Use of a clk2 inhibitor in the manufacture of a medicament for treating iga nephropathy

Abstract

The application belongs to the technical field of disease drug treatment, and particularly relates to application of a CLK2 inhibitor in preparation of a drug for treating IgA nephropathy. The CLK2 inhibitor (such as ganoderic acid A methyl ester) can significantly reduce 24hUTP, 24hmAlb, BUN and Scr levels of an IgA nephropathy model, and IGA deposition in the inside of a glomerulus, so as to achieve the purpose of effectively treating IgA nephropathy.

Description

Technical Field

[0001] This invention belongs to the field of disease drug treatment technology, specifically relating to the application of a CLK2 inhibitor in the preparation of a drug for treating IgA nephropathy. Background Technology

[0002] IgA nephropathy is the most common primary glomerular disease, characterized by predominantly IgA deposition in the glomerular mesangial area, with or without the deposition of other immunoglobulins. Currently, there are no effective drugs for treating IgA nephropathy in clinical practice.

[0003] The CLK (CDK-like kinase) family of protein kinases is a dual-specificity protein kinase that regulates intracellular signal transduction through phosphorylation of substrate proteins via tyrosine, serine / threonine residues. This family can be divided into four subtypes: CLK1, CLK2, CLK3, and CLK4. The CLK2 subtype has been found in most eukaryotes and regulates alternative RNA splicing by phosphorylating the domains of SR proteins. Studies have shown that CLK2 is a treatment option for osteoarthritis (Sun YQ, Hu TX, Zhang MD, et al. Structure-Guided Discovery of Potent and Selective CLK2 Inhibitors for the Treatment of Knee Osteoarthritis. J. Med. Chem. 2024, 67, 4603-4623), breast cancer (Riggs JR, Nagy M, Elsner J, et al. The Discovery of a Dual TTK Protein Kinase / CDC2-Like Kinase (CLK2) Inhibitor for the Treatment of Triple Negative Breast Cancer Initiated from a Phenotypic Screen. J. Med. Chem. 2017, 60, 8989-9002), and lung cancer (Hu TX, Huang JL, Chen R, et al. Discovery of CLKs inhibitors for the treatment of non-small cell lung cancer). Potential target protein of cancer.Eur.J.Med.Chem.2024,280,116952.

[0004] However, there are no reports of CLK2 inhibitors being used to treat IgA nephropathy. Summary of the Invention

[0005] This invention discovers that CLK2 inhibitors (such as methyl ganoderic acid A) can significantly reduce the levels of 24hUTP, 24hmAlb, BUN, and Scr in an IgA nephropathy model, as well as the deposition of IGA within the glomeruli, thereby achieving the goal of effectively treating IgA nephropathy.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions:

[0007] One aspect of this invention is the use of the CLK2 inhibitor of this invention in the preparation of a drug for treating IgA nephropathy.

[0008] Preferably, in the above applications, the CLK2 inhibitor is methyl ganoderic acid A.

[0009] Preferably, the above applications include one or more of the following: (i) the use of CLK2 inhibitors in the preparation of medicaments for reducing 24hUTP levels; (ii) the use of CLK2 inhibitors in the preparation of medicaments for reducing 24hmAlb levels; (iii) the use of CLK2 inhibitors in the preparation of medicaments for reducing BUN levels; (iv) the use of CLK2 inhibitors in the preparation of medicaments for reducing Scr levels; and (v) the use of CLK2 inhibitors in the preparation of medicaments for reducing IGA deposition within the glomeruli.

[0010] Preferably, in the above applications, the dosage form of the drug includes injections, oral liquids, patches, tablets, powders, or capsules.

[0011] Another aspect of the present invention provides the application of methyl ganoderic acid A in the preparation of health food, wherein the health food has one or more of the following functions: (i) the functional food has the function of maintaining a healthy 24hUTP level; (ii) the functional food has the function of maintaining a healthy 24hAlb level; (iii) the functional food has the function of maintaining a healthy BUN level; (iv) the functional food has the function of maintaining a healthy Scr level.

[0012] Preferably, in the above applications, the dosage form of the health food includes liquid, patch, tablet, powder or capsule.

[0013] In another aspect, the present invention provides the use of methyl ganoderic acid A in the preparation of or as a CLK2 inhibitor.

