Use of kavain C in the preparation of IDH1 inhibitors

Abstract

The present invention relates to the fields of clinical medicine and biopharmaceutical technology, and specifically relates to the application of flavokawain C in inhibiting IDH1 mutant tumor cells in the treatment of tumors. Through a large number of experimental studies, the present invention has found that flavokawain C has the function of selectively inhibiting IDH1 mutant tumor cells, can significantly reduce the 2-HG level in IDH1 mutant cells, and has no obvious toxic and side effects on normal cells. Based on the above clinical problems, the present invention aims to explore the application of flavokawain C compound as an IDH1 covalent inhibitor in anti-IDH1 mutant tumors, in order to provide new options and more effective solutions for the treatment of IDH1 mutant tumors.

Description

Technical Field

[0001] The present invention relates to the technical fields of clinical medicine and biopharmaceuticals, and particularly relates to the application of kavain C in the preparation of IDH1 inhibitors. Background Art

[0002] Isocitrate dehydrogenase 1 (IDH1) participates in the tricarboxylic acid cycle in cells and catalyzes the oxidative decarboxylation of isocitrate to generate α-ketoglutaric acid (α-KG). However, the mutated IDH1 acquires a new enzymatic activity and can reduce α-KG to 2-hydroxyglutaric acid (2-HG). The abnormal accumulation of 2-HG in cells interferes with normal cell metabolism and epigenetic regulation. It can competitively inhibit α-KG-dependent dioxygenases, such as histone demethylases and DNA demethylases, resulting in abnormal increases in histone and DNA methylation levels in cells. This abnormal epigenetic modification affects gene expression and promotes malignant transformation of cells, including promoting the proliferation of tumor cells, inhibiting apoptosis, enhancing the invasion and metastasis abilities of tumor cells, and promoting tumor angiogenesis, etc., thus playing a key role in the occurrence and development of tumors. IDH1 mutations are relatively common in various tumors, such as acute myeloid leukemia (AML), cholangiocarcinoma, glioma, etc. Therefore, IDH1 mutations have become an important target for tumor treatment, and research on IDH1 mutations is of great significance for the development of new anti-tumor therapies.

[0003] Although existing IDH1 inhibitors can inhibit the activity of mutant IDH1 enzymes to a certain extent and reduce the level of 2-HG, for some patients, tumor cells can still continue to proliferate or progress, resulting in unsatisfactory treatment effects, inability to effectively control tumor growth, and limited extension of the patient's survival period. Moreover, over time, tumor cells are prone to develop drug resistance to existing IDH1 inhibitors. The drug resistance mechanism is relatively complex and may be related to various situations such as secondary mutations of IDH1 gene mutations, changes in drug transporters, and adaptive changes in downstream signaling pathways. Once drug resistance occurs, the efficacy of the drug will significantly decline, leaving patients without effective treatment options.

[0004] Developing new, highly effective, and low-toxic IDH1 inhibitors faces many challenges, including the difficulty of drug molecule design, optimization of the pharmacokinetic properties of drugs, etc. Many potential compounds fail due to poor efficacy, excessive toxicity, or unsatisfactory drug metabolism characteristics after entering the pre-clinical research or clinical trial stage, resulting in relatively slow progress in the research and development of therapeutic drugs for IDH1 mutant tumors. In view of the above-mentioned defects and deficiencies of the existing technology, there is an urgent need to develop new IDH1-targeted treatment strategies. Summary of the Invention

[0005] Through a large number of experimental studies, the present invention has found that Flavokawain C has the function of selectively inhibiting IDH1 mutant tumor cells, can significantly reduce the level of 2-HG in IDH1 mutant cells, and has no obvious toxic and side effects on normal cells. Based on the above clinical problems, the present invention aims to explore the application of Flavokawain C compound as an IDH1 covalent inhibitor in anti-IDH1 mutant tumors, in order to provide new options and more effective solutions for the treatment of IDH1 mutant tumors.

[0006] Based on the above research, the present invention provides the application of Flavokawain C in the preparation of an IDH1 inhibitor.

