New application of medicine for intervening IPMK target spot

Abstract

The invention belongs to the technical field of medicines, and particularly relates to novel application of a medicine for intervening an IPMK target spot. The invention discovers that HIF2alpha and GATA3 jointly regulate IPMK and PI3K-AKT signal pathways to cause disease Th2 cell differentiation for the first time, and verifies that the prevention and treatment of type II immune diseases and diseases caused by Th2 / ILC2 differentiation disorder and the anti-tumor, anti-aging and anti-aging related diseases can be carried out by intervening an IPMK target spot for the first time. The invention provides a new potential target for clinically treating II-type immune diseases, tumors, diseases caused by Th2 / ILC2 differentiation disorder, aging resistance and related diseases, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a new use of a drug for intervening in the IPMK target. Background Art

[0002] Type II immune diseases are inflammations mediated mainly by Th2 cells, ILC2 cells and related cytokines. At present, type 2 inflammation has been recognized as the immunopathological mechanism of many diseases, which involve multiple systems such as the skin, respiratory, and digestive systems. Such reactions are characterized by sensitivity to a variety of allergens, rapid response, and distribution in multiple barrier tissues.

[0003] Asthma is a common type II allergic immune disease. Approximately 300 million people worldwide suffer from asthma, which is mainly manifested by the narrowing of the airways in the lungs, the overproduction of mucus, the remodeling of the tracheal wall, and disorders of the innate and adaptive immune systems, resulting in hyperreactivity of the bronchi to stimuli such as harmless antigens like pollen or cold air. Asthma patients often have symptoms such as shortness of breath, wheezing, and chest tightness. At present, the conventional clinical treatment methods mainly utilize glucocorticoid drugs and short-acting or long-acting β2-adrenergic agonists. Among them, the role of corticosteroid drugs is mainly to inhibit the inflammatory reaction in the bronchi, while the role of β2-adrenergic agonists is mainly to open the constricted bronchial smooth muscle. However, these treatment methods for asthma can only control the symptoms to a certain extent and cannot cure asthma fundamentally. Therefore, further studying the pathogenesis of asthma and finding new targets for treating asthma 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 can drive the immune and physiological characteristics of asthma, such as IgE-mediated inflammation, eosinophilia, airway hyperreactivity, and goblet cell hyperplasia. Th2 cells also interact with other T cell subsets such as Th17 cells and Th1 cells and jointly participate in the pathogenesis of asthma. In addition, the role of Th2 cells in asthma is not limited to immune responses. They can also serve as a bridge between inflammation and neuronal regulatory behavioral responses and play an important role in epithelial differentiation and injury repair. Therefore, Th2 cells are key factors in the pathogenesis of allergic asthma, and understanding their regulatory mechanisms and biological characteristics is crucial for developing new treatment strategies.

[0005] IPMK (inositol polyphosphate kinase) is a multifunctional enzyme that plays an important role in the nucleus and is involved in various cell signaling and metabolic regulation processes. IPMK plays a key role in the differentiation and function of Th1 and Th17 cells. For example, IPMK regulates the differentiation and effector functions of Th1 and Th17 cells by controlling the Akt-mTOR signaling pathway. In addition, IPMK deficiency leads to a significant attenuation of the immune responses of Th1 and Th17 cells, thereby affecting the host's resistance to certain pathogens.

[0006] Currently, there is no literature reporting the connection between IPMK and the differentiation and function of Th2 cells, nor the connection between IPMK and type II immune diseases. Summary of the Invention

[0007] In view of the problems of the prior art, the present invention provides a new use of a drug that intervenes in the IPMK target.

[0008] Use of a drug that intervenes in the IPMK target in the preparation of a drug for preventing and / or treating type II immune diseases and their complications, anti-tumor, anti-aging or anti-aging related diseases, wherein the drug that intervenes in the IPMK target is selected from IPMK inhibitors, drugs that inhibit IPMK gene expression, drugs that knockout or knockdown the IPMK gene, and drugs that degrade IPMK protein.

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

[0010] Preferably, the drug that inhibits IPMK gene expression is selected from circular RNAs, antisense nucleic acids, small interfering nucleic acids, nucleic acid aptamers, small activating nucleic acids, microRNAs, mRNA drugs, ribozymes.

[0011] Preferably, the drug that knocks out or knockdowns the IPMK gene can be a CRISPR / Cas9 gene editing system.

[0012] Preferably, the drug that degrades IPMK protein includes proteolysis agents, preferably the proteolysis agents are selected from PROTACs, molecular glues.

