Use of a small molecule compound in the preparation of a medicament for treating GMFB-mediated diseases

Abstract

The present invention relates to the use of small molecule compounds in the preparation of drugs for treating GMFB-mediated diseases. In the present invention, 6 small molecules that inhibit GMFB activity and have no effect on cell viability are screened. The affinity between the small molecules and GMFB is confirmed to be dose-dependent by biacore. Finally, small molecule compounds that inhibit human GMFB protein are obtained. Through luciferase reporter gene detection, it is proved that the above small molecule compounds can effectively inhibit the expression of GMFB. Through CCK8 cell proliferation toxicity detection, it is proved that the above small molecule compounds have low cytotoxicity to cell proliferation ability at the effective concentration administered to the subject. It shows that the use of the above small molecule compounds can effectively inhibit the activity of GMFB, and it has low toxicity and can be used as a therapeutic agent for treating GMFB-related diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field related to GMFB-mediated disease treatment, and in particular relates to the application of a small molecule compound in the preparation of a drug for treating GMFB-mediated diseases. Background Art

[0002] The human glial maturation factor beta (GMFB) gene is located on the long arm of human chromosome 14, has 6 introns and 7 exons, and is 7 kb in length. This protein is an acidic protein with an isoelectric point of pH 4.9, contains 142 amino acid residues, and has a molecular weight of approximately 17 kDa. It is widely present in vertebrates and is a highly conserved protein. However, knocking out this protein does not result in lethality. GMFB is expressed on the cell surface of cells such as astrocytes and thymic epithelial cell lines, and can cause changes in the signals and metabolism of glioblastoma cells. Therefore, GMFB may act as an intracellular and extracellular signaling molecule and affect signal transduction through autocrine or paracrine means.

[0003] GMFB is involved in the growth and differentiation factors of glial cells and neurons and is upregulated under some neuroinflammatory and neurodegenerative conditions. The mechanism of action of GMFB may be to mediate apoptosis and regulate the expression of superoxide dismutase, granulocyte-macrophage colony-stimulating factor, and neurotrophin. Therefore, this target may be a promising target for treating neuroinflammatory and neurodegenerative diseases. GMFB is mainly expressed in the central nervous system (CNS), and its expression has also been detected in various tissues such as the colon, thymus, and kidney. Its abnormal expression may be involved in the occurrence and development of various diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), neuroinflammation, age-related macular degeneration (AMD), chronic inflammatory demyelinating diseases, central nervous system injury, etc. Therefore, inhibiting the expression of GMFB can achieve the treatment or prevention of diseases or disorders caused by its abnormal expression by inhibiting intracellular inflammatory pathways.

[0004] Therefore, studying compounds that can inhibit the expression of GMFB is of great significance for treating or preventing diseases or disorders caused by abnormal GMFB expression. Summary of the Invention

[0005] In view of the current situation that there is no effective treatment for GMFB-related diseases on the market, the present invention provides the application of a small molecule compound in the preparation of a drug for treating GMFB-mediated diseases.

[0006] The object of the present invention can be achieved by the following technical solutions:

[0007] The present invention first provides the use of small molecule compounds in the preparation of drugs for treating GMFB-mediated diseases, and the small molecule compounds are selected from one or more of the following substances:

[0008]

[0009] In the present invention, the small molecule compounds are 6 small molecule compounds with backbone diversity and specific binding to human / mouse GMFB selected from 60,000 small molecules through bioinformatics tools such as MOE-Site Finder, Chemdiv, Stardrop, and protein ligand interface fingerprint (PLIF). The above six small molecule compounds are numbered DS-4, DS-5, DS-8, DS-19, DS-29, and DS-30 respectively.

[0010] In one embodiment of the present invention, the GMFB-mediated diseases include: diabetic retinopathy, diabetic osteoporosis, diabetic nephropathy, insulin resistance, neurodegenerative diseases with high GMFB expression, and tumors with high GMFB expression.

[0011] In one embodiment of the present invention, the neurodegenerative diseases with high GMFB expression include Alzheimer's disease, Parkinson's disease, etc.

[0012] In one embodiment of the present invention, the use of the small molecule compounds in the preparation of drugs for treating GMFB-mediated diseases that play the role of GMFB inhibitors.

[0013] In one embodiment of the present invention, the small molecule compounds bind to GMFB protein in a dose-dependent manner.

[0014] In one embodiment of the present invention, the small molecule compounds further include compounds modified based on the following basic small molecule structures or their pharmaceutically acceptable salts or solvates or one or more pharmaceutically acceptable excipients:

[0015]

[0016] In one embodiment of the present invention, the concentration range of the small molecule compounds in the drug is 50 μM - 200 μM.