[0007] The present invention also provides the application of Flavokawain C in the preparation of an anti-tumor drug, and the tumor is a tumor carrying mutant IDH1.

[0008] Preferably, the drug selectively inhibits the proliferation of tumor cells with IDH1-R132C mutation, and / or the drug selectively inhibits the proliferation of tumor cells with IDH1-R132H mutation. In in vitro cell experiments, Flavokawain C can selectively inhibit the proliferation of HT1080 tumor cells with IDH1-R132C mutation and TS603 tumor cells with IDH1-R132H mutation.

[0009] Preferably, the drug reduces the expression level of 2-HG in tumor cells.

[0010] Preferably, the drug reduces the methylation level of H3K9me3 in tumor cells, and / or the drug reduces the methylation level of H3K9me2 in tumor cells.

[0011] The present invention also provides a drug for treating IDH1 mutant tumors, and the drug contains Flavokawain C.

[0012] According to the drug of the specific embodiment of the present invention, the drug selectively inhibits the proliferation of tumor cells with IDH1-R132C mutation, and / or the drug selectively inhibits the proliferation of tumor cells with IDH1-R132H mutation.

[0013] According to the drug of the specific embodiment of the present invention, the drug reduces the production level of 2-HG in tumor cells.

[0014] According to the drug of the specific embodiment of the present invention, the drug reduces the methylation level of H3K9me3 in tumor cells, and / or the drug reduces the methylation level of H3K9me2 in tumor cells.

[0015] Preferably, the drug comprises a pharmaceutically acceptable carrier.

[0016] The above-mentioned drug can be prepared into oral preparations or injections; the oral preparations include but are not limited to capsules, tablets, granules, oral liquids; the injections include but are not limited to sterile powders for injection, aqueous injections, sodium chloride or glucose intravenous infusions.

[0017] In the above-mentioned drug, the oral preparation includes additives selected from at least one of fillers, diluents, disintegrants, binders, lubricants, glidants, surfactants, solvents, flavoring agents, stabilizers, colorants, and preservatives.

[0018] The fillers or diluents include saccharides such as lactose, sucrose, glucose, mannitol, sorbitol, dextrin; starches such as starch, pregelatinized starch, dextrin; celluloses such as microcrystalline cellulose, gum arabic, fenugreek gum, dextran; inorganic salts such as calcium sulfate, calcium hydrogen phosphate, medicinal calcium carbonate, light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, magnesium aluminum silicate.

[0019] The lubricants or glidants or anti-adhesives include stearic acid; metal salts of stearic acid such as calcium stearate or magnesium stearate; talc powder; colloidal silica; microcrystalline silica gel, hydrogenated vegetable oil; polyethylene glycol, lauryl sulfates such as sodium lauryl sulfate or magnesium lauryl sulfate; silicates such as silicon anhydride or hydrated silicate.

[0020] The binders include distilled water, ethanol with different concentrations, starch paste, hydroxypropyl methylcellulose, hydroxypropyl cellulose, polyvinylpyrrolidone, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, polyethylene glycol, and compounds similar to the above excipients.

[0021] The disintegrants include cellulose derivatives such as low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose or cross-linked sodium carboxymethyl cellulose; cross-linked polyvinylpyrrolidone; and chemically modified starches / celluloses, such as carboxymethyl starch or sodium carboxymethyl starch.

[0022] The surfactants include sodium dodecyl sulfate, sodium stearate sulfonate, etc.

[0023] The antioxidants include sodium bisulfite, sodium metabisulfite, sodium sulfite, anhydrous sodium sulfite, sodium thiosulfate, ascorbic acid, methionine, thiourea, phosphoric acid, citric acid, etc.

[0024] The preservatives or antibacterial agents include benzoic acid and sodium benzoate, sorbic acid, ethanol, parabens (nipagin esters), benzalkonium bromide, o-phenylphenol, benzyl alcohol, phenethyl alcohol, sodium propionate, sorbic acid, eucalyptus oil, cinnamon oil, and peppermint oil, etc.

