22 shows the experimental results of chromatin immunoprecipitation q-PCR and CUT&Tag experiments demonstrating that HIF2α protein can bind to the GATA3 promoter DNA sequence.

[0065] Figure 23 shows the experimental results of the CUT&Tag experiment demonstrating that HIF2α and GATA3 mainly bind to the promoter region of genes.

[0066] Figure 24 shows the experimental results of how HIF2α and GATA3 jointly maintain phospholipid metabolism and regulate T cell activation and differentiation.

[0067] Figure 25 shows the experimental results of HIF2α deficiency leading to a weakening of the PI3K-AKT signaling pathway in T cells responding to TCR stimulation.

[0068] Figure 26 shows the experimental results of HIF2α and GATA3 binding to the Ipmk promoter region to mediate transcriptional regulation.

[0069] Figure 27 shows the IPMK expression and AKT phosphorylation levels during Th2 differentiation compared to other CD4 groups. + The experimental results showed a significantly higher number of helper T cells.

[0070] Figure 28 shows the experimental results of Ipmk being specifically highly expressed in pathogenic Th2 cells, and the reduction of Ipmk expression levels in pathogenic Th2 cells due to HIF2α deficiency.

[0071] Figure 29 shows the experimental results of how overexpressing IPMK in HIF2α-deficient T cells can restore impaired Th2 differentiation.

[0072] Figure 30 shows the experimental results of how overexpressing IPMK in HIF2α-deficient T cells can restore weakened PI3K-AKT signaling and phosphatidylinositol levels.

[0073] Figure 31 shows the experimental results that treatment with the IPMK inhibitor vilazorone during in vitro differentiation can inhibit the differentiation of pathogenic Th2 cells, while having no effect on the differentiation of other helper T cells and other Th2 subsets.

[0074] Figure 32 shows the experimental results of how adding vilazorone to T cells during TCR stimulation can inhibit PI3K-AKT signaling.

[0075] Figure 33 shows the experimental results of reducing phosphatidylinositol levels in pathogenic Th2 cells by adding vilazorone during the exodifferentiation process.

[0076] Figure 34 shows the experimental results of reduced number of immune cells infiltrating the lungs and decreased tracheal wall thickening in mice treated with vilazodone after inducing asthma compared to the control group.

[0077] Figure 35 shows the CD45 infiltrates in the lungs of mice after vilazodone-induced asthma. +The experimental results showed that the relative proportions and absolute numbers of cells, eosinophils, and Th2 cells were lower than those in the control group mice.

[0078] Figure 36 shows the experimental results of reduced concentrations of IL-5 and IL-13 in the supernatant of bronchoalveolar lavage fluid in mice treated with vilazodone after inducing asthma compared to the control group. Detailed Implementation

[0079] In the following examples and experimental cases, reagents and raw materials not specifically described are all commercially available products.

[0080] Example 1 reveals that IPMK is a key regulatory target of HIF2a.

[0081] I. Experimental Methods

[0082] 1. Investigate the function of HIF2α in helper T cells

[0083] (1) Detection of HIF2α gene expression in Th0, Th1, Th2, Th17, and Treg cells:

[0084] The cells were isolated, and total RNA was extracted using the conventional phenol-chloroform method and reverse transcribed into cDNA. The differences in HIF2α mRNA expression levels in these cells were detected by qPCR.

[0085] (2) Overexpression of HIF2α during in vitro differentiation to detect the differentiation efficiency of various CD4-positive helper T cells:

[0086] In vitro, HIF2α-coated retroviral vectors were used to infect differentiating CD4-positive T cells, including Th1 (IL-2, IL-12, anti-IL-4), Th2 (IL-2, IL-4, anti-IFNgama), Th17 (IL-6, TGFβ, anti-IL-4, anti-IFNgama), and Treg (IL-2, TGFβ, anti-IL-4, anti-IFNgama). Cells were then stimulated to produce effector cytokines with PMA, inomycin, and monensin. The cells were stained with the corresponding flow cytometry antibodies, and the differentiation efficiency of different CD4-positive helper T cells was detected by flow cytometry.

