Kdm4c inhibitors and uses thereof

Abstract

This invention discloses a KDM4C inhibitor and its applications, relating to the field of biomedical technology. Specifically, this invention discloses the application of the KDM4C inhibitor in any of A1) to A5): A1) preparing a drug for the prevention or treatment of obesity; A2) preparing a drug for reducing weight; A3) preparing a drug for reducing food intake; A4) preparing a drug for reducing insulin sensitivity; A5) preparing a drug for enhancing glucose homeostasis. This is achieved through knockout... Kdm4c Inhibiting KDM4C gene activity or inhibiting KDM4C activity can significantly reduce obesity induced by a high-fat diet, thereby regulating the secretion of related hormones / adipokines, affecting food intake, improving insulin resistance, and enhancing insulin sensitivity, making it a potential target for the treatment of obesity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a KDM4C inhibitor and its application. Background Technology

[0002] Histone lysine demethylases 4C (KDM4C), also known as Jumonji domain-containing protein C (JMJD2C), can convert the trimethyl group at lysine residue 9 (Lys-9) or lysine residue 36 (Lys-36) on histone H3 to a dimethyl group via oxidation, thereby affecting heterochromatin formation, gene transcription regulation, and cell cycle regulation. Dysregulation of KDM4C expression is associated with many diseases. Current research on KDM4C mainly focuses on the occurrence, development, and treatment of cancer. Abnormal expression of KDM4C promotes the formation of cancers such as breast cancer, esophageal squamous cell carcinoma, and prostate cancer. However, research on KDM4C in other diseases is still scarce. Summary of the Invention

[0003] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes the application of KDM4C inhibitors.

[0004] The present invention also provides the application of at least one of disulfiram, rutin, and diosmin.

[0005] Application of the KDM4C inhibitor according to the first aspect of the present invention in any of A1) to A5): A1) To prepare drugs for the prevention or treatment of obesity; A2) Prepare drugs to reduce body weight; A3) Prepare drugs to reduce food intake; A4) Prepare drugs that reduce insulin sensitivity; A5) Prepare drugs that enhance glucose homeostasis.

[0006] The application of the present invention, according to embodiments thereof, has at least the following beneficial effects: This invention discovers knockout Kdm4c Inhibiting KDM4C gene activity or inhibiting KDM4C activity can significantly reduce obesity induced by a high-fat diet, thereby regulating the secretion of related hormones / adipokines, affecting food intake, improving insulin resistance, and enhancing insulin sensitivity, making it a potential target for the treatment of obesity.

[0007] According to some embodiments of the present invention, the KDM4C inhibitor includes inhibiting the activity of KDM4C and / or inhibiting... Kdm4cSubstances involved in gene expression. Inhibition of KDM4C activity refers to a decrease in KDM4C activity. For example, a decrease in KDM4C activity of at least 10%, 30%, 50%, 70%, or 90% compared to before inhibition. Kdm4c Gene expression refers to the process by which the KDM4C gene is prevented from being transcribed or reduced. Kdm4c Gene transcriptional activity, or making Kdm4c Genes are not expressed or are reduced Kdm4c Gene expression activity.

[0008] According to some embodiments of the present invention, the KDM4C inhibitor comprises at least one of nucleic acid molecules, carbohydrates, lipids, antibodies, small molecule compounds, peptides, proteins, gene editing vectors, lentiviruses, and adeno-associated viruses.

[0009] According to some embodiments of the present invention, the nucleic acid molecule includes at least one selected from microRNA, siRNA, shRNA, sgRNA, and antisense oligonucleotides. The nucleic acid molecule is used to target and interfere with / inhibit the expression of the KDM4C gene. The sgRNA is used to target and knock out exon nine of KDM4C.

[0010] According to some embodiments of the present invention, the antibody is an anti-KDM4C antibody or its active fragment.

[0011] According to some embodiments of the present invention, the small molecule compound includes at least one of disulfiram, rutin, and diosmin.

[0012] According to some embodiments of the present invention, the obesity is diet-induced obesity.

[0013] According to some embodiments of the present invention, the treatment of obesity manifests as at least one of weight reduction, reduced food intake, enhanced glucose homeostasis, and / or reduced insulin sensitivity.

[0014] According to some embodiments of the present invention, the drug is formulated into a pharmaceutically permissible dosage form.

[0015] According to some embodiments of the present invention, the dosage form of the drug includes at least one of pills, tablets, powders, capsules, granules, powders, drop pills, drops, sprays, injections, suspensions, ointments, gels, and suppositories.