[0025] The flavoring agents described above include sweeteners such as sodium saccharin, aspartame, syrup, stevioside, mannitol, sorbitol, mannose, galactose, maltose, fructose, glucose, sucrose, etc.; sour flavoring agents such as citric acid, malic acid or tartaric acid; and aromatic agents such as fennel oil, peppermint oil, menthol, peppermint water, cinnamon oil, lemon essence, lemon oil and spices of various flavors, etc.

[0026] For the usage method of the above-mentioned drug, it includes administering an effective amount of the above-mentioned drug to a subject. The administration method can be oral, intravenous injection or transdermal penetration, and is applied to the patient in need of treatment.

[0027] The pharmaceutically effective amount refers to the reasonable benefit / risk ratio that can be obtained by applying the drug for treatment and is sufficient to treat the disease. The level of the effective dose can be determined depending on some factors, including the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the administration time, the administration route, the excretion rate, the treatment cycle, the drugs used simultaneously, and other factors well-known in the medical field. The drug of the present invention can be administered as an independent therapeutic reagent or in combination with other therapeutic reagents. Moreover, the drug of the present invention can continuously or simultaneously add typical therapeutic reagents, and the composition can be administered once or multiple times. It is important to consider all the above factors and administer at the minimum dose that can produce the maximum effect without side effects, and the dose can be determined by a physician according to the patient's condition, age, etc.

[0028] Preferably, the concentration of flavokawain C used in in vitro cell experiments is 5 μmol / ml; in in vivo experiments, the concentration of flavokawain C used is 1 - 300 mg / kg. Preferably, the concentration of flavokawain C used is 10 - 50 mg / kg, and more preferably, the concentration of flavokawain C used is 20 mg / kg.

[0029] Advantages of the present invention:

[0030] Compared with the existing IDH1 mutation inhibitor AG120, flavokawain C can more significantly inhibit the proliferation of IDH1 mutant cells through covalent inhibition of IDH1 mutation. Flavokawain C can be used as an IDH1 mutation inhibitor. Further, flavokawain C can be used as a drug for treating cancer, providing a theoretical and clinical basis for the treatment of IDH1 mutant tumors. Description of the Drawings

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0032] Figure 1 shows the curve of the inhibitory effect of Flavokawain C and AG120 on the proliferation of IDH1 mutant cells.

[0033] Figure 2 shows the curve of the inhibitory effect of Flavokawain C on the proliferation of IDH1 mutant knockdown cells.

[0034] Figure 3 shows the safety evaluation of Flavokawain C on LO2 cells and BV2 cells.

[0035] Figure 4 shows the detection of the binding ability between Flavokawain C and IDH1-R132H.

[0036] Figure 5 shows the effect of Flavokawain C on the level of 2-HG in TS603 cells.

[0037] Figure 6 shows the effect of Flavokawain C on the methylation levels of H3K9me3 and H3K9me2 proteins in TS603 cells.

[0038] Figure 7 shows the effect of Flavokawain C on the proliferation of TS603 cell xenografts in nude mice.

[0039] Figure 8 shows the effect of Flavokawain C on the level of 2-HG in TS603 cell xenografts in nude mice.

[0040] Figure 9 shows the covalent binding mode between Flavokawain C and IDH1-R132H protein. Detailed implementation manners

[0041] To make the objectives, technical solutions and advantages of the present invention clearer, the following will describe the technical solutions of the present invention in detail. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. All other implementation manners obtained by those of ordinary skill in the art without creative efforts based on the embodiments of the present invention belong to the scope protected by the present invention.

[0042] Example 1 Inhibitory effect of Flavokawain C and AG120 on the proliferation of IDH1 mutant cells

[0043] (1) Experimental materials

[0044] HT1080 cells, TS603 cells, CCK-8 reagent, Flavokawain C (TargetMol), AG120 (Maclin), microplate reader (Biotek).

