Application of CLK2 inhibitor in preparation of medicine for treating IgA nephropathy

Abstract

The invention 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 be used for remarkably reducing the levels of 24h UTP, 24h mAlb, BUN and Scr of an IgA nephropathy model and IGA deposition in glomerulus, so that the aim of effectively treating the IgA nephropathy is fulfilled.

Description

Technical Field

[0001] The present invention belongs to the technical field of disease drug treatment, and in particular relates to the use of a CLK2 inhibitor in the preparation of a drug for treating IgA nephropathy. Background Art

[0002] IgA nephropathy is the most common primary glomerular disease, which refers to a primary glomerular disease characterized by the deposition of IgA or IgA in the glomerular mesangium, with or without the deposition of other immunoglobulins in the glomerular mesangium. Currently, there is a lack of effective drugs for the treatment of IgA nephropathy in clinical practice.

[0003] The protein kinase family CLK (CDK-like kinase) is a dual-specific protein kinase that regulates intracellular signal transduction by phosphorylating substrate proteins at tyrosine and serine / threonine residues, catalyzing the serine residues of proteins. The family can be divided into four subtypes: CLK1, CLK2, CLK3 and CLK4. The CLK2 subtype is found in most eukaryotic organisms, and it phosphorylates the domain of SR proteins to regulate RNA selective splicing. Studies have shown that CLK2 is a promising target for the treatment of 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. Eur. J. Med. Chem. 2024, 280, 116952).

[0004] However, there are no reports on the treatment of IgA nephropathy with CLK2 inhibitors. Summary of the invention

[0005] The present invention found that CLK2 inhibitors (such as ganoderic acid A methyl ester) can significantly reduce the 24hUTP, 24hmAlb, BUN and Scr levels of the IgA nephropathy model, as well as the IGA deposition inside the glomerulus, thereby achieving the purpose of effectively treating IgA nephropathy.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] In one aspect, the present invention provides a use of the CLK2 inhibitor of the present invention in the preparation of a medicament for treating IgA nephropathy.

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

[0009] Preferably, the above-mentioned application includes one or more of the following applications: (i) application of a CLK2 inhibitor in the preparation of a drug for reducing 24hUTP levels; (ii) application of a CLK2 inhibitor in the preparation of a drug for reducing 24hmAlb levels; (iii) application of a CLK2 inhibitor in the preparation of a drug for reducing BUN levels; (iv) application of a CLK2 inhibitor in the preparation of a drug for reducing Scr levels; (v) application of a CLK2 inhibitor in the preparation of a drug for reducing IGA deposition inside glomeruli.

[0010] Preferably, in the above application, the dosage form of the drug includes injection, oral solution, patch, tablet, powder or capsule.

[0011] On the other hand, the present invention provides an application of ganoderic acid A methyl ester in the preparation of health food, which has one or more of the following functions: (i) functional food has the function of maintaining a healthy level of 24hUTP; (ii) functional food has the function of maintaining a healthy level of 24hmAlb; (iii) functional food has the function of maintaining a healthy level of BUN; (iv) functional food has the function of maintaining a healthy level of Scr.

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

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

[0014] The beneficial effects of the present invention include: CLK2 inhibitors (such as ganoderic acid A methyl ester) can significantly reduce the 24hUTP, 24hmAlb, BUN and Scr levels of the IgA nephropathy model, as well as the IGA deposition inside the glomerulus, thereby achieving the purpose of effectively treating IgA nephropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the result of a new ligand-CLK2 binding mode and a unique and novel hydrogen bonding between ganoderic acid A methyl ester and CLK2. DETAILED DESCRIPTION

[0016] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0017] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0018] An embodiment of the present invention provides a use of the CLK2 inhibitor of the present invention in the preparation of a medicament for treating IgA nephropathy.

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

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

[0021] In some specific examples, the above-mentioned applications include one or more of the following applications: (i) application of a CLK2 inhibitor in the preparation of a drug for reducing 24hUTP levels; (ii) application of a CLK2 inhibitor in the preparation of a drug for reducing 24hmAlb levels; (iii) application of a CLK2 inhibitor in the preparation of a drug for reducing BUN levels; (iv) application of a CLK2 inhibitor in the preparation of a drug for reducing Scr levels; (v) application of a CLK2 inhibitor in the preparation of a drug for reducing IGA deposition inside glomeruli.

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

[0023] In some specific examples, in the above applications, the dosage form of the drug includes injection, oral solution, patch, tablet, powder or capsule.

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

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

[0026] In some specific examples, in the above applications, the dosage form of the health food includes liquid, patch, tablet, powder or capsule.

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

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

[0029] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0030] Example 1 Enzyme Selectivity of Ganoderic Acid Methyl Ester to CLK2 and CLK3

[0031] The Z'LYTETM kit (ThermoFisher, USA) was used to detect the inhibition rate of ganoderic acid A methyl ester on kinases using the FRET principle, and the compound was studied for its in vitro CLK2 and CLK3 enzyme inhibitory activity; specifically, ganoderic acid A methyl ester was diluted 4 times with DMSO to obtain a ganoderic acid A methyl ester solution, and the final starting concentrations of ganoderic acid A methyl ester were 64 μM, 16 μM, 4 μM, 1 μM, 0.25 μM, 00625 μM and 0.015625 μM; then ganoderic acid A methyl ester 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 minutes; 2 μL of kinase was added to each well of a 96-well reaction plate, incubated at 25°C for 40 minutes, and RLU (Relative Luminescence Index) was read using a BMG multifunctional plate reader. The signal intensity was used to characterize the activity of the kinase, and the IC of the compound was calculated using Prism10 software. 50 In addition, the CLK2 inhibitor Lorecivivint was used as a control.