[0016] According to some embodiments of the present invention, the method of administration of the drug is one or more of the following: intramuscular injection, subcutaneous injection, intravenous injection, oral administration, sublingual administration, and spray administration.

[0017] According to some embodiments of the present invention, the medicament comprises an active ingredient, a KDM4C inhibitor, and pharmaceutically acceptable excipients.

[0018] According to some embodiments of the present invention, the excipients include at least one of the following: diluent, buffer, suspension, emulsion, granule, encapsulating agent, excipient, filler, binder, spray, transdermal absorbent, humectant, disintegrant, absorption promoter, surfactant, colorant, flavoring agent, and adsorbent carrier.

[0019] According to some embodiments of the present invention, the KDM4C inhibitor is present in a concentration of 1 wt% to 99 wt% in the drug. For example, it can be 1 wt%, 5 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt%, 55 wt%, 60 wt%, 65 wt%, 70 wt%, 75 wt%, 80 wt%, 85 wt%, 90 wt%, 95 wt%, or 99 wt%.

[0020] The use of at least one of disulfiram, rutin, and diosmin in the preparation of a KDM4C inhibitor according to a second aspect of the present invention.

[0021] According to some embodiments of the present invention, the effective concentration of disulfiram is 10 μM-30 μM. For example, it can be 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM, or 30 μM.

[0022] According to some embodiments of the present invention, the effective concentration of rutin is 15 μM-30 μM. For example, it can be 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM or 30 μM.

[0023] According to some embodiments of the present invention, the effective concentration of diosmin is 15 μM-30 μM. For example, it can be 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM, or 30 μM.

[0024] The use of at least one of disulfiram, rutin, and diosmin according to a third aspect of the present invention in the preparation of a product for screening drugs for the treatment of obesity or drugs having KDM4C inhibitory activity.

[0025] According to some embodiments of the present invention, the product includes at least one of reagents and kits.

[0026] According to some embodiments of the present invention, the method of using the product for screening drugs with KDM4C inhibitory activity includes the following steps: KDM4C was incubated with the drug to be screened, and then the reaction substrate H3K9Me3 was added to initiate the enzyme reaction. The activity of KDM4C was then detected as inhibited. At least one of disulfiram, rutin, and diosmin was used as a positive control. The inhibition of KDM4C activity was detected by measuring the fluorescence intensity at a specific wavelength using an ELISA reader.

[0027] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0028] Figure 1 This is a diagram of the Kdm4c gene knockout protocol.

[0029] Figure 2 For PCR analysis to identify wild-type, Kdm4c + / - , Kdm4c - / - Electrophoresis image of mouse genotypes. Lanes from left to right: 2000 bp DNA marker band, 342 bp band (…). Kdm4c + / + Rats), stripeless ( Kdm4c + / + (mouse), 342bp band ( Kdm4c + / - (mouse), 743bp band ( Kdm4c + / - Rats), stripeless ( Kdm4c - / - (mouse), 743bp band ( Kdm4c - / - (Mouse), 2000 bp DNA marker band.

[0030] Figure 3The effect of KDM4C knockout on body weight in diet-induced obese (DIO) mice. A: Body weight changes in mice fed a high-fat diet (HFD) during the experimental period; B: Representative photographs of 30-week-old mice (fed a high-fat diet for 22 weeks); C: Average food intake of mice in each group fed a high-fat diet for 19 weeks. *** indicates highly significant differences (…). p <0.001); **** indicates a highly significant difference ( p <0.0001).

[0031] Figure 4 The graph shows the GTT results of blood glucose homeostasis in HFD-induced obese mice. A: Blood glucose-time curve; B: Area under the curve analysis results. ** indicates extremely significant differences. p <0.01); *** indicates a highly significant difference ( p <0.001).

[0032] Figure 5 The graph shows the ITT (Insulin-Tolerance Test) results for insulin sensitivity in HFD-induced obese mice. A: Blood glucose-time curve; B: Area under the curve analysis results. * indicates significant difference. p <0.05); ** indicates a highly significant difference ( p <0.01).

[0033] Figure 6 This is a schematic diagram of the detection principle of the in vitro drug screening enzyme activity detection system in Example 2.

[0034] Figure 7 The in vitro drug screening results for Example 2 (IC) 50 picture.