[0013] Preferably, the drug that intervenes in the IPMK target is an antibody-drug conjugate, and the antibody-drug conjugate is formed by connecting an antibody targeting target cells with an active ingredient that intervenes in the IPMK target through a linker; preferably, the target cells are Th2 cells and / or ILC2 cells.

[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 the differentiation of stem cells into Th2 cells, inhibits the differentiation of stem cell-like Th2 cell subsets into pathogenic Th2 cell subsets, and inhibits the secretion of effector cytokines by Th2 cells.

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

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

[0018] Preferably, 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, mesothelioma, leukemia, 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, testicular seminoma.

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

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

[0021] Use of an IPMK agonist or a drug overexpressing IPMK in the preparation of a drug for preventing and / or treating diseases caused by Th2 / ILC2 differentiation disorders.

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

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

[0024] Preferably, the IPMK agonist or the drug for overexpressing IPMK is an antibody-drug conjugate, which is formed by connecting an antibody targeting target cells with an active ingredient through a linker; the active ingredient has IPMK agonist activity or the activity of overexpressing IPMK; preferably, the target cells are Th2 cells and / or ILC2 cells.

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

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

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

[0028] Preferably, 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, mesothelioma, leukemia, 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, cutaneous basal cell carcinoma, testicular seminoma.

[0029] The present invention for the first time discovers that HIF2α and GATA3 jointly regulate the IPMK and PI3K-AKT signaling pathways to mediate the differentiation of pathogenic Th2 cells, and IPMK plays an important role in type II immune diseases, tumors, diseases caused by dysregulation of Th2 / ILC2 differentiation, aging and related diseases. The present invention for the first time verifies that the prevention and treatment of type II immune diseases, diseases caused by dysregulation of Th2 / ILC2 differentiation, as well as anti-tumor, anti-aging and anti-aging related diseases can be achieved by intervening in the IPMK target. The present invention provides a new potential target for clinically preventing and treating type II immune diseases, anti-tumor, anti-aging and related diseases, and treating diseases caused by dysregulation of Th2 / ILC2 differentiation. The present invention also provides new uses of old drugs such as vilazodone for preventing and treating type II immune diseases, especially for asthma and allergic rhinitis.

[0030] Definition:

[0031] "A drug for preventing and / or treating type II immune diseases and their complications" refers to a drug that can prevent type II immune diseases alone, treat type II immune diseases alone, prevent the complications of type II immune diseases alone, and treat the complications of type II immune diseases alone, can prevent type II immune diseases and the complications of type II immune diseases simultaneously, can treat type II immune diseases and the complications of type II immune diseases simultaneously, or can treat and prevent type II immune diseases or the complications of type II immune diseases.

[0032] IPMK inhibitor: refers to any substance having the effect of inhibiting the activity of IPMK. These substances can be small molecule compounds, polypeptides or antibodies, including molecules reported in the prior art that can inhibit the activity of IPMK, such as vilazodone, LI-2242, UNC7437, UNC9750, etc.

[0033] A drug for inhibiting the expression of IPMK gene: refers to any substance having the effect of inhibiting the expression of IPMK gene, including antisense nucleic acid (ASO), small interfering nucleic acid (siRNA), nucleic acid aptamer, small activating nucleic acid, microRNA, mRNA drug, ribozyme, etc.

[0034] A drug for knocking out or knocking down the IPMK gene: refers to any substance having the effect of knocking out or knocking down the IPMK gene, which can be a CRISPR / Cas9 gene editing system.

[0035] A drug for degrading IPMK: refers to any substance having the effect of degrading IPMK protein, such as a proteolytic agent. Proteolytic agents include Protac, molecular glue, etc.

[0036] IPMK agonist: refers to any substance that has the effect of inhibiting IPMK activity. These substances can be small molecule compounds, polypeptides or antibodies.

[0037] Drug for overexpressing IPMK: refers to a drug that makes the expression level of IPMK in cells or organisms higher than the normal state. These substances can be small molecule compounds, polypeptides, enzymes, nucleic acid drugs, and systems composed of them with gene editing or gene expression providing functions.

[0038] Type II immune diseases: refer to immune diseases mainly mediated by Th2 cells, ILC2 cells and related cytokines.

[0039] Th2 cells: refer to helper T cells type 2.

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

[0041] Obviously, based on the above content of the present invention, according to the common general knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can also be made.

[0042] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be understood as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Brief Description of the Drawings

[0043] Figure 1 Experimental results of high expression of HIF2α in Th2 cells compared with other types of CD4-positive helper T cells.