[0045] (2) Experimental methods

[0046] Cells were seeded at 8000 cells per well in 96-well plates and divided into a blank group, a series of gradient concentration Flavokawain C and AG120 administration groups. After the cells adhered, the medium was changed. In the administration groups, 200 μL of Flavokawain C or AG120 medium solutions with concentrations of 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.6 μM, and 0.8 μM were added. The blank group was given the same volume of 1640 medium containing 10% serum, and they were all treated for 48 hours. After 48 hours, the medium was changed to 10% cck8 medium solution. After 2 hours, the absorbance was measured at a wavelength of 480 nM.

[0047] The results were as Figure 1 shown. The IC 50 of Flavokawain C for HT1080 cells was 4.772 μM, and the IC 50 for TS603 cells was 7.916 μM; the IC 50 value of AG120 for HT1080 cells was greater than 50 μM.

[0048] Example 2 Construction of IDH1 knockdown cell line

[0049] (1) Experimental materials

[0050] TS603 cells, 293T cells, lentivirus packaging kit (Biorigin), pLV-U6-SHIDH1-CMV-EGFP (T2A)-PURO (Scilia Biotechnology), puromycin (Beyotime), RIPA lysis buffer (Shanghai Yeastar Biopharmaceutical Technology Co., Ltd.), IDH1 antibody (Proteintech, Wuhan), β-actin antibody (Aibotech, Wuhan), 0.22 μm PVDF membrane (Millipore, USA), goat anti-rabbit IgG secondary antibody (Beyotime, Shanghai), Omni-ECL chemiluminescence detection kit (Shanghai Yeastar Biopharmaceutical Technology Co., Ltd.).

[0051] (2) Experimental methods

[0052] Inoculate low-passage, healthy 293T cells into a 10 cm culture dish. When the cell density reaches about 80%, prepare for transfection. Configure the transfection reagent according to the lentivirus packaging kit instructions: 1000 μL of Opti-MEM, 7.5 μg of bone plasmid DNA, 7.5 μg of Easy Mix, and 37.5 μL of LipoX. After mixing, let it stand at room temperature for 15 min, and then add it to the cells. Shake well, add 10 μL of transfection enhancer, place it in the incubator for overnight transfection, and then discard the supernatant (sterilized). Add 10 mL of fresh medium, collect the supernatant after 36 h. Then add 10 mL and continue to culture for 24 h, and then collect the supernatant. Combine the two supernatants, centrifuge at 4000 rpm for 10 min at 4°C, collect the supernatant, and filter it through a 0.45 μm filter membrane. Aliquot and store at -80°C for long-term storage or at 4°C for use within one week.

[0053] Inoculate TS603 cells into a 6-well plate. When the cell confluence reaches 60 - 80%, add a 1:1 mixture of the virus packaging supernatant and the medium. Observe the cell status, change to the regular cell medium after 12 - 24 h of infection, and continue to culture for 24 h. Use puromycin to screen for stable transfected cell lines.

[0054] Verify the protein expression level by Western blot. Collect the cells with puromycin resistance, wash them twice with PBS, centrifuge to discard the supernatant, and collect the cells. Add RIPA cell lysate and protease inhibitor, place it on ice and ultrasonically disrupt it, centrifuge at 20000×g for 5 min at 4°C, collect the supernatant, add the loading buffer, and heat at 95°C for 10 min. Perform SDS-PAGE electrophoresis separation on the prepared loading solution, and then transfer it to a PVDF membrane. Block the PVDF membrane in 5% skim milk powder at room temperature for half an hour, then place it in the primary antibody incubation solution and incubate overnight at 4°C. Wash it in PBST for 5 minutes and repeat 3 times. Subsequently, place it in the secondary antibody incubation solution and incubate at room temperature for 30 min. Wash it in PBST for 5 minutes and repeat 3 times. Use a chemiluminescence kit to detect the target protein, analyze the PVDF membrane with a multifunctional imaging instrument, and quantify it with ImageJ software.

[0055] The results are as Figure 2 shown. Compared with the control group, the expression level of IDH1 was significantly decreased, indicating that the TS603 cell line with IDH1 knockdown was successfully constructed.