[0087] (3) Investigating the function of HIF2α in Th2 cells in type II immune diseases:

[0088] We integrated single-cell sequencing data from patients with allergic asthma and patients with chronic sinusitis and nasal polyps, and clustered Th2 cells into different subpopulations using UMAP dimensionality reduction. We then analyzed the characteristics of each Th2 subpopulation and the functional relationship between HIF2α and Th2 cell subpopulations.

[0089] (4) Comparison of the differentiation efficiency of various CD4-positive helper T cells in vitro between the control group and mice with T cell-specific HIF2α deficiency:

[0090] Two types of naturally occurring CD4-positive T cells from the spleen and lymph nodes of mice were obtained by magnetic bead sorting. In vitro differentiation experiments were conducted under specific cytokine and antibody conditions: Th1 (IL-2, IL-12, anti-IL-4), Th2 (IL-2, IL-4, anti-IFNgama), Th17 (IL-6, TGFβ, anti-IL-4, anti-IFNgama), and Treg (IL-2, TGFβ, anti-IL-4, anti-IFNgama). The cells were then stimulated to produce effector cytokines with PMA, inomycin, and monensin, and stained with corresponding flow cytometry antibodies. The differentiation efficiency of various CD4-positive helper T cells was detected by flow cytometry.

[0091] 2. In vivo study of the function of HIF2α in a mouse asthma model

[0092] (1) Construction of a mouse asthma model:

[0093] The allergen OVA and adjuvant alum were mixed and dissolved in PBS, and the mice were injected intraperitoneally on days 0, 7, and 14. The mice were then nebulized with 3% OVA from day 28 to day 34.

[0094] (2) After inducing asthma, lung inflammation was detected in T-cell HIF2α knockout mice:

[0095] The lung tissue sections were stained with H&E and observed for lung cell infiltration, bronchial wall morphology, and mucus production.

[0096] (3) After inducing asthma in T-cell HIF2α knockout mice, the number and composition of infiltrating immune cells in the lungs were examined:

[0097] Mice lungs were rinsed with PBS, and immune cells infiltrated in the alveoli and bronchi were removed. The number of cells was counted, and the immune cell types were detected by flow cytometry.

[0098] (4) After inducing asthma in T cell HIF2α knockout mice, the expression levels of Th2-related cytokines were detected:

[0099] CD4-positive helper T cells were obtained from bronchial lavage fluid by magnetic bead sorting. RNA was extracted and reverse transcribed into cDNA. The expression levels of GATA3, IL-4, IL-5, and IL-13 were detected. The expression levels of IL-4, IL-5, and IL-13 in the supernatant of bronchial lavage fluid were detected by ELISA.

[0100] 3. The interleukin-4-GATA3 signaling pathway induces HIF2α expression.

[0101] (1) Detecting how HIF2α is induced:

[0102] Interleukin-4 stimulated normal T cells and GATA3 knockout T cells, and total RNA and total protein were extracted from the cells. Real-time quantitative PCR and Western blotting were used to detect HIF2α induction.

[0103] (2) Investigating the interaction between GATA3 and HIF2α:

[0104] In 293T cells, reporter plasmids containing the HIF2α promoter sequence and plasmids expressing GATA3 or lacking DNA-binding function of GATA3 protein were transfected, and relative luciferase activity was detected using a microplate reader.

[0105] GATA3 was overexpressed in the Jurkat cell line, and the nuclear protein was extracted and incubated with conserved or mutated sequences of GATA3 in the HIF2α promoter region. The binding of GATA3 to HIF2α promoter DNA was studied by gel electrophoresis migration assay.

[0106] The CUT&Tag assay was used to enrich the regulatory targets of GATA3, and IGV was used to investigate the binding peak of GATA3 on the HIF2α gene.

[0107] 4. Investigate the genes regulated by HIF2α and GATA3 and the functions enriched in these genes.

[0108] (1) Using CUT & Tag to study genes regulated by HIF2α and GATA3:

[0109] Stem-like Th2 and pathogenic Th2 were sorted, incubated with antibodies against HIF2α and GATA3 respectively, and the chromatin regions bound by HIF2α and GATA3 were cleaved using transposomal enzymes. After protein digestion, DNA sequencing was performed, and the sequencing results were compared with a reference genome to obtain genes regulated by HIF2α and GATA3.