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

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

[0046] Figure 4 Experimental results of heterogeneity of Th2 cells in asthma and sinusitis patients.

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

[0048] Figure 6 Experimental results of inhibition of in vitro differentiation of Th2 cells by HIF2α deficiency.

[0049] Figure 7 Experimental results showing that the deletion of HIF2α reduces the infiltrating cells in the lungs of asthmatic mice.

[0050] Figure 8 Experimental results showing that the deletion of HIF2α causes a decrease in the total number of cells in BALF and the lungs.

[0051] Figure 9 Experimental results showing that the deletion of HIF2α reduces the infiltration of immune cells in the BALF of asthmatic mice.

[0052] Figure 10 Experimental results showing that the deletion of HIF2α reduces the infiltration of immune cells in the lungs of asthmatic mice.

[0053] Figure 11 Experimental results showing that the deletion of HIF2α inhibits the differentiation of Th2 cells in the BALF of asthmatic mice.

[0054] Figure 12 Experimental results showing that the deletion of HIF2α inhibits the differentiation of Th2 cells in the lungs of asthmatic mice.

[0055] Figure 13 Experimental results showing that the deletion of HIF2α inhibits the expression of Th2 cell-related effector molecules.

[0056] Figure 14 Experimental results showing that the deletion of HIF2α reduces the infiltration of eosinophils in the BALF.

[0057] Figure 15 Experimental results showing that the deletion of HIF2α reduces the infiltration of eosinophils in the lungs.

[0058] Figure 16 Experimental results showing that HIF2α is no longer induced in T cells of GATA3 knockout mice after stimulation with IL-4 compared with control mice.

[0059] Figure 17 Experimental results showing that the luciferase reporter system and EMSA experiments prove that GATA3 can bind to the HIF2α promoter DNA sequence.

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

[0061] Figure 19 Experimental results showing that the co-overexpression of HIF2α and GATA3 in 293T cells increases the expression levels of both.

[0062] Figure 20The experimental results demonstrated by Western blot that the expression level of GATA3 in T cells decreased in T cell-specific HIF2α knockout mice compared with that in the control group mice.

[0063] Figure 21 The experimental results demonstrated by the luciferase reporter system that the HIF2α protein can bind to the GATA3 promoter DNA sequence.

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

[0065] Figure 23 The experimental results demonstrated by CUT&Tag that HIF2α and GATA3 mainly bind to the promoter region of genes.

[0066] Figure 24 The experimental results that HIF2α and GATA3 jointly maintain phospholipid metabolism and regulate T cell activation and differentiation.

[0067] Figure 25 The experimental results that HIF2α deficiency leads to a weakened PI3K-AKT signaling pathway intensity when T cells respond to TCR stimulation.

[0068] Figure 26 The experimental results that both HIF2α and GATA3 can bind to the Ipmk promoter region to mediate its transcriptional regulation.

[0069] Figure 27 The experimental results that during Th2 differentiation, the expression of IPMK and the level of AKT phosphorylation are significantly higher than those in other CD4 + helper T cells.

[0070] Figure 28 The experimental results that Ipmk is specifically highly expressed in pathogenic Th2 cells and HIF2α deficiency leads to a decrease in the Ipmk expression level in pathogenic Th2 cells.

[0071] Figure 29 The experimental results that overexpression of IPMK in HIF2α-deficient T cells can rescue the blocked Th2 differentiation.

[0072] Figure 30 The experimental results that overexpression of IPMK in HIF2α-deficient T cells can rescue the weakened PI3K-AKT signaling and phosphatidylinositol levels.

[0073] Figure 31The experimental results show that adding the IPMK inhibitor vilazodone during in vitro differentiation can inhibit the differentiation of pathogenic Th2 cells, but has no effect on the differentiation of other helper T cells and other Th2 subsets.

[0074] Figure 32 The experimental results show that adding vilazodone during TCR stimulation of T cells can inhibit the PI3K-AKT signal.

[0075] Figure 33 The experimental results show that adding vilazodone during in vitro differentiation can reduce the phosphatidylinositol level in pathogenic Th2 cells.

[0076] Figure 34 The experimental results show that after inducing asthma in mice treated with vilazodone, the number of immune cells infiltrating the lungs is reduced compared to control mice, and the degree of thickening of the tracheal wall is reduced.

[0077] Figure 35 For the CD45 + cells, eosinophils and the relative proportion and absolute number of Th2 cells in the lungs of mice induced with asthma after treatment with vilazodone are reduced compared to control mice.

