Use of mln4924 in the preparation of a medicament for n4bp1 deficiency-induced immune activation-related diseases
Patent Information
- Application Number
- CN202611041024.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-07-14
- Publication Date
- 2026-08-21
AI Technical Summary
[0005]针对现有技术中缺乏针对N4BP1缺陷诱导免疫激活状态的有效小分子用途方案的问题，本发明提供MLN4924在制备相关疾病药物中的应用，尤其提供其在伴随IL-17信号通路异常活化和Act1核定位增强的炎症性皮肤疾病中的应用
[0018] This application discloses that MLN4924 can significantly inhibit the expression of abnormally elevated downstream target genes of the IL-17 signaling pathway in N4BP1 deficiency, IL-17 stimulation models, and Act1 overexpression models. The mechanism is that MLN4924 inhibits the function of Act1 protein by suppressing its nuclear localization and accumulation. Furthermore, topical application of MLN4924 significantly alleviates IMQ-induced psoriatic-like skin lesions in N4BP1-deficient mice. Therefore, MLN4924 can be used to prepare drugs for the prevention or treatment of N4BP1 deficiency-induced immune activation-related diseases, especially inflammatory skin diseases accompanied by abnormal IL-17 activation.
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Figure CN122604793A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of pharmaceutical technology, and in particular to the application of MLN4924 in the preparation of drugs for diseases related to N4BP1 deficiency-induced immune activation. Background Technology
[0002] N4BP1 (NEDD4 binding protein 1) is an important negative regulator of the body's immune response. Studies have shown that N4BP1 deficiency can lead to abnormally elevated levels of target genes related to the IL-17 signaling pathway and increase susceptibility to inflammatory skin lesions. In the context of reduced or absent N4BP1 function, inflammatory signals are amplified, chemokines are upregulated, and skin barrier damage is exacerbated, suggesting that N4BP1 deficiency is closely related to the development and progression of immune activation-related diseases.
[0003] Act1 (NF-κB activator 1) is a key adaptor protein in the IL-17 signaling pathway. Aberrant activation of Act1 can induce NF-κB and MAPK signaling cascades, further promoting the expression of inflammatory target genes such as CXCL1, CCL20, CCL2, MMP9, CXCL8, and S100A8, thereby exacerbating local inflammatory damage. Therefore, intervention targeting Act1 function is an important entry point for controlling N4BP1 deficiency-related immune overactivation.
[0004] Current treatment strategies targeting the IL-17 axis are mainly based on biologics, which suffer from limitations in administration methods, high costs, and complex long-term management. Although some small molecules have been shown to exert inhibitory effects by interfering with Act1-related interactions, there is still a lack of drug use protocols with clear mechanisms of action and in vitro and in vivo experimental support in the context of abnormal immune activation induced by N4BP1 deficiency. Summary of the Invention
[0005] To address the lack of effective small molecule applications for N4BP1-deficient induced immune activation in existing technologies, this invention provides the application of MLN4924 in the preparation of drugs for related diseases, particularly its application in inflammatory skin diseases accompanied by abnormal activation of the IL-17 signaling pathway and enhanced nuclear localization of Act1.
[0006] To achieve the above objectives, this application provides the following technical solution:
[0007] Application of MLN4924 in the preparation of drugs for the treatment and / or prevention of N4BP1 deficiency-induced immune activation-related diseases.
[0008] Preferably, the MLN4924 is used to exert a pharmacological effect by inhibiting the nuclear localization and / or nuclear enrichment of Act1 protein.
[0009] Preferably, the drug is used to downregulate the expression of one or more downstream target genes of the IL-17 signaling pathway.
[0010] Preferably, the target gene includes at least one of CXCL1, CCL20, CCL2, MMP9, CXCL8 and S100A8.
[0011] Preferably, the N4BP1 deficiency-induced immune activation-related diseases are diseases related to abnormal activation of the IL-17 signaling pathway.
[0012] Preferably, the disease includes one or more of psoriasis, psoriasis-like dermatitis, and inflammatory bowel disease.
[0013] This application also provides a medicament for treating immune activation-related diseases, the medicament comprising MLN4924.
[0014] Preferably, the drug also includes other medically acceptable excipients.
[0015] Preferably, the drug is used to inhibit Act1 nuclear localization and downregulate abnormal activation of the IL-17 signaling pathway.
[0016] Preferably, the drug is one of an injection, a powder, or a cell treatment working solution.