[0056] Example 3 Inhibitory effect of Flavokawain C on the proliferation of IDH1 mutant knockdown cells

[0057] (1) Experimental materials

[0058] Sh_IDH1 TS603 cells, CCK-8 reagent, Flavokawain C (Shanghai TargetMol), microplate reader (Biotek).

[0059] (2)Experimental method

[0060] Inoculate Sh_IDH1 TS603 cells at 8000 cells per well in a 96-well plate, and divide them into a blank group and a series of gradient concentration Flavokawain C administration groups. After the cells adhere to the wall, change the medium. In the administration groups, add 200 μL of Flavokawain C medium solutions with concentrations of 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.6 μM, and 0.8 μM respectively. The blank group is given the same volume of 1640 medium containing 10% serum, and treat for 48 hours simultaneously. After 48 hours, change to a 10% cck8 medium solution, and measure the absorbance at a wavelength of 480 nM after 2 hours.

[0061] The results are as Figure 2 shown. The IC 50 of Flavokawain C against Sh_IDH1 TS603 cells after 48 hours is 36.07 μM.

[0062] Example 4 Evaluation of the cytotoxicity of Flavokawain C

[0063] (1)Experimental materials

[0064] BV2 cells, CCK-8 reagent, Flavokawain C (Shanghai TargetMol), microplate reader (Biotek).

[0065] (2)Experimental method

[0066] Inoculate the cells at 8000 cells per well in a 96-well plate, and divide them into a blank group and a series of gradient concentration Flavokawain C administration groups. After the cells adhere to the wall, change the medium. In the administration groups, add 200 μL of Flavokawain C medium solutions with concentrations of 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.6 μM, and 0.8 μM respectively. The blank group is given the same volume of 1640 medium containing 10% serum, and treat for 48 hours simultaneously. After 48 hours, change to a 10% cck8 medium solution, and measure the absorbance at a wavelength of 480 nM after 2 hours.

[0067] The results are as Figure 3As shown, the IC 50 value of Flavokawain C against BV2 cells after 48 hours is greater than 200 μM, indicating good safety.

[0068] Example 5 Expression of IDH1-R132H Recombinant Protein

[0069] (1) Experimental Materials

[0070] BL21(DE3) competent Escherichia coli strain (Nanjing Novoprotein Biological Co., Ltd.), BCA kit (Beijing Solarbio Science & Technology Co., Ltd.), IPTG (Shanghai Beyotime Biotechnology Co., Ltd.), kanamycin (Shanghai Beyotime Biotechnology Co., Ltd.), His-tag purification resin (Shanghai Beyotime Biotechnology Co., Ltd.), tryptone (Thermo Fisher Scientific, USA), yeast extract (Thermo Fisher Scientific, USA), agar powder (Hubei Bergi Biology Co., Ltd.), imidazole (Shanghai Aladdin Biochemical Technology Co., Ltd.), pET-28a+ vector (Beijing Tsingke Biotechnology Co., Ltd.).

[0071] (2) Experimental Methods

[0072] Mutate arginine (R) at position 132 of human IDH1 (NM_001282386.1, CDS region) to histidine (R132H), and then clone it into the NdeI / Xho I sites of the pET-28a+ vector to obtain a recombinant expression vector.

[0073] Transform the recombinant expression vector into Escherichia coli BL21(DE3), and form monoclonal colonies on Luria-Bertani (LB) solid medium containing kanamycin. Culture and induction: Pick monoclonal colonies and culture them in 300 mL of LB medium on a shaker at 37°C for 12 h. Induce with 0.4 mmol / L of isopropyl-D-thiogalactoside (IPTG) at 16°C for 6 h. Centrifuge at 4000 rpm for 20 min to remove cell debris in the supernatant. Resuspend with PBS and sonicate the bacterial cells on ice. After centrifugation at 4000 rpm for 30 min, load the supernatant onto the pre-equilibrated His-tag purification resin. Wash the miscellaneous proteins with 20 mM imidazole, and then elute the protein with 200 mM imidazole. Determine the protein concentration using a BCA kit, and detect the protein purity by SDS-PAGE combined with Coomassie Brilliant Blue. Replace the imidazole solution with ultrafiltration using a PBS solution, and concentrate the IDH1-R132H recombinant protein to 10 mg / ml. Store and use at 4°C.