[0078] Figure 36 The experimental results show that after inducing asthma in mice treated with vilazodone, the concentrations of IL-5 and IL-13 in the supernatant of bronchoalveolar lavage fluid are reduced compared to control mice. Detailed implementation methods

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

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

[0081] I. Experimental methods

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

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

[0084] Isolate the above cells, extract total RNA by the conventional phenol-chloroform method and reverse transcribe it into cDNA, and detect the difference in the expression level of HIF2α mRNA in these cells by qPCR.

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

[0086] In vitro, various CD4-positive T cells in the process of differentiation are infected with HIF2α coated with a retroviral vector, 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), Treg (IL-2, TGFβ, anti-IL-4, anti-IFNgama). Then, the cells are stimulated with PMA, ionomycin, monensin, etc. to produce effector cytokines, stained with corresponding flow antibodies, and the differentiation efficiency of various CD4-positive helper T cells is detected by flow cytometry.

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

[0088] Integrate the single-cell sequencing data of patients with allergic asthma and patients with chronic rhinosinusitis with nasal polyps. Cluster Th2 cells into different subsets by UMAP dimensionality reduction method, and analyze the characteristics of each Th2 subset and the functional relationship between HIF2α and Th2 cell subsets.

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

[0090] Use magnetic beads to sort natural CD4-positive T cells in the spleens and lymph nodes of two types of mice, and conduct in vitro differentiation experiments 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), Treg (IL-2, TGFβ, anti-IL-4, anti-IFNgama). Then, the cells are stimulated with PMA, ionomycin, monensin, etc. to produce effector cytokines, stained with corresponding flow antibodies, and the differentiation efficiency of various CD4-positive helper T cells is detected by flow cytometry.

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

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

[0093] Mix the allergen OVA and adjuvant alum and dissolve them in PBS, and intraperitoneally inject the mice on days 0, 7, and 14. Nebulize the mice with 3% OVA from day 28 to day 34.

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

[0095] The lungs were sectioned, and after H&E staining, the infiltration of lung cells, the morphology of the bronchial wall, and the production of mucus were observed.

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

[0097] The lungs of mice were lavaged with PBS, and the immune cells infiltrating at the alveolar bronchi of the lungs were taken out, the cell number was counted, and the types of immune cells 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] Magnetic beads were used to sort CD4-positive helper T cells in the bronchoalveolar lavage fluid of the lungs. After extracting RNA, it was reverse transcribed into cDNA, and 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 the bronchoalveolar lavage fluid of the lungs were detected by ELISA.

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

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

[0102] Interleukin-4 was used to stimulate normal T cells and T cells with GATA3 knocked out. Total cellular RNA and total protein were extracted, and the induction of HIF2α was detected by real-time fluorescence quantitative PCR and immunoblotting.

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

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

[0105] GATA3 was overexpressed in the Jurkat cell line, nuclear proteins were extracted, and incubated with the conserved sequence or mutant sequence of GATA3 in the HIF2α promoter region. Gel electrophoresis mobility shift assay was used to study the binding of GATA3 to the HIF2α promoter DNA.

[0106] CUT&Tag experiment was used to enrich the regulatory targets of GATA3, and IGV was used to explore the binding peaks of GATA3 on the HIF2α gene.

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

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

[0109] Sort stem-like Th2 and pathogenic Th2, incubate them with antibodies against HIF2α and GATA3 respectively, use the transposase to cut the chromatin regions bound by HIF2α and GATA3, perform DNA sequencing after digesting the proteins, and then compare the sequencing results to the reference genome to obtain the genes regulated by HIF2α and GATA3.

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

[0111] Take the intersection of the genes regulated by HIF2α and GATA3, detect whether there is a synergistic effect between them, perform pathway analysis on the genes regulated by them alone and the genes regulated synergistically, and determine the functions exerted by HIF2α and GATA3 alone and the functions exerted synergistically.

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

[0113] (1) Use the sequencing data of CUT&Tag to explore the peaks enriched by HIF2α on the GATA3 gene:

[0114] Use IGV to explore the binding peaks of HIF2α on the GATA3 gene.

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

[0116] Transfect the overexpression plasmids of HIF2α and GATA3 by the calcium phosphate method to express them separately and co-express them in 293T cells, collect cell samples and extract proteins, and explore the expression of HIF2α and GATA3 by immunoblotting.

[0117] (3) Detect the expression of GATA3 between control mice and mice with HIF2α knocked out in T cells:

[0118] Use magnetic beads to sort CD4-positive T cells from control mice and mice with HIF2α knocked out in T cells, stimulate them with interleukin-4, extract proteins, and explore the expression of GATA3 between the two groups of cells by immunoblotting.