[0110] (2) Investigating the functions of genes regulated by HIF2α and GATA3:

[0111] By taking the intersection of genes regulated by HIF2α and GATA3, we can detect whether there is a synergistic effect between them. We can also perform pathway analysis on genes regulated individually and genes regulated synergistically to determine the functions of HIF2α and GATA3 individually and synergistically.

[0112] 5. Investigate the mechanism by which HIF2α regulates GATA3.

[0113] (1) Using CUT&Tag sequencing data, investigate the peak enriched by HIF2α on the GATA3 gene:

[0114] Investigate the binding peak of HIF2α on the GATA3 gene using IGV.

[0115] (2) Construct expression vectors containing mouse HIF2α and GATA3 respectively, and investigate whether HIF2α affects the expression of GATA3:

[0116] HIF2α and GATA3 overexpression plasmids were transfected using the calcium phosphate method, resulting in their separate and co-overexpression in 293T cells. Cell samples were collected and proteins were extracted. Immunoblotting was used to investigate the expression of HIF2α and GATA3.

[0117] (3) Detection of GATA3 expression between control mice and T cell knockout HIF2α mice:

[0118] CD4-positive T cells from control mice and HIF2α knockout mice were separated using magnetic beads. After stimulation with interleukin-4, proteins were extracted, and immunoblotting was used to investigate the expression of GATA3 between the two groups of cells.

[0119] (4) Detect the binding of HIF2α to the GATA3 promoter:

[0120] In 293T cells, reporter plasmids containing the GATA3 promoter sequence and plasmids expressing HIF2α or lacking the binding domain were transfected, and relative luciferase activity was detected using a microplate reader.

[0121] Stem-like Th2 and pathogenic Th2 were sorted, incubated with anti-HIF2α antibody, and the proteins were digested to obtain DNA. The enrichment of the GATA3 promoter was detected by real-time quantitative PCR.

[0122] 6. Investigate the co-regulation of IPMK by HIF2α and GATA3.

[0123] (1) Detection of the effect of HIF2α deficiency on PI3K-AKT signaling during T cell activation:

[0124] In vitro, isolated WT and CD4 Cre + Epas1 f / f mice CD4 +T cells were first stimulated with TCR and IL-4 for 24 hours to upregulate HIF2α expression, and then restimulated with TCR for 0, 5, and 10 minutes. Total cellular protein was extracted, and the phosphorylation levels of key molecules in the PI3K-AKT signaling pathway, such as AKT, S6K, and FOXO1, were detected by Western blotting.

[0125] (2) Validating the regulation of IPMK by HIF2α and GATA3 using CUT & Tag sequencing data:

[0126] The binding peaks of HIF2α and GATA3 at the Ipmk promoter were verified using IGV. The expression levels of Ipmk in various Th2 subpopulations with WT and HIF2α deletions were verified using single-cell sequencing data.

[0127] II. Experimental Results

[0128] 1. HIF2α is highly expressed in pathogenic Th2 cells.

[0129] Compared to Th0, Th1, Th17, and Treg cells, Th2 cells showed the highest expression level of the HIF2α gene (Figure 1). Overexpression of HIF2α during in vitro differentiation promoted Th2 cell differentiation without affecting the differentiation of other helper T cells (Figure 2). Th2 cells exhibited the highest HIF2α gene expression level in type II immune diseases (Figure 3). Th2 cells in type II immune diseases showed heterogeneity, with the highest HIF2α gene expression level observed in pathogenic Th2 cells (Figure 4). The HIF2α gene expression level was positively correlated with the enrichment fraction of the pathogenic gene set (Figure 5).

[0130] The above experimental results indicate that the HIF2α gene is expressed at the highest level in pathogenic Th2 cells.

[0131] 2. HIF2α deficiency can inhibit Th2 cell differentiation.

[0132] To further verify the effect of the HIF2α gene on Th2 cells, the differentiation efficiency of various CD4-positive helper T cells after HIF2α knockout was examined. The results showed that the in vitro differentiation of Th2 cells was inhibited, while the in vitro differentiation of other helper T cells was not affected (as shown in Figure 6).