[0074] Example 6 Detection of the Binding Affinity between Flavokawain C and IDH1-R132H Protein

[0075] (1) Experimental Materials

[0076] SMAP probe (Gator, USA), Biotin (Beijing Biolegend Technology Co., Ltd.), Flavokawain C (TargetMol, Shanghai), Gator plus biomembrane interferometer (Gator, USA).

[0077] (2)Experimental method

[0078] Mix the biotin reagent with the IDH1-R132H recombinant protein in a 2-fold molar amount and incubate in the dark at room temperature for 2 h. Remove the unbound biotin by ultrafiltration.

[0079] Pre-wet the SMAP probe in PBS buffer (0.05% BSA, 0.01% Tween-20) for 10 min. Take a protein microplate, add 250 μL of the biotinylated protein solution and the control solution to each well, with a total of 5 wells, and place one probe in each well. The probe immobilizes the biotinylated protein (10 μg / mL), and the analyte is a gradient-diluted Flavokawain C solution. Use the Gatorplus biomembrane interferometer to determine the binding affinity of Flavokawain C to recombinant IDH1.

[0080] The parameters are set as follows: temperature 25 °C; baseline 120 sec, 1000 rpm; loading 160 sec, 400 rpm; baseline 120 sec, 1000 rpm; binding 300 sec, 400 rpm; dissociation 600 sec, 400 rpm; binding mode 1:1.

[0081] When loading the biotin-labeled IDH1 protein, stop loading the protein when the signal is greater than 0.6 nm and not saturated. After the baseline is stable, place the probe in the drug solution for the detection of binding force. The background control is a set of repeated probes without protein loading, which is used to deduct the small molecule background signal. All data are statistically analyzed using Gator Bio data analysis software. According to the ratio of K off and K on , calculate the equilibrium dissociation constant (K d value).

[0082] The results are as Figure 4 shown. The binding force of Flavokawain C to the IDH1-R132H recombinant protein is 8.16×10 -7 M, indicating a strong binding ability.

[0083] Example 7 Effect of Flavokawain C on the level of 2-HG in TS603 cells

[0084] (1)Experimental materials

[0085] D-2-Hydroxyglutaric acid (D2HG) ELISA kit (Shanghai Hengyuan Biotechnology), Flavokawain C (Shanghai Taoshu Biotechnology), and microplate reader (Biotek).

[0086] (2) Experimental methods

[0087] Culture TS603 cells to appropriate density, add Flavokawain C and incubate for 24 hours. Discard the culture medium, wash with PBS 2-3 times, digest with trypsin, and collect the cells. Extract the cell precipitate with butanol:methanol:water (5:25:70 V:V:V) and store at -20℃ for later use. Equilibrate the kit at room temperature for 15-30 minutes, and dilute the 30-fold concentrated washing solution. Set up standard wells, blank wells, and wells to be tested. Add 50μl of different concentrations of standard products to the standard wells, add 40μl of diluent and then 10μl of sample to the wells to be tested, and mix after adding the sample. Add 50μl of enzyme labeling reagent to each well except the blank well, seal the plate and incubate at 37℃ for 60 minutes. Remove the membrane, discard the liquid, and shake dry. Wash 5 times and pat dry. Add 50μl of color developer A and B to each well in turn, and color at 37℃ in the dark for 15 minutes. Add 50μl stop solution and measure the OD value at 450nm (within 15 minutes). Draw a standard curve, find out or calculate the concentration based on the sample OD value and multiply it by 5 to get the actual concentration.

[0088] The results are as follows Figure 5 As shown, Flavokawain can inhibit 2-HG production in TS603 cells.