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

[0120] Transfect the reporter plasmid containing the GATA3 promoter sequence and the plasmid expressing HIF2α or HIF2α protein lacking the binding domain in 293T cells, and detect the relative luciferase activity using a microplate reader.

[0121] Sort stem-like Th2 and pathogenic Th2, incubate with anti-HIF2α antibody, digest the protein to obtain DNA, and detect the enrichment of the GATA3 promoter by real-time fluorescence quantitative PCR.

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

[0123] (1) Detect the effect of HIF2α deletion on the PI3K-AKT signal during T cell activation:

[0124] In vitro, isolate WT and CD4Cre + Epas1 f / f mice CD4 + T cells are first stimulated with TCR and IL-4 for 24 hours to upregulate HIF2α expression, and then the cells are restimulated with TCR for 0, 5, and 10 minutes. Extract the total cell protein and detect the phosphorylation levels of key molecules in the PI3K-AKT signaling pathway such as AKT, S6K, and FOXO1 using WB.

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

[0126] Use IGV to verify the binding peaks of HIF2α and GATA3 on the Ipmk promoter. Verify the expression levels of Ipmk in each Th2 subset with WT and HIF2α deletion in single-cell sequencing data.

[0127] II. Experimental Results

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

[0129] Compared with Th0, Th1, Th17, and Treg cells, the expression level of the HIF2α gene in Th2 cells is relatively the highest (as Figure 1 shown). Overexpression of HIF2α during in vitro differentiation can promote the differentiation of Th2 cells, but has no effect on the differentiation of other helper T cells (as Figure 2 shown). The expression level of the HIF2α gene in Th2 cells is relatively the highest in type II immune response diseases (as Figure 3as shown). In type II immune response diseases, Th2 cells are heterogeneous, and the expression level of the HIF2α gene is highest in pathogenic Th2 cells (as Figure 4 shown). The expression level of the HIF2α gene is positively correlated with the enrichment score of the pathogenic gene set (as Figure 5 shown).

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

[0131] 2. Deletion of HIF2α can inhibit the differentiation of Th2 cells

[0132] To further verify the effect of the HIF2α gene on Th2 cells, the differentiation efficiency of various CD4+ helper T cells after knocking out HIF2α in T cells was investigated. 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 Figure 6 shown).

[0133] The above experimental results indicate that the HIF2α gene plays a positive regulatory role in the differentiation of Th2 cells. When the function of Th2 cells is disordered, it will lead to the occurrence of the autoimmune disease asthma. Therefore, the effect of HIF2α deletion on asthma disease was further investigated below.

[0134] 3. Deletion of HIF2α can effectively relieve the inflammatory symptoms in a mouse asthma model

[0135] The in vivo experimental results showed that the number of immune cells infiltrating the lungs of mice with HIF2α deletion in T cells was lower than that of the control group mice after inducing asthma, and the degree of tracheal wall thickening was reduced (as Figures 7 to 10 shown). The relative proportion and absolute number of Th2 cells infiltrating the lungs of mice with HIF2α deletion in T cells were lower than those of the control group mice after inducing asthma (as Figures 11 to 12 shown). The expression levels of the Th2 transcription factor GATA3, cytokines IL-4, IL-5, and IL-13 in CD4+ T cells in the lungs of mice with HIF2α deletion in T cells were lower than those of the control group mice after inducing asthma, and the concentrations of IL-4, IL-5, and IL-13 in the supernatant of bronchoalveolar lavage fluid were lower than those of the control group mice (as Figure 13 shown). The relative proportion and absolute number of eosinophils infiltrating the lungs of mice with HIF2α deletion in T cells were lower than those of the control group mice after inducing asthma (as Figures 14 to 15 shown).

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

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

[0138] Compared with the control group of mice, after knocking out GATA3, HIF2α in T cells could no longer be induced by interleukin-4 (as Figure 16 shown). GATA3 could bind to the promoter region of HIF2α and transcriptionally regulate the expression of HIF2α (as Figures 17 to 18 ).

[0139] The experimental results showed that HIF2α was induced by interleukin-4 and GATA3 pathway during Th2 differentiation.

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

[0141] Overexpressing HIF2α and GATA3 simultaneously in 293T cells would increase the expression levels of both (as Figure 19 shown). Compared with the control group of mice, after specifically knocking out HIF2α in T cells, the expression level of GATA3 in T cells decreased (as Figure 20 shown). The results of CUT&Tag experiment and luciferase reporter system demonstrated that HIF2α could bind to the GATA3 gene (as Figures 21 to 22 ).