[0017] In one embodiment of the present invention, there is provided the use of the small molecule compound in the preparation of a medicament for treating GMFB-mediated diseases by acting as a GMFB inhibitor, wherein the following small molecule compounds, or compounds further modified based on the following basic small molecule structure, or pharmaceutically acceptable salts or solvates thereof, or one or more pharmaceutically acceptable excipients are directly involved in binding to the pocket1 region of GMFB:

[0018]

[0019] In one embodiment of the present invention, there is provided the use of the small molecule compound in the preparation of a medicament for treating GMFB-mediated diseases by acting as a GMFB inhibitor, wherein the following small molecule compounds, or compounds further modified based on the following basic small molecule structure, or pharmaceutically acceptable salts or solvates thereof, or one or more pharmaceutically acceptable excipients are directly involved in binding to the pocket2 region of GMFB:

[0020]

[0021] The present invention also provides a medicament for treating GMFB-mediated diseases, the medicament comprising a GMFB inhibitor or a compound further modified based on the basic structure of the GMFB inhibitor as the medicinal ingredient; the GMFB inhibitor is selected from one or more of the following substances:

[0022]

[0023] In one embodiment of the present invention, the medicament for treating GMFB-mediated diseases further comprises: its pharmaceutically acceptable salt or solvate, or one or more pharmaceutically acceptable excipients.

[0024] In one embodiment of the present invention, in the medicament for treating GMFB-mediated diseases, the concentration range of the GMFB inhibitor or the compound further modified based on the basic structure of the GMFB inhibitor as the medicinal ingredient is 50 μM - 200 μM.

[0025] The present invention also provides a method for treating GMFB-related diseases, the method comprising administering to a subject in need thereof an effective amount of a medicament comprising a GMFB inhibitor as the medicinal ingredient.

[0026] The present invention screens a small molecule compound library, screens 6 small molecules that inhibit GMFB activity and have no effect on cell viability, confirms the dose-dependence of the affinity between the small molecules and GMFB by biacore, and finally obtains small molecule compounds that inhibit human GMFB protein. The six small molecule compounds are numbered DS-4, DS-5, DS-8, DS-19, DS-29, and DS-30 respectively.

[0027] Through luciferase reporter gene assay, it was demonstrated that the above-mentioned small molecule compounds could effectively inhibit the expression of GMFB.

[0028] Through CCK8 cell proliferation cytotoxicity assay, it was demonstrated that the above-mentioned small molecule compounds had low cytotoxicity to cell proliferation ability at the effective concentration of the object administration.

[0029] The research of the present invention found that the GMFB small molecule inhibitor could significantly inhibit the activity of NFkappa B caused by the overexpression of GMFB in 293T cells. It was detected by CCK8 experiment that each inhibitor had little effect on the activity of 293T cells. The affinity between the inhibitor and GMFB showed a dose-dependent relationship (Kd values: DS-4 = 0.00005078, DS-5 = 0.0001616, DS-8 = 0.00001398, DS-19 = 0.00001809, DS-29 = 0.00006275, DS-30 = 0.0000221), indicating that the use of this small molecule inhibitor could effectively inhibit the activity of GMFB, with low toxicity, and could be used as a therapeutic agent for treating GMFB-related diseases. Brief Description of the Drawings

[0030] Figure 1 : Showing the inhibitory effect of 4 small molecule compounds on the expression of GMFB detected by luciferase reporter gene method at the acting concentration of 50 μM. (n≥3, Mean±SD, significant difference compared with the control group is indicated as *, ****P<0.0001, significant difference compared with the GMFB group is indicated as #, ##P<0.01, #P<0.0001) Figure 1 : Showing the inhibitory effect of 2 small molecule compounds on the expression of GMFB detected by luciferase reporter gene method at the acting concentration of 25 μM. (n≥3, Mean±SD, significant difference compared with the control group is indicated as *, ****P<0.0001, significant difference compared with the GMFB group is indicated as #, ##P<0.01, #P<0.0001)

[0031] Figure 2 : Showing the inhibitory effect of 2 small molecule compounds on the expression of GMFB detected by luciferase reporter gene method at the acting concentration of 25 μM. (n≥3, Mean±SD, significant difference compared with the control group is indicated as *, ****P<0.0001, significant difference compared with the GMFB group is indicated as #, ##P<0.01, #P<0.0001) Figure 1 : Showing the inhibitory effect of 2 small molecule compounds on the expression of GMFB detected by luciferase reporter gene method at the acting concentration of 25 μM. (n≥3, Mean±SD, significant difference compared with the control group is indicated as *, ****P<0.0001, significant difference compared with the GMFB group is indicated as #, ##P<0.01, #P<0.0001)

[0032] Figure 3 : Showing the CCK8 cell proliferation cytotoxicity assay Figure 1 The effect of six small molecule compounds on cell activity is shown. It shows that the absorbance of CCK8 is higher than 50% of the DMSO control group under the action of six small molecule compounds, proving that these 6 small molecule compounds have low cytotoxicity to cells. Detailed Description of the Invention

[0033] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0034] Among them, the small molecule compounds to be studied in the present invention were purchased from Shanghai TaoSu Biochemical Technology Co., Ltd., and their specific information is shown in Table 1.