[0089] Example 8 Flavokawain C downregulates the methylation levels of H3K9me3 and H3K9me2 proteins in TS603 cells

[0090] (1) Experimental materials

[0091] TS603 cells, RIPA lysis buffer (Shanghai Yazyme Biopharmaceutical Technology Co., Ltd.), H3K9me3 antibody (Biyuntian), H3K9me2 antibody (Wuhan Sanying Biotechnology), β-actin antibody (Wuhan Abotek), 0.22 μm PVDF membrane (Millipore, USA), goat anti-rabbit IgG secondary antibody (Shanghai Biyuntian), Omni-ECL chemiluminescence detection kit (Shanghai Yazyme Biopharmaceutical Technology Co., Ltd.).

[0092] (2) Experimental methods

[0093] Culture TS603 cells to an appropriate density, add Flavokawain C and incubate for 24 hours. Discard the culture medium, wash 2 - 3 times with PBS, digest with trypsin, and collect the cells. Add RIPA cell lysate and protease inhibitor, place on ice and sonicate, centrifuge at 20000×g for 5 min at 4°C, collect the supernatant, add loading buffer, and incubate at 95°C for 10 min. Perform SDS-PAGE electrophoresis separation on the prepared loading solution, and then transfer it to a PVDF membrane. Block the PVDF membrane in 5% non-fat milk powder at room temperature for half an hour, then place it in the primary antibody incubation solution and incubate overnight at 4°C. Wash in PBST for 5 minutes and repeat 3 times. Subsequently, place it in the secondary antibody incubation solution and incubate at room temperature for 30 min. Wash in PBST for 5 minutes and repeat 3 times. Use a chemiluminescence kit to detect the target protein, analyze the PVDF membrane with a multifunctional imager, and quantify it with ImageJ software.

[0094] The results are as Figure 6 shown that Flavokawain C can inhibit the methylation levels of H3K9me3 and H3K9me2 in TS603 cells.

[0095] Example 9 Flavokawain C inhibits the proliferation of xenograft tumors of TS603 cells in nude mice

[0096] (1) Experimental materials

[0097] TS603 cells, BALB / c mice (Speywood), Flavokawain C (TargetMol), AG120 (Maclin), sodium carboxymethylcellulose (Solarbio).

[0098] (2) Experimental methods

[0099] Collect TS603 cells in good condition in physiological saline and inject 2×10 6 cells into the right axilla of 5-week-old BALB / c mice to form xenograft tumors. Start drug administration one week after tumor inoculation. After preparing 0.5% sodium carboxymethylcellulose with physiological saline, prepare 30 mg / kg and 20 mg / kg Flavokawain C suspension, 50 mg / kg AG120 suspension as the positive drug, and the solvent control suspension. Administer the drugs intraperitoneally every day for 2 consecutive weeks.

[0100] After drug treatment, use vernier calipers and other tools to measure the long and short diameters of the xenograft tumors in nude mice every 3 - 4 days, and record the data to calculate the tumor volume.

[0101] The calculation formula is V = (a×b²) / 2, where V represents the tumor volume, a is the long diameter of the tumor, and b is the short diameter of the tumor. One day after the last administration, the mice were sacrificed, blood was collected, the tumors were dissected, weighed, and photographed.

[0102] The results are as Figure 7 shown. Both 30 mg / kg and 20 mg / kg Flavokawain and 50 mg / kg AG120 could significantly inhibit the growth of xenograft tumors in nude mice, and there was no significant difference in the inhibitory effect on xenograft tumors in TS603 nude mice between 30 mg / kg Flavokawain C and 50 mg / kg AG120.

[0103] Example 10 Flavokawain C down-regulates the levels of 2-HG in xenograft tumors and blood of TS603 cells in nude mice

[0104] (1) Experimental materials

[0105] D-2-Hydroxyglutaric acid (D2HG) ELISA kit (Shanghai Hengyuan Bio), microplate reader (Biotek).