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

[0033] Table 1 Enzyme activity of ganoderic acid A methyl ester on CLK2 and CLK3

[0034]

[0035] The experimental results in Table 1 show that ganoderic acid A methyl ester has a strong inhibitory activity on CLK2, IC 50 The value was 0.69 μM, indicating that the compound ganoderic acid A methyl ester is a CLK2 inhibitor; however, the inhibitory activity of ganoderic acid A methyl ester on CLK3 is very weak, IC 50 The value is >20 μM, so ganoderic acid A methyl ester is a selective CLK2 inhibitor.

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

[0037] Download the CLK2 protein structure and perform docking experiments (PDB: 6FYK). The results are as follows Figure 1As shown, the results showed that the triterpene compound ganoderic acid A methyl ester can form three unique and novel hydrogen bonds with Asp327, Leu169 and Lys292 of CLK2 protein; ganoderic acid A methyl ester can form Alkyl and Pi-Alkyl interactions with Phe174, Val227, Leu246, Ala191, Val177, Phe243, Val326 and Leu297 of CLK2 protein, that is, a new mode of ganoderic acid A methyl ester-CLK2 binding and a unique and novel hydrogen bond interaction, which can be used for further structural optimization and drug design.

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

[0039] The miR-23b- / - mice were selected to simulate the IgA nephropathy model (the model construction method refers to the literature "Journal of Clinical Nephrology", 2024, 24: 1017-1027), and C57BL / 6J mice (provided by Beijing Huafukang Biotechnology Co., Ltd.) were selected as the blank control group; mice of the same age were kept in sterile individual 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 ganoderic acid A methyl ester group, with 10 mice in each group; in addition, 10 C57BL / 6J mice were used as a blank control group; the mice were uniformly raised until 20 weeks of age and gavage was started, among which the ganoderic acid A methyl ester group was gavaged 30 mg / Kg per day, and the intervention was continuously administered for 8 weeks; the model group and the blank control group were given the same amount of distilled water; after 8 weeks of intervention, the mouse urine was collected in a metabolic cage, and blood and kidneys were collected.

[0040] 24h urine was collected at the urine collection port at the lower end of the metabolic cage, centrifuged for 10 minutes, and the supernatant was taken to detect urine indicators. The urine indicators 24hUTP (24-hour urine protein quantification) and 24hmAlb (24-hour urine microalbumin) were detected by automatic biochemical analyzer (Shenzhen Mindray Bio-Medical Electronics Co., Ltd., model: BS-2000); the mouse serum indicators were detected by microplate method (China Enzyme Biological Company, catalog number ml092663), and the changes in serum BUN (blood urea nitrogen) and Scr (serum creatinine) were detected to evaluate renal function indicators; the deposition of IGA was analyzed by immunofluorescence staining experiment (refer to the literature "Chinese Pharmacological Bulletin", 2024, 40, 2037-2041), and the IOD / Area value (Area is the area of ​​the selected range, and IOD is the sum of the fluorescence density) was calculated to evaluate whether the drug reduces the deposition of IGA inside the glomerulus. The test results are shown in Table 2.

[0041]

[0042] It can be seen from Table 2 that compared with the blank control group, the 24hUTP, 24hmAlb, BUN and Scr of the model group were significantly increased, and the IGA deposition in the glomerulus was serious; compared with the model group, after the administration of ganoderic acid A methyl ester, 24hUTP, 24hmAlb, BUN, Scr and IGA deposition in the glomerulus were significantly reduced. According to the results of animal experiments, ganoderic acid A methyl ester can be used to prepare anti-IgA nephropathy drugs.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. Use of CLK2 inhibitors in the preparation of drugs for treating IgA nephropathy.

2. The use according to claim 1, characterized in that: The CLK2 inhibitor is ganoderic acid A methyl ester.

3. The use according to claim 1 or 2, characterized in that: Applications include one or more of the following: (i) Use of a CLK2 inhibitor in the preparation of a medicament for reducing 24hUTP levels; (ii) Use of a CLK2 inhibitor in the preparation of a medicament for reducing 24hmAlb levels; (iii) Use of a CLK2 inhibitor in the preparation of a medicament for reducing BUN levels; (iv) Use of a CLK2 inhibitor in the preparation of a medicament for reducing Scr levels; (v) Use of a CLK2 inhibitor in the preparation of a medicament for reducing IGA deposition in glomeruli.

4. The use according to claim 1 or 2, characterized in that: The drug is available in the form of an injection, oral solution, patch, tablet, powder or capsule.

5. The use according to claim 3, characterized in that: The drug is available in the form of an injection, oral solution, patch, tablet, powder or capsule.

6. Application of ganoderic acid A methyl ester in the preparation of health food, the health food having one or more of the following functions: (i) Functional foods have the function of maintaining healthy levels of 24hUTP; (ii) Functional foods have the function of maintaining healthy levels of 24hmAlb; (iii) Functional foods have the function of maintaining healthy BUN levels; (iv) Functional foods have the function of maintaining healthy levels of Scr.

7. The use according to claim 6, characterized in that: The dosage forms of health foods include liquids, patches, tablets, powders or capsules.

8. Use of ganoderic acid A methyl ester as or in the preparation of CLK2 inhibitors.

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