[0017] Compared with the prior art, this application has at least the following beneficial effects:
[0018] This application discloses that MLN4924 can significantly inhibit the expression of abnormally elevated downstream target genes of the IL-17 signaling pathway in N4BP1 deficiency, IL-17 stimulation models, and Act1 overexpression models. The mechanism is that MLN4924 inhibits the function of Act1 protein by suppressing its nuclear localization and accumulation. Furthermore, topical application of MLN4924 significantly alleviates IMQ-induced psoriatic-like skin lesions in N4BP1-deficient mice. Therefore, MLN4924 can be used to prepare drugs for the prevention or treatment of N4BP1 deficiency-induced immune activation-related diseases, especially inflammatory skin diseases accompanied by abnormal IL-17 activation. Attached Figure Description
[0019] Figure 1 : MLN4924 upregulated target genes after N4BP1 knockout reversal;
[0020] Figure 2 MLN4924 reverses the upregulated target genes of IL-17 treatment;
[0021] Figure 3 The target genes upregulated after N4BP1 knockout reversal in MLN4924 are not related to MAPK;
[0022] Figure 4 MLN4924 treatment did not downregulate total Act1 protein;
[0023] Figure 5 MLN4924 inhibits nuclear localization of Act1 protein;
[0024] Figure 6 MLN4924 inhibits the upregulation of IL-17 target genes induced by Act1 overexpression;
[0025] Figure 7 MLN4924 showed significant inhibitory effects in cells with varying Act1 protein expression levels.
[0026] Figure 8 MLN4924 can inhibit IL-17 target genes that are overregulated by Act1 through Act1 nuclear localization;
[0027] Figure 9 MLN4924 alleviates psoriasis-like skin lesions induced by IMQ in N4BP1-deficient mice. Detailed Implementation
[0028] This application provides the use of MLN4924 in the preparation of drugs for the treatment and / or prevention of N4BP1 deficiency-induced immune activation-related diseases, through which MLN4924 exerts its pharmacological effects by inhibiting the nuclear localization and / or nuclear enrichment of Act1 protein.
[0029] MLN4924 is a small molecule inhibitor with potential applications in inflammation regulation. The molecular formula of MLN4924 is C2. 21 H 25 N5O4S
[0030] Structural formula:
[0031]
[0032] In one embodiment, the drug is used to downregulate the expression of one or more downstream target genes of the IL-17 signaling pathway, the target genes including at least one of CXCL1, CCL20, CCL2, MMP9, CXCL8 and S100A8.
[0033] In one embodiment, the N4BP1 deficiency-induced immune activation-related disease is a disease associated with abnormal activation of the IL-17 signaling pathway. Specifically, the disease includes one or more of psoriasis, psoriatic dermatitis, and inflammatory bowel disease.
[0034] This application also provides a medicament for treating immune activation-related diseases, the medicament comprising MLN4924, and in one embodiment, the medicament further comprising other medically acceptable excipients.
[0035] The drug is used to inhibit Act1 nuclear localization and downregulate abnormal activation of the IL-17 signaling pathway.
[0036] The above content will be explained in conjunction with specific verification experiments below.
[0037] In this application, N4BP1 KO cells were treated with MLN4924, and the changes in IL-17 target gene expression were detected at different treatment times. Furthermore, the cells were pretreated with MLN4924 and then stimulated with IL-17 to analyze the effect of IL-17 stimulation on the induced expression of target genes.
[0038] MLN4924 was used to treat HEK293T cells in control and N4BP1 KO cells to detect the phosphorylation levels of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (P38), as well as the expression level and subcellular localization of Act1. Nucleocytoplasmic separation experiments were conducted to observe the changes in the distribution of Act1 between the nucleus and cytoplasm after MLN4924 treatment.
[0039] Act1 was overexpressed in HEK293T cells, and the mRNA and protein levels of IL-17 target genes were detected. After treatment with MLN4924, changes in target gene and Act1 expression were analyzed. By transfecting different doses of Act1 plasmid and treating with MLN4924, it was further clarified whether the effect of MLN4924 depends on the total amount of Act1 protein.
[0040] In HEK293T cells overexpressing Act1, nucleocytoplasmic separation was performed after treatment with MLN4924 to detect Act1 protein distribution. Changes in Act1 localization and IL-17 target gene expression were analyzed using MLN4924 or nuclear export inhibitors. The effects of MLN4924 and nuclear export inhibitors on target gene expression were also verified in N4BP1 knockout cells.
[0041] In WT and N4BP1 KO mice with IMQ-induced psoriasis model, MLN4924 was applied topically three days before modeling to assess the degree of skin damage and the expression level of IL-17 target genes.
[0042] The N4BP1 defect models used in this application include established N4BP1 knockout HaCaT cells, N4BP1 knockout NIH3T3 cells, and N4BP1 gene knockout mouse models; MLN4924 can be prepared as a 10 μM cell treatment working solution, and local administration to animals can be carried out at 10 μmol / kg.