[0035] Figure 8 For DSF pair Kdm4c Effect of diet-induced obesity (DIO) on body weight in knockout mice. A: Wild-type mice and Kdm4c mice in different treatment groups fed with HFD during the experiment. - / - Figure B: Body weight of wild-type mice under different treatment groups fed with HFD during the experiment; Kdm4c - / - C: Changes in body weight of mice; D: Daily food intake of mice in each group after 9 weeks of HFD feeding; E: Representative photographs of mice in each group after 9 weeks of HFD feeding; F: ITT curves of insulin sensitivity in mice under different HFD treatments; Glucose-time curves of ITT measurement. * indicates significant differences. p <0.05); ** indicates a highly significant difference ( p <0.01); *** indicates a highly significant difference ( p<0.001); **** indicates a highly significant difference ( p <0.0001) Detailed Implementation The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0036] Unless otherwise specified, "room temperature" in this invention means (25±5)℃. For examples where specific conditions are not specified, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0037] Unless otherwise specified, substrate H3K9Me3 was purchased from Sangon Biotech (Shanghai) Co., Ltd.; formaldehyde dehydrogenase (FDH) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; rutin and diosmin were purchased from MedChemexpress Biotechnology, Inc. (USA); disulfiram was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; and dimethyl sulfoxide (DMSO) was purchased from Anaiji (Shanghai) Pharmaceutical Chemical Co., Ltd.

[0038] Unless otherwise specified, all gene-modified mice were purchased from Cyagen Biosciences Co., Ltd. C57BL / 6 mice (8 All 13-week-old male mice were housed in SPF-grade conditions at the Laboratory Animal Center of Sun Yat-sen University (Shenzhen Campus) (basic conditions included a 12-hour light / dark cycle, room temperature of 20-26°C, and humidity of 40%-60%). Unless otherwise specified, the mice had free access to food and water during the rearing process. Animal experiments in this study were approved by the Laboratory Animal Management and Use Committee (IACUC) of Sun Yat-sen University and conducted in accordance with the Laboratory Animal Operation Manual and the Animal Ethics Committee of Sun Yat-sen University.

[0039] Unless otherwise specified, the amino acid sequence of the recombinant protein KDM4C is shown in SEQ ID NO: 1. It was obtained by expression using the Bac-to-bac insect expression system via the following method: (1) The coding sequence of the recombinant protein KDM4C was inserted into the pfastbac1 vector. The constructed plasmid was transformed into DH10bac competent cells and screened by blue-white screening and PCR on plates containing kanamycin, tetracycline, gentamicin, IPTG, and X-gal. Successfully recombinant clones were selected and inoculated into liquid LB medium to extract the viral genome vector. The extracted viral genome vector was transfected into adherent Sf9 cells using Cellfectin 2000 liposomes and cultured at 27°C for 4 days. After 5 days, the supernatant was collected as the first-generation virus P1. The gene coding sequence of P1 was processed according to 1... 2% (v / v) of the virus was inoculated into suspension cultured Sf9 cells and incubated at 27°C for 4 days. The supernatant was collected by centrifugation as the second-generation virus P2. The second-generation virus P2 was further amplified to generate P3 virus, which was then added to 5... 10% serum should be stored at 4°C.

[0040] (2) Dilute the suspension-cultured High Five cells to a density of 1×10⁻⁶. 6 2×10 6 cells / ml, according to 1 4% (v / v) of cells were inoculated with P3 virus. Cells were cultured at 27°C with constant temperature and shaking for 48 days. After 60 h, centrifuge at 2000 × g for 25 minutes, discard the supernatant, and freeze the settled cells with liquid nitrogen and store at -80 ℃.

[0041] (3) Remove the frozen cells, add lysis buffer, and thaw at room temperature. After partial thawing, transfer to a 4°C chromatography cabinet and continue dissolving in a magnetic stirrer until complete, adding 1% of 100 mM PMSF protease inhibitor during this process. Use a high-pressure homogenizer to homogenize the completely dissolved bacterial solution, cycling 2-3 times until the solution changes from viscous milky white to translucent. Centrifuge at 16000×g for 30 min at 4°C, collect the supernatant, and bind it with nickel beads at 4°C for 1 h. Centrifuge at 3000×g for 3 min, discard the supernatant, resuspend in 5 column volumes of lysis buffer, and wash twice. Transfer to a gravity column, and after the flow-through is drained, add 10 mL of elution buffer to elute the target protein.