[0035] Table 1: Chemical formulas and chemical structures of small molecule compounds DS-4, DS-5, DS-8, DS-19, DS-29 and DS-30

[0036]

[0037]

[0038] Preparation and storage of compound solution:

[0039] DMSO stock solution, the stock solution is 100 mM, the sample to be tested is 10 mM, stored at -20 °C within 3 months, and stored at -80 °C for more than three months.

[0040] Examples

[0041] 1. Luciferase reporter gene method

[0042] 1.1 Reagents: Use the Luciferase Reporter Assay Kit (Promega) and pNFκB-luc (reporter gene plasmid) (Beyotime). The experimental method is based on the Promega detection kit instructions.

[0043] Inoculation: Take 293T cells in the logarithmic growth phase and inoculate them into a 96-well plate at a cell density of 5x10 4 cells per well, with a total volume of 100 μl per well, and culture in a 37 °C incubator for 24 h.

[0044] 1.2 Transfection: Transfect 50 ng of human-derived GMFB wild-type plasmid (pc3.1(+)-hGMFB) and pNFκB-luc reporter gene plasmid into each well and continue to culture for 24 h. After 24 h, remove the cell culture medium, wash with 1xPBS, and then add 50 μM DS-2, 50 μM DS-4, 50 μM DS-8, 25 μM DS-19, 50 μM DS-29, 25 μM DS-30 to the experimental wells, with at least 3 replicate wells, and incubate for 24 h.

[0045] 1.3 Determination of Luciferase activity:

[0046] 1.3.1 Use the lysis buffer 5xPLB provided in the kit and dilute it to 1x. Remove the cell culture medium, wash away the residual culture medium with PBS, and add 1xPLB. Shake the 96-well plate on a shaker at room temperature for 15 min to fully lyse the cells. Place it at 4 °C and centrifuge at 100 g for 30 s. The supernatant is used for activity measurement. Add 40 μl of LARII (luciferase substrate) and 10 μl of cell lysate into a paper tube, mix well 2 - 3 times with a pipette tip, and measure and read the value in a luminometer, which is the firefly luciferase value.

[0047] Figure 1 Showing the inhibitory effect of 4 small molecule compounds on GMFB expression detected by the luciferase reporter gene method at a working concentration of 50 μM. (n≥3, Mean±SD, significant difference compared with the control group is indicated as *, ****P<0.0001, significant difference compared with the GMFB group is indicated as #, ##P<0.01, #P<0.0001). Figure 1 Showing the inhibitory effect of 2 small molecule compounds on GMFB expression detected by the luciferase reporter gene method at a working concentration of 25 μM. (n≥3, Mean±SD, significant difference compared with the control group is indicated as *, ****P<0.0001, significant difference compared with the GMFB group is indicated as #, ##P<0.01, #P<0.0001)

[0048] Figure 2 Showing the inhibitory effect of 2 small molecule compounds on GMFB expression detected by the luciferase reporter gene method at a working concentration of 25 μM. (n≥3, Mean±SD, significant difference compared with the control group is indicated as *, ****P<0.0001, significant difference compared with the GMFB group is indicated as #, ##P<0.01, #P<0.0001) Figure 1 Showing the inhibitory effect of 2 small molecule compounds on GMFB expression detected by the luciferase reporter gene method at a working concentration of 25 μM. (n≥3, Mean±SD, significant difference compared with the control group is indicated as *, ****P<0.0001, significant difference compared with the GMFB group is indicated as #, ##P<0.01, #P<0.0001)

[0049] Figure 3 Showing the effect of six small molecule compounds on cell viability detected by CCK8 cell proliferation toxicity assay Figure 1 The effect of six small molecule compounds on cell viability, showing that the absorbance of CCK8 is higher than 50% of the DMSO control group under the action of six small molecule compounds, proving that these 6 small molecule compounds have low cytotoxicity to cells.

[0050] The study found that the GMFB small molecule inhibitor can significantly inhibit the activity of NFkappa B caused by the overexpression of GMFB in 293T cells. It was detected by the CCK8 experiment that each inhibitor has little effect on the activity of 293T cells. The affinity between the inhibitor and GMFB shows a dose-dependent relationship (Kd values: DS-4 = 0.00005078, DS-5 = 0.0001616, DS-8 = 0.00001398, DS-19 = 0.00001809, DS-29 = 0.00006275, DS-30 = 0.0000221), indicating that the use of this small molecule inhibitor can effectively inhibit the activity of GMFB, with low toxicity, and can be used as a therapeutic agent for treating GMFB-related diseases.

[0051] The above description of the embodiments is provided to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. A drug for treating GMFB-mediated diseases, characterized in that, the drug uses a GMFB inhibitor as a medicinal ingredient; the GMFB inhibitor is selected from one or more of the following substances: 、 ； the GMFB-mediated diseases are diabetic osteoporosis or diabetic retinopathy.

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