Application of kavaenin C in preparation of IDH1 inhibitor

Abstract

The invention relates to the technical field of clinical medicine and biological pharmacy, in particular to application of Flavokawain C in inhibiting IDH1 mutant tumor cells in tumor treatment. A large number of experimental studies show that the Flavokawain C has the function of selectively inhibiting IDH1 mutant tumor cells, can significantly reduce the 2-HG level in the IDH1 mutant cells, and has no obvious toxic or side effect on normal cells. Based on the clinical problems, the invention aims to explore the application of the Flavokawain C compound as an IDH1 covalent inhibitor in anti-IDH1 mutant tumors, so as to provide a new choice and a more effective solution for the treatment of the IDH1 mutant tumors.

Description

Technical Field

[0001] The present invention relates to the technical fields of clinical medicine and biopharmaceuticals, and in particular to application of kava pepper C in the preparation of an IDH1 inhibitor. Background Art

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

[0003] Although existing IDH1 inhibitors can inhibit the activity of IDH1 mutant enzymes to a certain extent and reduce 2-HG levels, 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 patient survival. And as treatment time goes on, tumor cells are prone to develop resistance to existing IDH1 inhibitors. The resistance mechanism is relatively complex and may be related to multiple situations such as secondary mutations of the IDH1 gene mutation, changes in drug transporters, and adaptive changes in downstream signaling pathways. Once resistance occurs, the efficacy of the drug will be significantly reduced, and patients will lose effective treatment options.

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

[0005] After a large number of experimental studies, the present invention 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 side effects on normal cells. Based on the above clinical problems, the present invention aims to explore the application of Flavokawain C compounds as IDH1 covalent inhibitors in the treatment of 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 use of kava pepper C in the preparation of IDH1 inhibitors.

[0007] The present invention also provides the use of kava pepper C in the preparation of anti-tumor drugs, wherein the tumor is a tumor carrying a mutated 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, wherein the drug comprises kava piperine C.

[0012] According to a 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 a specific embodiment of the present invention, the drug reduces the production level of 2-HG in tumor cells.

[0014] According to a 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 medicament comprises a pharmaceutically acceptable carrier.

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

[0017] In the above-mentioned medicine, the oral preparation includes additives, and the additives are 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 sugars such as lactose, sucrose, glucose, mannitol, sorbitol, and dextrin; starches such as starch, pregelatinized starch, and dextrin; celluloses such as microcrystalline cellulose, gum arabic, fenugreek gum, and dextran; and inorganic salts such as calcium sulfate, calcium hydrogen phosphate, medicinal calcium carbonate, light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminosilicate.

[0019] The lubricant, glidant or anti-adhesive agent includes stearic acid; metal stearate such as calcium stearate or magnesium stearate; talc; colloidal silica; micro powder silica gel, hydrogenated vegetable oil; polyethylene glycol, lauryl sulfate such as sodium lauryl sulfate or magnesium lauryl sulfate; silicate such as silicic anhydride or silicate hydrate, etc.

[0020] The adhesive comprises distilled water, ethanol of different concentrations, starch slurry, hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, polyvinyl pyrrolidone, polyethylene glycol and compounds similar to the above excipients.

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

[0022] The surfactant includes sodium lauryl sulfate, sodium stearate sulfonate and the like.

[0023] The antioxidants include sodium bisulfite, sodium pyrosulfite, sodium sulfite, dried sodium sulfite, sodium thiosulfate, ascorbic acid, methionine (methionine), thiourea, phosphoric acid, citric acid and the like.

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

[0025] The flavoring agents include sweeteners such as saccharin sodium, 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 aromatics such as fennel oil, peppermint oil, menthol, peppermint water, cinnamon oil, lemon essence, lemon oil and spices of various flavors.

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

[0027] Pharmaceutically effective amount refers to a reasonable benefit / risk ratio that can be obtained by applying drug treatment, a quantity sufficient to treat the disease. The level of effective dose can be determined depending on some factors, including the patient's disease type, severity, activity of the drug, sensitivity to the drug, application time, route of administration, excretion rate, treatment cycle, drugs used simultaneously, and other factors known in the medical field. The drug of the present invention can be used as an independent therapeutic agent, or combined with other therapeutic agents. In addition, the drug of the present invention can be added with typical therapeutic agents continuously or simultaneously, and the composition can be applied once or multiple times. It is important to consider all of the above factors and apply the minimum dose that can produce the maximum effect without side effects, and the dosage can be determined by the physician according to the patient's condition, age, etc.

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

[0029] Beneficial effects of the present invention: Compared with the existing IDH1 mutation inhibitor AG120, Flavokawain C can more significantly inhibit the proliferation of IDH1 mutant cells through its covalent inhibition of IDH1 mutation. Flavokawain C can be used as an IDH1 mutation inhibitor. Furthermore, Flavokawain C can be used as a drug for treating cancer, providing a theoretical and clinical basis for the treatment of IDH1 mutation tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] FIG1 shows the inhibitory curve of Flavokawain C and AG120 on the proliferation of IDH1 mutant cells.

[0032] FIG2 shows the curve of the inhibition of proliferation of IDH1 mutant knockdown cells by Flavokawain C.