[0042] MEVAEVESPLNPSCKIMTFRPSMEEFREFNKYLAYMESKGAHRAGLAKVIPPKEWKPRQCYDDIDNLLIPAPIQQMVTGQSGLFTQYNIQKKAMTVKEFRQLANSGKYCTPRYLDYEDLERKYWKNLTFVAPIYGADINGSIYDEGVDEWNIARLNTVLDVVEEECGISIEGVNTPY LYFGMWKTTFAWHTEDMDLYSINYLHFGEPKSWYAIPPEHGKRLERLAQGFFPSSSQGCDAFLRHKMTLISPSVLKKYGIPFDKITQEAGEFMITFPYGYHAGFNHGFNCAESTNFATVRWIDYGKVAKLCTCRKDMVKISMDIFVRKFQPDRYQLWKQGKDIYTIDHTKPTP (SEQ ID NO: 1).

[0043] Unless otherwise specified, the procedure for the GTT glucose tolerance test is as follows: Mice were fasted overnight for 15 hours (18:00-9:00) and then injected intraperitoneally with glucose at 2 g / kg body weight. Blood was collected from the tail tip, and blood glucose was measured using a handheld glucometer at time points before glucose injection (0 minutes) and at 15, 30, 60, 90, and 120 minutes after glucose injection.

[0044] Unless otherwise specified, the procedure for the ITT insulin tolerance test is as follows: Mice were fasted for 6 hours (9:00-15:00) and then injected intraperitoneally with insulin at 0.75 U / kg body weight. Blood was collected from the tail tip, and blood glucose was measured using a handheld glucometer at time points before insulin injection (0 minutes) and at 15, 30, 60, 90, and 120 minutes after glucose injection.

[0045] Example 1 All mice used in this experiment were wild-type mice or Kdm4c mice. + / - Mice, Kdm4c - / -Genotypes of mice were identified by PCR analysis. The AdeptTect mouse tissue rapid PCR kit (from Aikerui Biotechnology) was used for genotype identification. 100 μL of Mouse Tissue Lysis Buffer and 1 μL of Proteinase K were added to the mouse tail sample, vortexed, and incubated at 60℃ for 5 min, then at 98℃ for 2 min. After rapid centrifugation, the supernatant was used for PCR. After PCR, electrophoresis was performed on a 1% agarose gel. The gel was then developed and photographed using a gel imaging system. Genotypes were determined based on the molecular weight of the DNA bands (judgment criteria: ...). Kdm4c + / + 342 bp; Kdm4c - / - (743bp).

[0046] The primers used for mouse genotyping are: Kdm4c-F:5'-TGAAGGCTCTGTGCCCTAGTA-3' (SEQ ID NO: 2); Kdm4c-KOR:5'TGTGTGTGTACACGACAGAACTAATC-3' (SEQ ID NO: 3); Kdm4c-WTR:5'-GGGAAGGCTGAGACAGAACAC-3' (SEQ ID NO: 4).

[0047] The results of the identification are as follows Figure 2 As shown.

[0048] The experimental animals were divided into two groups, one of which was wild-type C57BL / 6 mice. Kdm4c + / + 9 in total, and another group of 9 in total. Kdm4c Gene knockout ( Kdm4c - / - Nine mice were used. They were fed a high-fat diet (60 kcal% Fat; Research Diets formulas D12492; Research Diets, Inc., New Brunswick, NJ) from 8 weeks of age for 22 weeks, and were weighed weekly. Some wild-type mice died during the experiment; the number of wild-type mice at the time of GTT and ITT tests was 7. GTT analysis was performed at 25 weeks of age; ITT analysis was performed at 26 weeks of age.

[0049] like Figure 3 As shown. Under high-fat diet feeding conditions, Kdm4c Knockout significantly reduced the body weight and food intake of DIO mice.

[0050] like Figure 4 As shown. In the GTT analysis, Kdm4c Knockout significantly enhances glucose homeostasis in DIO mice.

[0051] like Figure 5 As shown. In the ITT experiment, compared with wild-type mice, Kdm4c - / - The mice exhibited higher insulin sensitivity.

[0052] Example 2 The level of NADH produced indirectly reflects the strength of enzyme activity. The detection principle is as follows: Figure 6 As shown in Table 1, disulfiram (DSF), rutin, and diosmin were used as inhibitors. A control group (100% activity, with the inhibitor replaced by an equal volume of solvent), a background group (0% activity, with the substrate H3K9Me3 replaced by an equal volume of HEPES buffer), and a positive group (inhibitor replaced by SD70) were set up. Following the system shown in Table 1, the inhibitors were added to the 96-well plates, followed by the enzyme mixture. The mixture was incubated at room temperature for 15 minutes. The substrate mixture was then added, and the relative fluorescence intensity (RFU) was measured using a microplate reader at excitation and emission wavelengths of 355 nm and 460 nm, respectively. Nonlinear regression analysis was performed on the measured data using GraphPad Prism software to calculate the IC50 of each compound. 50 value.