[0014] The beneficial effects of this invention include: CLK2 inhibitors (such as methyl ganoderic acid A) can significantly reduce the levels of 24hUTP, 24hmAlb, BUN and Scr in IgA nephropathy models, as well as the deposition of IGA in the glomeruli, thereby achieving the goal of effectively treating IgA nephropathy. Attached Figure Description

[0015] Figure 1 This is the result of a new mode of ligand-CLK2 binding between methyl ganoderic acid A and CLK2, as well as a unique and novel hydrogen bonding interaction. Detailed Implementation

[0016] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0017] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0018] This invention provides an application of the CLK2 inhibitor of this invention in the preparation of a drug for treating IgA nephropathy.

[0019] In some specific examples, the CLK2 inhibitor in the above applications is methyl ganoderic acid A.

[0020] Specifically, the structural formula of methyl ganoderic acid A is shown below: Methyl ganoderic acid is a known natural product.

[0021] In some specific examples, the above applications include one or more of the following: (i) the use of CLK2 inhibitors in the preparation of medicaments for reducing 24hUTP levels; (ii) the use of CLK2 inhibitors in the preparation of medicaments for reducing 24hmAlb levels; (iii) the use of CLK2 inhibitors in the preparation of medicaments for reducing BUN levels; (iv) the use of CLK2 inhibitors in the preparation of medicaments for reducing Scr levels; and (v) the use of CLK2 inhibitors in the preparation of medicaments for reducing IGA deposition within the glomeruli.

[0022] It should be noted that CLK2 inhibitors (such as methyl ganoderic acid A) can significantly reduce the levels of 24hUTP, 24hmAlb, BUN, and Scr in IgA nephropathy models, as well as IGA deposition within the glomeruli, thereby achieving the goal of effectively treating IgA nephropathy.

[0023] In some specific examples, the dosage forms of the drugs in the above applications include injections, oral liquids, patches, tablets, powders, or capsules.

[0024] This invention also provides an application of methyl ganoderic acid A in the preparation of health food, which has one or more of the following functions: (i) the functional food has the function of maintaining 24hUTP health level; (ii) the functional food has the function of maintaining 24hmAlb health level; (iii) the functional food has the function of maintaining BUN health level; (iv) the functional food has the function of maintaining Scr health level.

[0025] It should be noted that methyl ganoderic acid A can maintain 24hUTP, 24hmAlb, BUN and Scr at healthy levels, so it can be prepared into health food products.

[0026] In some specific examples, the dosage forms of health foods mentioned above include liquids, patches, tablets, powders, or capsules.

[0027] This invention also provides the use of methyl ganoderic acid A in the preparation of or as a CLK2 inhibitor.

[0028] It should be noted that methyl ganoderic acid A can selectively inhibit CLK2 and form a new ligand-CLK2 binding mode with CLK2, exhibiting a unique and novel hydrogen bonding effect.

[0029] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0030] Example 1: Enzyme selectivity of methyl ganoderic acid A for CLK2 and CLK3

[0031] The inhibitory rate of methyl ganoderic acid A on kinases was detected using the Z'LYTE™ kit (ThermoFisher, USA) based on the FRET principle. The in vitro inhibitory activities of the compound on CLK2 and CLK3 enzymes were studied. Specifically, methyl ganoderic acid A was serially diluted 4-fold with DMSO to obtain methyl ganoderic acid A solutions. The final initial concentrations of methyl ganoderic acid A were 64 μM, 16 μM, 4 μM, 1 μM, 0.25 μM, 0.0625 μM, and 0.015625 μM. Then, methyl ganoderic acid A was added to kinase reaction buffer (50 mM Hepes, 10 mM MgCl2, 0.01% Brij, 1 mM EGTA, 2 mM DTT) and incubated at 25°C for 20 min. 2 μL of kinase was added to each well of a 96-well plate and incubated at 25°C for 40 min. The RLU (Relative Luminous Intake) was read using a BMG multi-functional plate reader. The luminescence unit (FMU) signal, with signal intensity used to characterize the degree of kinase activity, was used to calculate the IC50 of the compound using Prism10 software. 50 Value; additionally, the CLK2 inhibitor Lorecivivint was set as a control.

[0032] The test results are shown in Table 1 below.

[0033] Table 1. Enzyme activities of methyl ganoderic acid A on CLK2 and CLK3.

[0034]

[0035] The experimental results in Table 1 show that methyl ganoderic acid A has strong inhibitory activity against CLK2, IC50... 50 The value was 0.69 μM, indicating that methyl ganoderic acid A is a CLK2 inhibitor; however, methyl ganoderic acid A has very weak inhibitory activity against CLK3, with an IC50 value of 0.69 μM. 50 With a value >20μM, methyl ganoderic acid A is a selective CLK2 inhibitor.