[0142] 6. Co-regulatory function of HIF2α and GATA3

[0143] The CUT&Tag experiment demonstrated that HIF2α and GATA3 mainly bind to the promoter regions of genes, transcriptionally regulate gene expression, and have a synergistic regulatory function. They co-regulate 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), genes related to cell metabolism (Capn2, Npc2, Hmgcs1, Anxa11, Dgat1, Pmm1, Srebf2, Insig1, Cyp11a1, Fabp5, Fdps, Pparg), genes related to cell signaling and transduction (Rgs1, Adarb1, Gm2a, 2610507B11Rik, Vmp1, Ormld3, Rps6ka5, Dusp1, Rasgrp1, Tasp1, Ppp3ca, Dusp16, Crebl2, Itpr2, Dctn6), and some genes with less research reports (2610507B11Rik, AI467606, 1700061F12Rik, B4galnt4, Smco4, Cnot6l, Hip1, Nt5c3, Ttc39c). Through pathway enrichment analysis, it was found that these co-regulated genes were mainly enriched in phospholipid metabolism, T cell activation and differentiation, and signal transduction pathways (such as Figures 23 to 24 ).

[0144] The above experimental results showed that HIF2α and GATA3 co-regulate phospholipid metabolism in Th2 cells and HIF2α and GATA3 co-regulate IPMK.

[0145] In summary, in this example, through in vitro and in vivo experiments, it was found that the expression level of the HIF2α gene was the highest in pathogenic Th2 cells and played a positive regulatory role in the differentiation of Th2 cells. After knocking out the HIF2α gene, the infiltration of immune cells in the lungs of asthmatic mice decreased and the asthmatic inflammatory symptoms were alleviated. Further research found that HIF2α and GATA3 co-regulate IPMK and the PI3K-AKT signaling pathway.

[0146] Example 2 Mechanistic study of IPMK on Th2 differentiation

[0147] I. Experimental methods

[0148] 1. Explore the regulatory effect of IPMK on Th2 differentiation

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

[0150] Induce and differentiate CD4 + T cells in vitro. During the in vitro differentiation process, add the following to each culture medium: Th1 (IL-2, IL-12, anti-IL-4), Th2 (IL-2, IL-4, anti-IFNgama), Th17 (IL-6, TGFβ, anti-IL-4, anti-IFNgama), Treg (IL-2, TGFβ, anti-IL-4, anti-IFNgama). After culturing for 5 days, collect the cells and perform TCR stimulation for 0, 5, and 10 minutes. Stain with IPMK and pAKT flow antibodies, and use flow cytometry to detect the expression of IPMK and the phosphorylation level of AKT in various CD4 + T cells.

[0151] Sort stem-like Th2, pathogenic Th2, and Ikzf2 + Epas1 f / f Th2 cells from the dLNs of WT and CD4Cre + mice with induced asthma. Extract cellular RNA and reverse transcribe it to obtain cDNA, and use qPCR to detect the expression level of Ipmk in different Th2 cell subsets when HIF2α is absent.

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

[0153] In vitro, use a retroviral vector to coat IPMK and infect various CD4-positive T cells that are in the process of differentiation, namely Th1 (IL-2, IL-12, anti-IL-4), Th2 (IL-2, IL-4, anti-IFNgama), Th17 (IL-6, TGFβ, anti-IL-4, anti-IFNgama), Treg (IL-2, TGFβ, anti-IL-4, anti-IFNgama). Then, stimulate the cells with PMA, inomycin, monensin, etc. to produce effector cytokines, stain with the corresponding flow antibodies, and use flow cytometry to detect the differentiation efficiency of various CD4-positive helper T cells and Th2 subset cells.

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

[0155] In vitro, use a retroviral vector to coat IPMK and infect WT or HIF2α-deficient CD4 +T cells were stimulated with TCR for 10 minutes, total cellular proteins were extracted, and the phosphorylation levels of AKT, S6K, and FOXO1 were detected by WB.

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

[0157] Treat with vilazodone in vitro CD4 + T cells were stimulated with TCR for 10 minutes, total cellular proteins were extracted, and the phosphorylation levels of AKT, S6K, and FOXO1 were detected by WB.

[0158] Induce Th2 cell differentiation in vitro (IL-2, IL-4, anti-IFNgama), divided into 3 groups: WT group, HIF2α deletion group, and vilazodone treatment group. Use flow cytometry to sort pathogenic Th2 cells differentiated in vitro and perform lipid metabolism sequencing to analyze the levels of phosphatidylinositol in cells of each group.