[0133] The above experimental results indicate that the HIF2α gene plays a positive regulatory role in Th2 cell differentiation. Disorders in Th2 cell function can lead to the development of asthma, an allergic disease. Therefore, the following investigation will further examine the role of HIF2α deficiency in asthma.

[0134] 3. HIF2α deficiency can effectively alleviate inflammatory symptoms in a mouse asthma model.

[0135] In vivo experiments showed that mice lacking HIF2α T cells had a lower number of immune cells infiltrating the lungs and a reduced degree of tracheal wall thickening after asthma induction compared to control mice (Figures 7-10). The relative proportion and absolute number of Th2 cells infiltrating the lungs were also lower in mice lacking HIF2α T cells after asthma induction compared to control mice (Figures 11-12). The expression levels of Th2 transcription factor GATA3 and cytokines IL-4, IL-5, and IL-13 in CD4-positive T cells in the lungs of mice lacking HIF2α T cells were lower than in control mice after asthma induction, and the concentrations of IL-4, IL-5, and IL-13 in the alveolar bronchial lavage fluid supernatant were also lower in mice lacking HIF2α T cells after asthma induction compared to control mice (Figure 13). The relative proportion and absolute number of eosinophils infiltrating the lungs were also lower in mice lacking HIF2α T cells after asthma induction compared to control mice (Figures 14-15).

[0136] The above experimental results indicate that HIF2α deficiency can reduce the infiltration of immune cells in the lungs of asthmatic mice and alleviate asthma inflammatory symptoms.

[0137] 4. The interleukin-4-GATA3 signaling pathway induces the expression of the HIF2α gene.

[0138] Compared with the control group mice, knockout of GATA3 prevented HIF2α in T cells from being induced by interleukin-4 (as shown in Figure 16). GATA3 can bind to the promoter region of HIF2α and transcribe HIF2α expression (as shown in Figures 17 and 18).

[0139] The results of this experiment indicate that HIF2α is induced by the interleukin-4 and GATA3 pathways during Th2 differentiation.

[0140] 5. HIF2α positively regulates Th2 differentiation by transcriptionally regulating GATA3 expression.

[0141] Simultaneous overexpression of HIF2α and GATA3 in 293T cells increased the expression levels of both (Figure 19). Compared with control mice, T cell-specific knockout of HIF2α reduced the expression level of GATA3 in T cells (Figure 20). CUT&Tag experiments demonstrated that the luciferase reporter system showed that HIF2α can bind to the GATA3 gene (Figures 21-22).

[0142] 6. Synergistic regulatory function of HIF2α and GATA3

[0143] CUT&Tag experiments demonstrated that HIF2α and GATA3 primarily bind to the promoter regions of genes, transcriptionally regulating gene expression. HIF2α and GATA3 exhibit synergistic regulatory functions, co-regulating genes related to inflammation and immune responses (Il1r2, Il10, Ifngr1, Ipmk, Peli1, Socs3, Fos, Jun, Il2ra, Ccr1, Tnfrsf8, Il10ra, Flt3l), genes related to T cell development and differentiation (Aff4, Gata3, Foxp1, Maf, Grap2, Tmem109, Rps6ka1), and genes related to cell metabolism (Capn2, Npc2, Hmgcs1, Anxa11, Dgat). 1. Genes related to cell signaling and transduction (Rgs1, Adarb1, Gm2a, 2610507B11Rik, Vmp1, Ormld3, Rps6ka5, Dusp1, Rasgrp1, Tasp1, Ppp3ca, Dusp16, Crebl2, Itpr2, Dctn6), and some less frequently reported genes (2610507B11Rik, AI467606, 1700061F12Rik, B4galnt4, Smco4, Cnot6l, Hip1, Nt5c3, Ttc39c). Pathway enrichment analysis revealed that these co-regulated genes were mainly enriched in phospholipid metabolism, T cell activation and differentiation, and signal transduction pathways (Figures 23-24).

[0144] The above experimental results indicate that HIF2α and GATA3 jointly regulate phospholipid metabolism in Th2 cells and that HIF2α and GATA3 jointly regulate IPMK.