[0036] Example 2: A novel ligand-CLK2 binding mechanism and unique hydrogen bonding between methyl ganoderic acid A and CLK2.

[0037] Download the CLK2 protein structure and perform docking experiments (PDB: 6FYK). The results are as follows: Figure 1As shown, the results indicate that the triterpenoid compound methyl ganoderic acid A can form three unique and novel hydrogen bond interactions with Asp327, Leu169, and Lys292 of the CLK2 protein; methyl ganoderic acid A can also form Alkyl and Pi-Alkyl interactions with Phe174, Val227, Leu246, Ala191, Val177, Phe243, Val326, and Leu297 of the CLK2 protein. These novel hydrogen bond interactions and new binding modes of methyl ganoderic acid A-CLK2 can be further optimized for structural design and drug development.

[0038] Example 3: In vivo activity of methyl ganoderic acid, a novel triterpenoid CLK2 inhibitor, against IgA nephropathy.

[0039] A miR-23b- / - mouse model of IgA nephropathy was selected (model construction method referred to the literature "Clinical Nephrology Journal", 2024, 24:1017-1027). C57BL / 6J mice (provided by Beijing Huafukang Biotechnology Co., Ltd.) were selected as the blank control group. Mice of the same age were housed in sterile, individually ventilated cages under specific pathogen-free conditions and fed with water and sterile feed. The miR-23b- / - mice were divided into a model group and a methyl ganoderic acid A group, with 10 mice in each group; in addition, 10 C57BL / 6J mice served as the blank control group. The mice were uniformly housed until 20 weeks of age and then administered the drug by gavage. The methyl ganoderic acid A group was administered 30 mg / kg by gavage daily for 8 consecutive weeks. The model group and the blank control group were given the same amount of distilled water. After 8 weeks of intervention, urine was collected from the mice using metabolic cages, and blood and kidney samples were collected.

[0040] Urine was collected over 24 hours from the collection port at the lower end of the metabolic cage. After centrifugation for 10 minutes, the supernatant was used to detect urinary parameters. 24hUTP (24-hour urinary protein quantification) and 24hmAlb (24-hour urinary microalbumin) were detected using an automated biochemical analyzer (Shenzhen Mindray Bio-Medical Electronics Co., Ltd., model: BS-2000). Serum parameters were detected using a microplate method (China Enzyme-Linked Biotechnology Co., Ltd., catalog number ml092663), measuring changes in serum BUN (blood urea nitrogen) and Scr (serum creatinine) to evaluate renal function. Immunofluorescence staining (referencing the literature "Chinese Journal of Pharmacology and Toxicology", 2024, 40, 2037-2041) was used to analyze IGA deposition, and the IOD / Area value (Area being the area of ​​the selected region, IOD being the sum of fluorescence densities) was calculated to evaluate whether the drug reduced IGA deposition within the glomeruli. The results are shown in Table 2.

[0041]

[0042] Table 2 shows that, compared with the blank control group, the model group had significantly elevated 24hUTP, 24hmAlb, BUN, and Scr, and severe IgA deposition within the glomeruli. Compared with the model group, after administration of methyl ganoderic acid A, 24hUTP, 24hmAlb, BUN, Scr, and IgA deposition within the glomeruli were all significantly reduced. Based on the animal experimental results, methyl ganoderic acid A can be used to prepare drugs for treating IgA nephropathy.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Use of a CLK2 inhibitor for the manufacture of a medicament for the treatment of IgA nephropathy, wherein the CLK2 inhibitor is ganodermic acid A methyl ester.

2. Use according to claim 1, characterized in that, The use includes one or more of the following uses: (i) Use of a CLK2 inhibitor for the manufacture of a medicament for reducing 24h UTP levels; (ii) Use of a CLK2 inhibitor for the manufacture of a medicament for reducing 24h mAlb levels; (iii) Use of a CLK2 inhibitor for the manufacture of a medicament for reducing BUN levels; (iv) Use of a CLK2 inhibitor for the manufacture of a medicament for reducing Scr levels; (v) Use of a CLK2 inhibitor for the manufacture of a medicament for reducing intraglomerular IGA deposition.

3. Use according to claim 1 or 2, characterized in that, The dosage form of the medicament includes an injection, an oral liquid, a tablet, a powder, or a capsule.

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