[0043] The specific experimental steps are as follows:
[0044] Experiment 1: Cell Culture
[0045] NIH3T3 cells, HEK293T cells, and HeLa cells were cultured in DMEM complete medium (containing a mixture of 10% fetal bovine serum, 1% penicillin, and streptomycin) at 37°C in a 5% CO2 incubator. The medium was changed every 2 days. HaCaT cells were cultured in 1640 complete medium. When the cells reached a confluence density of 80%–90%, they were digested with trypsin, collected, and passaged at a ratio of 1:3.
[0046] Experiment 2: Construction of Stable Cell Lines
[0047] To construct a stable cell line, cells were first seeded in 6-well plates and transfected after adhesion. Plasmid and Lipofectamine 2000 were mixed at a volume ratio of 1:1 to 1:2, incubated at room temperature for 5 min, and then a suitable amount of low-serum medium was added, followed by incubation for 20 min to form a transfection complex. The medium was then changed, and the mixture was added to the cells and gently mixed. The cells were cultured for 6-8 h, followed by a medium change. Green fluorescence expression was observed 48-72 h after transfection, and positive cells were selected stepwise using low to high concentrations of puromycin. After all WT cells in the control group died, the cells were expanded by changing the medium. Once the cells reached confluence, they were cryopreserved, and the knockout effect was verified by Western blot.
[0048] Experiment 3, Western Blot:
[0049] After aspirating the culture medium, wash the cells once with PBS, then digest for about 2 minutes. Add culture medium to stop the digestion and mix well by pipetting. Transfer to a centrifuge tube, centrifuge, and discard the supernatant. Add lysis buffer prepared at RIPA:PMSF = 100:1, place on ice, and lyse on a shaker at 200 rpm for 30 minutes. After lysis, centrifuge at 12000 rpm for 15 minutes, collect the supernatant into a new EP tube, add 5× loading buffer, mix well, and heat at 95°C for 5 minutes. Then perform SDS-PAGE electrophoresis. First, pass through a stacking gel at 80 V. After the markers are clearly separated, increase the V to 120 V until the bromophenol blue is close to the bottom of the gel. Transfer the protein to an NC membrane at 300 mA for 1 hour. Block the membrane with 5% skim milk powder for 2 hours, add primary antibody, and incubate overnight at 4°C. The next day, wash the membrane with TBST, add the corresponding secondary antibody, incubate at room temperature for 2 hours, wash again, and then develop.
[0050] Experiment 4, qPCR:
[0051] After lysis with TRIzol and thorough mixing, intracellular RNA was released. Chloroform was added, the mixture was shaken, and allowed to separate into layers. The upper aqueous phase was transferred to a new tube, and an equal volume of isopropanol was added to precipitate RNA. The precipitate was obtained by centrifugation, washed with 75% ethanol to remove impurities and salt ions, dried, and dissolved in RNase-free water. The concentration and purity were then measured. Following the kit instructions, genomic DNA was first removed, followed by reverse transcription to synthesize cDNA. cDNA, specific primers, amplification reagents, and RNase-free water were added to the qPCR reaction system. Amplification was performed according to the set program, and the expression of the target gene was relatively quantified based on the Ct value and an internal reference gene.
[0052] Experiment 5, IF:
[0053] Sterile cell slides were placed in 24-well plates, and prepared single-cell suspensions were seeded onto the slides at an appropriate density for culture. Once cell confluence reached 40%-60%, inhibitors or stimulating factors were added for continued culture. The culture medium was then discarded, and the cells were washed three times with PBS, fixed with 4% paraformaldehyde at room temperature for 20 min, washed again with PBS, and permeabilized with PBS containing 0.1% Triton X-100 for 10-15 min. After permeabilization, the cells were blocked with 5% bovine serum albumin for 1 h, followed by the addition of proportionally diluted primary antibody and incubation overnight in a humidified chamber at 4°C. The next day, the primary antibody was recovered and thoroughly washed, and the corresponding fluorescently labeled secondary antibody was added, followed by incubation at room temperature in the dark for 1 h. The cell nuclei were then stained with Hoechst stain in the dark, washed with PBS, and fitted with an anti-fluorescence quenching mounting medium. The slides were then inverted and mounted on a glass slide, and finally observed and imaged under a laser confocal microscope through a suitable channel.
[0054] Experiment 6: Animal Culture and IMQ Modeling
[0055] In animal experiments, 8-10 week old, similarly sized, and same-sex WT and N4BP1 KO mice were selected. The fur on the experimental area of the back was first shaved clean. Then, the mice were randomly assigned to groups, and 62.5 mg of imiquimod cream was applied evenly to the bare back skin at a fixed time each day for 6-8 consecutive days. The mice's mental state and changes in skin erythema, scaling, and thickening were continuously observed. From day 1 to 3 after IMQ modeling, MLN4924 or Iguratimod was administered locally according to the experimental protocol. Twenty-four hours after the last treatment, the mice were sacrificed, and skin tissue from the treatment site on the back was collected. A portion of the samples was rapidly placed in cryovials, flash-frozen on dry ice, and then stored at −80°C for subsequent protein or RNA extraction.