[0145] In summary, this study, through in vitro and in vivo experiments, revealed that the HIF2α gene is expressed at the highest level in pathogenic Th2 cells and plays a positive regulatory role in Th2 cell differentiation. Knocking out the HIF2α gene reduced the infiltration of immune cells in the lungs of asthmatic mice and alleviated asthma inflammation symptoms. Further research found that HIF2α and GATA3 jointly regulate the IPMK and PI3K-AKT signaling pathways.

[0146] Example 2: Study on the mechanism of IPMK in Th2 differentiation

[0147] I. Experimental Methods

[0148] 1. Investigate the regulatory role of IPMK on Th2 differentiation.

[0149] (1) Verify the expression level of IPMK in Th2 cells:

[0150] CD4 in vitro + T cell differentiation was induced by adding the following to the culture media during in vitro differentiation: Th1 (IL-2, IL-12, anti-IL-4), Th2 (IL-2, IL-4, anti-IFNgama), Th17 (IL-6, TGFβ, anti-IL-4, anti-IFNgama), and Treg (IL-2, TGFβ, anti-IL-4, anti-IFNgama). After 5 days of culture, cells were collected and stimulated with TCR for 0, 5, and 10 minutes. Staining was performed using IPMK and pAKT flow cytometry antibodies, and various CD4+ cells were detected by flow cytometry. + Expression of IPMK and phosphorylation level of AKT in T cells.

[0151] WT and CD4 Cre from an induced asthma model + Epas1 f / f Stem-like Th2, pathogenic Th2, and Ikzf2 were sorted from mouse dLN. + Th2 cells. Cellular RNA was extracted and reverse transcribed to obtain cDNA. The expression level of Ipmk in different Th2 cell subsets with HIF2α deficiency was detected by qPCR.

[0152] (2) To investigate whether overexpression of IPMK can compensate for the Th2 differentiation inhibition caused by HIF2α deficiency:

[0153] In vitro, retroviral vectors were used to coat IPMK to infect various CD4-positive T cells that were undergoing differentiation, including WT or HIF2α-deficient cells: Th1 (IL-2, IL-12, anti-IL-4), Th2 (IL-2, IL-4, anti-IFNgama), Th17 (IL-6, TGFβ, anti-IL-4, anti-IFNgama), and Treg (IL-2, TGFβ, anti-IL-4, anti-IFNgama). Cells were then stimulated to produce effector cytokines with PMA, inomycin, and monensin, and stained with the corresponding flow cytometry antibodies. The differentiation efficiency of various CD4-positive helper T cells and Th2 subsets was detected by flow cytometry.

[0154] (3) Investigating whether IPMK overexpression can compensate for the weakening of PI3K-AKT signal caused by HIF2α deficiency:

[0155] In vitro, IPMK was used to infect WT or HIF2α-deficient CD4 cells with a retroviral vector. +T cells were stimulated with TCR for 10 minutes, and total cellular protein was extracted. The phosphorylation levels of AKT, S6K, and FOXO1 were detected by Western blotting.

[0156] (4) Verify whether the IPMK inhibitor vilazorone can attenuate PI3K-AKT signaling:

[0157] Treatment with vilazorone in vitro CD4 + T cells were stimulated with TCR for 10 minutes, and total cellular protein was extracted. The phosphorylation levels of AKT, S6K, and FOXO1 were detected by Western blotting.

[0158] Th2 cells were induced to differentiate in vitro (IL-2, IL-4, anti-IFNgama) and divided into three groups: WT group, HIF2α-deficient group, and vilazorone-treated group. Pathogenic Th2 cells differentiated in vitro were sorted by flow cytometry and lipid metabolism sequencing was performed to analyze the level of phosphatidylinositol in each group.

[0159] (5) Verify whether the IPMK inhibitor vilazorone can lead to a reduction in Th2 cell differentiation:

[0160] During in vitro differentiation, vilazone was added to each culture medium, and the differentiation efficiency of various CD4-positive helper T cells was detected. Th1 (IL-2, IL-12, anti-IL-4), Th2 (IL-2, IL-4, anti-IFNgama), Th17 (IL-6, TGFβ, anti-IL-4, anti-IFNgama), and Treg (IL-2, TGFβ, anti-IL-4, anti-IFNgama) cells were stimulated with PMA, inomycin, and monensin to produce effector cytokines. The cells were then stained with the corresponding flow cytometry antibodies, and the differentiation efficiency of various CD4-positive helper T cells and Th2 subsets was detected using flow cytometry.