[0106] (2) Experimental methods

[0107] The same position of the xenograft tumors in nude mice was excised, weighed, ultrasonically extracted with butanol: methanol: water (5:25:70 V:V:V), the supernatant was taken after centrifugation at 6000 rpm for 10 minutes, and the levels of 2-HG in xenograft tumors and blood of TS603 cells in nude mice were detected by the same method as before.

[0108] The results are as Figure 8 shown. Both 30 mg / kg and 20 mg / kg Flavokawain and 50 mg / kg AG120 could significantly inhibit the level of 2-HG in xenograft tumors in nude mice.

[0109] Example 11 Flavokawain C covalently binds to the cysteine of IDH1-R132H protein

[0110] (1) Experimental materials

[0111] 5-TAMRA azide (Xi'an Ruixi Bio), IAA-yne (Xi'an Ruixi Bio), CuSO4 (Shanghai Aladdin), TCEP (Shanghai Macklin), TBTA (Shanghai Macklin), acetone (Sinopharm Chemical Reagent), tert-butanol (Shanghai Aladdin), Flavokawain C (Shanghai TargetMol).

[0112] (2) Experimental methods

[0113] Set up a blank control group, an IAA-yne probe control group, an IAA + IAA-yne competition control group, and a Flavokawain C + IAA-yne competition group.

[0114] Probe labeling: Prepare 5 μg of IDH1-R132H recombinant protein for each group.

[0115] In the blank group, only IDH1-R132H protein and the control solvent were added. In the IAA-yne probe control group, IDH1 protein was added.

[0116] In the IAA competition group, IDH1 protein and IAA were added.

[0117] In the Flavokawain C competition group, IDH1-R132H protein and Flavokawain C were added.

[0118] After preparing the above systems, place them in EP tubes. Place the EP tubes in a shaker at 37°C and incubate for 2 h. Then, add the IAA-yne probe and continue to incubate in the shaker at 37°C for 1 h. Keep the total volume of the system at 40 μL.

[0119] Prepare the click reagent: Prepare the click reagent according to 72 μL of tert-butanol, 18 μL of 1.7 mM TBTA, 30 μL of 50 mM CuSO4, 9 μL of 10 mM 5-TAMRA azide, and 30 μL of 50 mM TCEP. Prepare it freshly before use and mix well.

[0120] Click reaction: According to the ratio of adding 12 μL of the click reagent to every 100 μL of the system, quickly add it to the sample, mix well, and incubate in a shaker at 29°C for 1 h. Protein precipitation with acetone: After the click reaction, add 1 mL of cold acetone, place it at -20°C for more than 30 min. Centrifuge at 20000×g for 5 min, discard the supernatant, and let the acetone evaporate completely.

[0121] SDS-PAGE: Add 20 μL of protein loading buffer to each tube, denature at 95°C for 5 min, centrifuge, and take the supernatant for SDS-PAGE electrophoresis. Take the protein gel for fluorescence imaging. Finally, perform Coomassie Brilliant Blue staining and take pictures for record.

[0122] The results are as Figure 9 shown. Flavokawain C can competitively bind to IDH1-R132H protein with IAA-YNE, indicating that Flavokawain C binds to the cysteine site of IDH1-R132H in a covalent manner.

[0123] As described above, it is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claimed rights.

Claims

1. The use of kava pepper C in the preparation of anti-tumor drugs, characterized in that: The tumor is a tumor carrying a mutated IDH1, the mutated IDH1 is an IDH1-R132C mutation and / or an IDH1-R132H mutation, and the tumor is a fibrosarcoma or a glioma.

2. The use according to claim 1, characterized in that: The drug selectively inhibits the proliferation of tumor cells with IDH1-R132C mutation, and / or, the drug selectively inhibits the proliferation of tumor cells with IDH1-R132H mutation.

3. The use according to claim 1, characterized in that: The drug reduces the expression level of 2-HG in tumor cells.

4. The use according to claim 2, characterized in that: The drug reduces the methylation level of H3K9me3 in tumor cells, and / or the drug reduces the methylation level of H3K9me2 in tumor cells.

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