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

[0160] Add vilazodone to each culture medium during in vitro differentiation, and detect the differentiation efficiency of various CD4+ helper T 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), Treg (IL-2, TGFβ, anti-IL-4, anti-IFNgama), then stimulate the cells with PMA, ionomycin, monensin, etc. to produce effector cytokines, stain with corresponding flow antibodies, and use flow cytometry to detect the differentiation efficiency of various CD4+ helper T cells and Th2 subset cells.

[0161] II. Experimental results

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

[0163] HIF2α deletion inhibits the PI3K-AKT signaling pathway, and the phosphorylation levels of AKT, S6K, and FOXO1 induced by TCR stimulation decreased significantly after HIF2α deletion (as Figure 25 ). CUT&Tag sequencing analysis showed that HIF2α and GATA3 jointly bind to the promoter region of Ipmk in pathogenic Th2 (as Figure 26 ). During Th2 differentiation, the expression of IPMK and the phosphorylation level of AKT were higher than those of other CD4+ Helper T cells were significantly higher (such as Figure 27 ). Ipmk was highly specifically expressed in pathogenic Th2 cells, and the deletion of HIF2α led to a decrease in the expression level of Ipmk in pathogenic Th2 (such as Figure 28 ). Overexpression of IPMK in T cells lacking HIF2α could compensate for the blocked Th2 differentiation, weakened PI3K-AKT signaling, and phosphatidylinositol levels (such as Figures 29 to 30 ). Treatment with the IPMK inhibitor vilazodone during in vitro differentiation could inhibit the differentiation of pathogenic Th2 cells, but had no effect on the differentiation of other helper T cells and other Th2 subsets (such as Figure 31 ). Adding vilazodone during TCR stimulation of T cells could inhibit PI3K-AKT signaling. At the same time, adding vilazodone during in vitro differentiation could reduce the phosphatidylinositol level in pathogenic Th2 cells (such as Figures 32 to 33 ).

[0164] The above experimental results showed that overexpression of IPMK could compensate for the blocked Th2 differentiation caused by the deletion of HIF2α, while treatment with the IPMK-specific inhibitor could inhibit the differentiation of pathogenic Th2. This indicated that IPMK was a potential target for the prevention and / or treatment of asthma.

[0165] Example 3 The IPMK-specific inhibitor vilazodone can alleviate asthma inflammatory symptoms

[0166] I. Experimental methods

[0167] 1. Explore the therapeutic effect of the IPMK-specific inhibitor vilazodone on asthma:

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

[0169] A sensitizer OVA and adjuvant alum were mixed and dissolved in PBS, and mice were intraperitoneally injected on days 0, 7, and 14. From day 28 to day 34, mice were nebulized with 3% OVA, and before each nebulization, mice were given the drug vilazodone by intraperitoneal injection at a dose of 5 mg / kg of body weight.

[0170] (2) After treatment with vilazodone, detect the pulmonary inflammation in mice:

[0171] The lungs were sectioned and observed for pulmonary cell infiltration and bronchial wall morphology after H&E staining.

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

[0173] The lungs of mice were lavaged with PBS, and the immune cells infiltrating in the lungs and alveolar bronchi were taken out. The cell count was performed, and the types of immune cells were detected by flow cytometry.

[0174] (4) After treatment of mice with vilazodone, 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 of the lungs were detected by ELISA.

[0176] II. Experimental Results

[0177] The experimental results showed that the number of immune cells infiltrating in the lungs of mice induced with asthma after treatment with vilazodone was lower than that of the control group mice, and the degree of tracheal wall thickening was reduced (as Figure 34 ). After treatment with vilazodone, the relative proportion and absolute number of CD45 + cells, eosinophils, and Th2 cells infiltrating in the lungs of mice induced with asthma were lower than those of the control group mice (as Figure 35 ). In addition, after treatment with vilazodone, the concentrations of IL-5 and IL-13 in the supernatant of bronchoalveolar lavage fluid of mice induced with asthma were lower than those of the control group mice (as Figure 36 ).

[0178] In summary, this example proved that the IPMK specific inhibitor vilazodone has a therapeutic effect on asthma.