[0161] II. Experimental Results

[0162] HIF2α and GATA3 mediate the differentiation of pathogenic Th2 cells by co-regulating IPMK expression:

[0163] HIF2α deficiency inhibits the PI3K-AKT signaling pathway, and the phosphorylation levels of AKT, S6K, and FOXO1 induced by TCR stimulation are significantly reduced after HIF2α deficiency (Figure 25). CUT&Tag sequencing analysis showed that HIF2α and GATA3 jointly bind to the promoter region of Ipmk in pathogenic Th2 cells (Figure 26). During Th2 differentiation, IPMK expression and AKT phosphorylation levels are higher than those of other CD4 cells. +Helper T cells showed significantly higher expression levels (Figure 27). Ipmk was specifically highly expressed in pathogenic Th2 cells, and HIF2α deficiency led to decreased Ipmk expression levels in pathogenic Th2 cells (Figure 28). Overexpression of IPMK in HIF2α-deficient T cells could restore impaired Th2 differentiation and weakened PI3K-AKT signaling and phosphatidylinositol levels (Figures 29-30). Treatment with the IPMK inhibitor vilazone during in vitro differentiation inhibited the differentiation of pathogenic Th2 cells, but had no effect on the differentiation of other helper T cells and other Th2 subsets (Figure 31). Adding vilazone during TCR stimulation of T cells inhibited PI3K-AKT signaling. Simultaneously, adding vilazone during in vitro differentiation reduced phosphatidylinositol levels in pathogenic Th2 cells (Figures 32-33).

[0164] The above experimental results indicate that overexpression of IPMK can compensate for the Th2 differentiation inhibition caused by HIF2α deficiency, and treatment with IPMK-specific inhibitors can inhibit pathogenic Th2 differentiation. This suggests that IPMK is a potential target for the prevention and / or treatment of asthma.

[0165] Example 3: The IPMK-specific inhibitor vilazorone can alleviate asthma inflammatory symptoms.

[0166] I. Experimental Methods

[0167] 1. To investigate the therapeutic effect of vilazorone, a specific inhibitor of IPMK, on ​​asthma:

[0168] (1) Construction of a mouse asthma model and treatment with vilazorone:

[0169] The allergen OVA and adjuvant alum were mixed and dissolved in PBS. Mice were injected intraperitoneally on days 0, 7, and 14. From day 28 to 34, mice were nebulized with 3% OVA. Before each nebulization, mice were given vilazone via intraperitoneal injection at a dose of 5 mg / kg of body weight.

[0170] (2) After treatment with vilazorone, the lung inflammation in mice was assessed:

[0171] The lung tissue sections were stained with H&E and the lung cell infiltration and bronchial wall morphology were observed.

[0172] (3) After treatment with vilazorone, the number and composition of infiltrating immune cells in the lungs of mice were detected:

[0173] Mice lungs were irrigated with PBS, and immune cells infiltrated in the lungs and alveoli-bronchial regions were removed. The number of cells was counted, and the immune cell types were detected by flow cytometry.

[0174] (4) After treatment with vilazone, the expression levels of Th2-related cytokines were detected:

[0175] The expression levels of IL-4, IL-5, and IL-13 in the supernatant of bronchoalveolar lavage fluid were detected by ELISA.

[0176] II. Experimental Results

[0177] Experimental results showed that the number of immune cells infiltrating the lungs of mice treated with vilazodone after inducing asthma was reduced compared to the control group, and the degree of tracheal wall thickening was also decreased (Figure 34). After vilazodone treatment, the number of CD45 cells infiltrating the lungs of asthma-induced mice was reduced. + The relative proportions and absolute numbers of cells, eosinophils, and Th2 cells were reduced compared to the control group (Figure 35). Furthermore, vilazone treatment resulted in lower concentrations of IL-5 and IL-13 in the bronchoalveolar lavage fluid supernatant of asthmatic mice compared to the control group (Figure 36).

[0178] In summary, this embodiment demonstrates that the IPMK-specific inhibitor vilazorone has a therapeutic effect on asthma.