[0056] II. Experimental Conclusions
[0057] 1. Please refer to Figure 1 and Figure 2 The inhibitor MLN4924 significantly suppressed the upregulation of target genes induced by N4BP1 knockout in a time-dependent manner. Furthermore, the inhibitory effect of MLN4924 remained significant in both wild-type and IL-17-stimulated conditions.
[0058] 2. For example Figure 3 and Figure 4 As shown, after MLN4924 treatment, p-ERK and p-P38 were downregulated in the short term (1-8 h) and significantly increased in the long term (24 h), mainly located in the nucleus; the total amount of Act1 protein decreased in 24 h, while its nuclear protein amount was significantly reduced in the short term and remained at low expression.
[0059] 3. Please refer to Figure 5 After treatment with MLN4924, the amount of Act1 nuclear protein decreased significantly in a short period of time and remained at a low level.
[0060] 4. Act1 overexpression strongly upregulates IL-17 target genes, an effect that can be significantly inhibited by MLN4924. Even at different Act1 expression levels, MLN4924 can bring target gene expression close to basal levels, suggesting that its effect is independent of the total Act1 protein (see...). Figure 6 , Figure 7 ).
[0061] 5. MLN4924 and nuclear export inhibitors exhibit similar effects, both rapidly and persistently reducing the localization of Act1 in the cell nucleus and downregulating the expression of target genes (see...). Figure 8 ).
[0062] 6. Topical application of MLN4924 significantly alleviated IMQ-induced psoriatic-like skin lesions and upregulation of IL-17 target genes in mice, with more pronounced effects in N4BP1 KO mice (see [link to study]). Figure 9 ).
[0063] In summary, this application presents MLN4924, which significantly inhibits the expression of abnormally elevated downstream target genes of the IL-17 signaling pathway in N4BP1 deficiency, IL-17 stimulation models, and Act1 overexpression models. The mechanism involves MLN4924 inhibiting Act1 protein function by suppressing its nuclear localization and accumulation. Furthermore, topical application of MLN4924 significantly alleviates IMQ-induced psoriatic-like skin lesions in N4BP1-deficient mice. Therefore, MLN4924 can be used to prepare drugs for the prevention or treatment of N4BP1 deficiency-induced immune activation-related diseases, especially inflammatory skin diseases accompanied by abnormal IL-17 activation.
Claims
1. Application of MLN4924 in the preparation of drugs for the treatment and / or prevention of N4BP1 deficiency-induced immune activation-related diseases.
2. The use of MLN4924 according to claim 1 in the preparation of drugs for treating and / or preventing N4BP1 deficiency-induced immune activation-related diseases, characterized in that: The MLN4924 is used to exert its pharmacological effect by inhibiting the nuclear localization and / or nuclear enrichment of Act1 protein.
3. The use of MLN4924 according to claim 1 in the preparation of drugs for treating and / or preventing N4BP1 deficiency-induced immune activation-related diseases, characterized in that: The drug is used to downregulate the expression of one or more downstream target genes of the IL-17 signaling pathway.
4. The use of MLN4924 according to claim 3 in the preparation of drugs for treating and / or preventing N4BP1 deficiency-induced immune activation-related diseases, characterized in that: The target genes include at least one of CXCL1, CCL20, CCL2, MMP9, CXCL8, and S100A8.
5. The use of MLN4924 according to claim 1 in the preparation of drugs for treating and / or preventing N4BP1 deficiency-induced immune activation-related diseases, characterized in that: The N4BP1 deficiency-induced immune activation-related diseases are diseases associated with abnormal activation of the IL-17 signaling pathway.
6. The use of MLN4924 according to claim 1 in the preparation of drugs for treating and / or preventing N4BP1 deficiency-induced immune activation-related diseases, characterized in that: The diseases mentioned include one or more of psoriasis, psoriasis-like dermatitis, and inflammatory bowel disease.
7. A medicament for treating immune activation-related diseases, characterized in that: The drug contains MLN4924.
8. A medicament for treating immune activation-related diseases according to claim 7, characterized in that: The drug also includes other medically acceptable excipients.
9. A medicament for treating immune activation-related diseases according to claim 8, characterized in that: The drug is used to inhibit Act1 nuclear localization and downregulate abnormal activation of the IL-17 signaling pathway.
10. A medicament for treating immune activation-related diseases according to claim 7, characterized in that: The drug is one of the following: injection, powder, or cell treatment working solution.