[0179] In short, through single-cell sequencing data, the present invention found that Th2 cells are heterogeneous in patients with allergic asthma and patients with chronic rhinosinusitis with nasal polyps, and the transcription factor HIF2α is highly expressed in pathogenic Th2 cells. When interleukin-4 stimulates T cells, it can strongly induce the expression of the HIF2α gene (manifested as an increase in the mRNA level). Specific knockout of HIF2α in T cells can inhibit the differentiation of naive CD4-positive T cells into Th2 cells in vitro, but has no effect on the differentiation of other helper T cells.

[0180] Furthermore, through CUT&Tag experiments, it was found that HIF2α and GATA3 jointly regulate phospholipid metabolism in Th2 cells, and HIF2α can positively feedback regulate the expression of GATA3 to promote the differentiation of Th2 cells. Further, we found that during Th2 differentiation, HIF2α and GATA3 can synergistically regulate phosphatidylinositol kinase IPMK and upregulate the PI3K-AKT signaling pathway. In CD4 T cells lacking HIF2α + , overexpression of IPMK can compensate for the inhibited differentiation of pathogenic Th2 cells and the PI3K-AKT signal. Treatment of cells with the IPMK specific inhibitor vilazodone can specifically inhibit the differentiation of pathogenic Th2 cells.

[0181] Based on the above experimental results, targeting IPMK can treat and prevent diseases mediated mainly 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 dysdifferentiation, and anti-aging and related diseases.

[0182] To further verify whether asthma can be treated by targeting IPMK, the specific inhibitor vilazodone of IPMK was used to treat asthmatic mice, and it was found that the inflammatory symptoms of the mice treated with the drug were alleviated.

[0183] Thus, the present invention demonstrates that type II immune diseases, prevention and treatment of diseases caused by Th2 / ILC2 dysdifferentiation, as well as anti-tumor, anti-aging and anti-aging related diseases can be achieved by intervening in the IPMK target.

Claims

1. Use of a drug that intervenes in IPMK targets in the preparation of a drug for preventing and / or treating type II immune diseases and their complications, anti-tumor, anti-aging or anti-aging related diseases, characterized in that: The drug that intervenes in the IPMK target is selected from IPMK inhibitors, drugs that inhibit IPMK gene expression, drugs that knock out or knock down IPMK genes, and drugs that degrade IPMK.

2. The use according to claim 1, characterized in that: The IPMK inhibitor is selected from small molecule compounds, polypeptides, antibodies, preferably vilazodone, LI-2242, UNC7437, UNC9750, quercetin, and chlorogenic acid.

3. The use according to claim 1, characterized in that: The drug for inhibiting 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, ribozyme.

4. The use according to claim 1, characterized in that: The drug for knocking out or knocking 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 degrader, and preferably the protein degrader is selected from PROTAC and molecular glue.

6. The use according to claim 1, characterized in that: The drug for intervening the IPMK target is an antibody-drug conjugate, which is formed by connecting an antibody targeting the target cell and an active ingredient for intervening the IPMK target via 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 drug for treating type II immune diseases is a drug 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 the differentiation of stem cells into Th2 cells, inhibits the differentiation of stem cell-like Th2 cell subpopulations into pathogenic Th2 cell subpopulations, and inhibits the secretion of effector cytokines by Th2 cells.

9. The use according to any one of claims 1 to 6, characterized in that: 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 granulomatosis with polyangiitis, food allergies, 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: The 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 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 cancer, leukocyte cancer, esophageal cancer, breast cancer, muscle cancer, connective tissue cancer, intestinal cancer, lung cancer, adrenal cancer, thyroid cancer, kidney cancer or bone cancer; or it is glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma, choriocarcinoma, basal cell carcinoma of the skin, testicular seminoma.

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

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

14. Use of an IPMK agonist or a drug that overexpresses IPMK in the preparation of a drug for preventing and / or treating 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, polypeptide 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 regulating enhancer-induced overexpression, drugs for regulating transcription-induced overexpression, drugs for regulating transcription element-induced overexpression, small molecule drug response systems for overexpressing genes, Cre-loxp systems, Flp-frt systems, and Dre-rox systems.

17. The use according to claim 14, characterized in that: The IPMK agonist or the drug that overexpresses IPMK is an antibody-drug conjugate, which is formed by connecting an antibody targeting a target cell to 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 helminth infection, obesity, obesity-related complications, primary immune thrombocytopenia, 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 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 cancer, leukocyte cancer, esophageal cancer, breast cancer, muscle cancer, connective tissue cancer, intestinal cancer, lung cancer, adrenal cancer, thyroid cancer, kidney cancer or bone cancer; or it is glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma, choriocarcinoma, basal cell carcinoma of the skin, testicular seminoma.

Images