[0179] In summary, this invention, through single-cell sequencing data, reveals heterogeneity in Th2 cells among patients with allergic asthma and chronic sinusitis with nasal polyps, and that the transcription factor HIF2α is highly expressed in pathogenic Th2 cells. Interleukin-4 stimulation of T cells strongly induces HIF2α gene expression (manifested as an increase in mRNA levels). Specific knockout of HIF2α in T cells inhibits the differentiation of naturally occurring CD4-positive T cells into Th2 cells in vitro, but has no effect on the differentiation of other helper T cells.

[0180] Furthermore, CUT&Tag experiments revealed that HIF2α and GATA3 jointly regulate phospholipid metabolism in Th2 cells, and HIF2α can positively feedback-regulate GATA3 expression, promoting Th2 cell differentiation. We further discovered that during Th2 differentiation, HIF2α and GATA3 synergistically regulate phosphatidylinositol kinase IPMK and upregulate the PI3K-AKT signaling pathway. In HIF2α-deficient cells… CD4 + Overexpression of IPMK in T cells can restore suppressed pathogenic Th2 differentiation and PI3K-AKT signaling. Treatment of cells with the IPMK-specific inhibitor vilazone specifically inhibits the differentiation of pathogenic Th2 cells.

[0181] Based on the above experimental results, IPMK as a target can treat and prevent diseases mediated by Th2 cells, ILC2 cells and related cytokines, including but not limited to: type II immune diseases, tumors, prevention and treatment of diseases caused by Th2 / ILC2 differentiation disorders, as well as anti-aging and related diseases.

[0182] To further verify whether IPMK can be used as a target for asthma treatment, asthmatic mice were treated with vilazodone, a specific inhibitor of IPMK. The results showed that the inflammatory symptoms of the mice treated with the drug were relieved.

[0183] Therefore, this invention demonstrates that type II immune diseases, diseases caused by Th2 / ILC2 differentiation disorder, as well as anti-tumor, anti-aging, and anti-aging-related diseases can be prevented and treated by intervening in the IPMK target.

Claims

1. The use of a drug targeting IPMK in the preparation of drugs for the prevention and / or treatment of type II immune diseases and their complications, anti-tumor, anti-aging, or anti-aging-related diseases, characterized in that: The drugs that intervene in the IPMK target are selected from IPMK inhibitors, drugs that inhibit IPMK gene expression, drugs that knock out or knock down the IPMK gene, and drugs that degrade IPMK.

2. The use according to claim 1, characterized in that: The IPMK inhibitor is selected from small molecule compounds, peptides, and antibodies, with verazordone, LI-2242, UNC7437, UNC9750, quercetin, and chlorogenic acid being preferred.

3. The use according to claim 1, characterized in that: The drugs that inhibit IPMK gene expression are selected from circular RNA, antisense nucleic acid, small interfering nucleic acid, nucleic acid aptamer, small activating nucleic acid, micronucleic acid, mRNA drugs, and ribozymes.

4. The use according to claim 1, characterized in that: The drug used to knock out or knock down the IPMK gene is the CRISPR / Cas9 gene editing system.

5. The use according to claim 1, characterized in that: The drug for degrading IPMK protein includes a protein degrading agent, preferably selected from PROTAC and molecular gels.

6. The use according to claim 1, characterized in that: The drug that intervenes in the IPMK target is an antibody-drug conjugate, which is formed by linking an antibody targeting the target cell with an active ingredient that intervenes in the IPMK target through a linker; preferably, the target cell is a Th2 cell and / or an ILC2 cell.

7. The use according to any one of claims 1 to 6, characterized in that: The drugs mentioned for treating type II immune diseases are drugs for treating Th2 cell-mediated immune diseases and / or ILC2 cell-mediated immune diseases.

8. The use according to claim 7, characterized in that: The drug is a drug that inhibits stem cell differentiation into Th2 cells, inhibits the differentiation of stem cell-like Th2 cell subsets into pathogenic Th2 cell subsets, and inhibits Th2 cells from secreting effector cytokines.