[0053] Table 1

[0054] The results are as follows Figure 7 As shown, the IC of disulfiram 50 IC50 with 11.41 μM rutin 50 IC50 with 16.27 μM diosmin 50 Both at 20.26 μM exhibited good KDM4C inhibitory activity. Furthermore, disulfiram and rutin showed better inhibitory activity than the standard positive compound SD70 (IC50). 50 (17.01μM) showed better KDM4C inhibition.

[0055] Example 3 The experimental animals were divided into four groups. One group consisted of wild-type ( Kdm4c + / + Four mice were used, all fed a high-fat diet (60kcal% Fat; derived from Ruide Biotechnology); one group consisted of... Kdm4c Knockout ( Kdm4c - / - Four mice were used, all fed a high-fat diet; one group consisted of wild-type mice. Kdm4c+ / + Four mice were used, all fed a high-fat diet supplemented with 150 mg / kg DSF; one group consisted of... Kdm4c Knockout ( Kdm4c - / - Four mice were used, all fed a high-fat diet supplemented with 150 mg / kg DSF. The experiment began at 8 weeks of age and continued for 9 weeks, with mice weighed weekly. An ITT test was performed at week 9 of the high-fat diet.

[0056] like Figure 8 As shown. Under high-fat diet conditions, compared with wild-type mice, Kdm4c - / - Mice, administered DSF Kdm4c + / + Mice, administered DSF Kdm4c - / - The mice all experienced significant reductions in body weight and food intake. Kdm4c - / - Mice, administered DSF Kdm4c + / + Mice, administered DSF Kdm4c - / - There was no significant difference in the body weight of the mice. In the ITT experiment, Kdm4c - / - Mice, administered DSF Kdm4c + / + Mice, administered DSF Kdm4c - / - Mice showed higher insulin sensitivity compared to wild-type mice, and were given DSF. Kdm4c + / + Mice, administered DSF Kdm4c - / - There was no significant difference in insulin sensitivity among mice.

[0057] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. Application of KDM4C inhibitors in any of A1) to A5): A1) To prepare drugs for the prevention or treatment of obesity; A2) Prepare drugs to reduce body weight; A3) Prepare drugs to reduce food intake; A4) Prepare drugs that reduce insulin sensitivity; A5) Prepare drugs that enhance glucose homeostasis.

2. The application according to claim 1, characterized in that, The KDM4C inhibitor includes at least one of the following: nucleic acid molecules, carbohydrates, lipids, antibodies, small molecule compounds, peptides, proteins, gene editing vectors, lentiviruses, and adeno-associated viruses.

3. The application according to claim 2, characterized in that, The nucleic acid molecule includes at least one of microRNA, siRNA, shRNA, sgRNA, and antisense oligonucleotide; and / or, the small molecule compound includes at least one of disulfiram, rutin, and diosmin.

4. The application according to claim 1, characterized in that, The treatment for obesity involves at least one of the following: weight reduction, reduced food intake, enhanced glucose homeostasis, and / or reduced insulin sensitivity.

5. The application according to claim 1, characterized in that, The drug includes the active ingredient KDM4C inhibitor and pharmaceutically acceptable excipients.

6. The application according to claim 1, characterized in that, The dosage form of the drug includes at least one of the following: pills, tablets, powders, capsules, granules, powders, drop pills, drops, sprays, injections, suspensions, ointments, gels, and suppositories.

7. The application according to claim 1, characterized in that, The KDM4C inhibitor is present in a concentration of 1 wt% to 99 wt% in the drug.

8. The use of at least one of disulfiram, rutin, and diosmin in the preparation of KDM4C inhibitors.

9. The application according to claim 8, characterized in that, The effective concentration of the disulfiram is 10 μM-30 μM; and / or, the effective concentration of rutin is 10 μM-30 μM; and / or, the effective concentration of diosmin is 10 μM-30 μM.

10. The use of at least one of disulfiram, rutin, and diosmin in the preparation of products for screening drugs for the treatment of obesity or drugs with KDM4C inhibitory activity.

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