9. The use according to any one of claims 1 to 6, characterized in that: The type II immune diseases mentioned include atopic dermatitis, chronic spontaneous urticaria, nodular prurigo, bullous pemphigoid, chronic sinusitis with or without nasal polyps, allergic rhinitis, asthma, allergic bronchopulmonary aspergillosis, chronic obstructive pulmonary disease, eosinophilic granulomatous polyangiitis, food allergy, eosinophilic esophagitis, allergic conjunctivitis, ulcerative colitis, lichen planus, and lymphedema.

10. The use according to any one of claims 1 to 6, characterized in that: Complications of the type II immune disease include tissue fibrosis and organ fibrosis.

11. The use according to any one of claims 1 to 6, characterized in that: The tumors include prostate cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, gastric cancer, endometrial cancer, uterine cancer, brain cancer, liver cancer, bladder cancer, ovarian cancer, testicular cancer, head cancer, neck cancer, skin cancer, mesothelial carcinoma, leukocyte carcinoma, esophageal cancer, breast cancer, muscle cancer, connective tissue cancer, intestinal cancer, lung cancer, adrenal cancer, thyroid cancer, kidney cancer, or bone cancer; or glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma, choriocarcinoma, basal cell carcinoma of the skin, and seminoma of the testis.

12. The use according to any one of claims 1 to 6, characterized in that: The aging process includes nervous system aging, immune aging, tissue aging, cardiovascular system aging, and skin aging.

13. The use according to any one of claims 1 to 6, characterized in that: The age-related diseases include sarcopenia, chronic low-grade inflammation, Alzheimer's disease, Parkinson's disease, neuromyelitis optica, chronic infection, age-related obesity, and cardiac fibrosis.

14. Use of IPMK agonists or drugs that overexpress IPMK in the preparation of drugs for the prevention and / or treatment of diseases caused by Th2 / ILC2 differentiation disorders.

15. The use according to claim 14, characterized in that: The IPMK agonist is a small molecule compound, peptide, or antibody that can activate IPMK.

16. The use according to claim 14, characterized in that: The drugs for overexpressing IPMK include drugs for T-cell directed overexpression, drugs for adeno-associated virus-mediated overexpression, drugs for lentivirus-mediated overexpression, drugs for adenovirus-mediated overexpression, drugs for retrovirus-mediated overexpression, drugs for enhancer-induced overexpression, drugs for transcription-induced overexpression, drugs for transcription element-induced overexpression, small molecule drug response systems for overexpressing genes, the Cre-loxp system, the Flp-frt system, and the Dre-rox system.

17. The use according to claim 14, characterized in that: The IPMK agonist or IPMK overexpressing drug is an antibody-drug conjugate, which is formed by linking an antibody targeting the target cell with an active ingredient via a linker; the active ingredient has IPMK agonist activity or IPMK overexpression activity; preferably, the target cell is a Th2 cell and / or an ILC2 cell.

18. The use according to claim 14, characterized in that: Diseases caused by Th2 / ILC2 differentiation disorder include worm infections, obesity, obesity-related complications, primary immune thrombocytopenic purpura, rheumatoid arthritis, systemic lupus erythematosus, rheumatoid arthritis, type I diabetes, multiple sclerosis, chronic thyroiditis, and tumors.

19. The use according to claim 18, characterized in that: The obesity-related complications include diseases caused by abnormal accumulation of lipid droplets.

20. The use according to claim 19, characterized in that: The diseases caused by abnormal lipid droplet accumulation include fatty liver, insulin resistance, diabetes, cardiovascular and cerebrovascular diseases, hyperlipidemia, chronic kidney disease, atherosclerosis, coronary heart disease, heart failure, myocardial lipotoxicity, and metabolic syndrome.

21. The use according to claim 18, characterized in that: The tumors include prostate cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, gastric cancer, endometrial cancer, uterine cancer, brain cancer, liver cancer, bladder cancer, ovarian cancer, testicular cancer, head cancer, neck cancer, skin cancer, mesothelial carcinoma, leukocyte carcinoma, esophageal cancer, breast cancer, muscle cancer, connective tissue cancer, intestinal cancer, lung cancer, adrenal cancer, thyroid cancer, kidney cancer, or bone cancer; or glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma, choriocarcinoma, basal cell carcinoma of the skin, and seminoma of the testis.

Images