[0033] FIG3 shows the safety evaluation of Flavokawain C on LO2 cells and BV2 cells.

[0034] FIG4 shows the binding ability detection of Flavokawain C and IDH1-R132H.

[0035] FIG5 shows the effect of Flavokawain C on the level of 2-HG in TS603 cells.

[0036] FIG6 shows the effect of Flavokawain C on the methylation levels of H3K9me3 and H3K9me2 proteins in TS603 cells.

[0037] FIG. 7 shows the effect of Flavokawain C on the proliferation of TS603 cell transplanted tumors in nude mice.

[0038] FIG8 shows the effect of Flavokawain C on 2-HG levels in TS603 cell xenografts in nude mice.

[0039] FIG. 9 shows the covalent binding mode of Flavokawain C to IDH1-R132H protein. DETAILED DESCRIPTION

[0040] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0041] Example 1 Inhibitory effects of Flavokawain C and AG120 on proliferation of IDH1 mutant cells (1) Experimental materials HT1080 cells, TS603 cells, CCK-8 reagent, Flavokawain C (Shanghai Taoshu Biotechnology), AG120 (Shanghai MacLean), and microplate reader (Biotek).

[0042] (2) Experimental methods The cells were seeded in 96-well plates at 8000 cells per well and divided into blank group, series gradient concentration Flavokawain C and AG120 treatment groups. After the cells attached to the wall, the culture medium was replaced. The treatment group was added with 200 μL of Flavokawain C or AG120 culture medium solution with concentrations of 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.6 μM, 0.8 μM, and the blank group was given the same volume of 1640 culture medium containing 10% serum and treated for 48 hours. After 48 hours, it was replaced with 10% cck8 culture medium solution, and the absorbance was detected at a wavelength of 480 nM after 2 hours.

[0043] The results are as follows Figure 1 As shown, the IC of Flavokawain C on HT1080 cells after 48 hours 50 The IC value for TS603 cells is 4.772 μM. 50 The IC value of AG120 for HT1080 cells is 7.916 μM. 50 Values ​​greater than 50 μM.

[0044] Example 2 Construction of IDH1 knockdown cell line (1) Experimental materials TS603 cells, 293T cells, lentiviral packaging kit (Bio-Gene), pLV-U6-SHIDH1-CMV-EGFP (T2A)-PURO (Scilia Biotechnology), puromycin (Biyuntian), RIPA lysis buffer (Shanghai Yazyme Biotechnology Co., Ltd.), IDH1 antibody (Wuhan Proteintech), 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 Biotechnology Co., Ltd.).

[0045] (2) Experimental methods Inoculate low-passage, good-condition 293T cells in a 10 cm culture dish and prepare for transfection when the cell density is about 80%. Prepare transfection reagents according to the instructions of the lentiviral packaging kit: Opti-MEM 1000 μL, bone plasmid DNA 7.5 μg, Easy Mix 7.5 μg, LipoX 37.5 μL. Mix well and place at room temperature for 15 min before adding to the cells. Shake well, add 10 μL of transfection enhancer, place in an incubator for transfection overnight and discard the supernatant (sterilized). Add 10 mL of fresh culture medium and collect the supernatant after 36 hours. Then add 10 mL and continue to culture for 24 hours before collecting the supernatant. Combine the two supernatants, centrifuge at 4°C, 4000rpm for 10 min, collect the supernatant, and filter with a 0.45 μm filter. Aliquot and store at -80°C for a long time or at 4°C for use within one week.

[0046] Inoculate TS603 cells in a 6-well plate. When the cell confluence is 60-80%, add a mixture of virus packaging supernatant and culture medium in a ratio of 1:1. Observe the cell status, change to regular cell culture medium 12-24 hours after infection, and continue to culture for 24 hours. Use puromycin to screen stable cell lines.

[0047] Western blot protein expression verification. Puromycin-resistant cells were collected, washed twice with PBS, centrifuged and discarded the supernatant, and the cells were collected. RIPA cell lysis buffer and protease inhibitors were added and ultrasonically disrupted on ice. Centrifuged at 20,000 × g for 5 min at 4°C, the supernatant was collected, and the loading buffer was added for 10 min at 95°C. The prepared loading solution was separated by SDS-PAGE electrophoresis and then transferred to a PVDF membrane. After blocking the PVDF membrane in 5% skim milk powder at room temperature for half an hour, it was placed in the primary antibody incubation solution at 4°C overnight. It was placed in PBST for 5 minutes and repeated 3 times. It was then placed in the secondary antibody incubation solution at room temperature for 30 min. It was placed in PBST for 5 minutes and repeated 3 times. The target protein was detected using a chemiluminescence kit, and the PVDF membrane was analyzed by a multifunctional imager and quantified by ImageJ software.

[0048] The results are as follows Figure 2 As shown, compared with the control group, the expression level of IDH1 was significantly reduced, indicating that the TS603 cell line with IDH1 knockdown had been successfully constructed.

[0049] Example 3: Inhibitory effect of Flavokawain C on proliferation of IDH1 mutant knockdown cells (1) Experimental materials Sh_IDH1 TS603 cells, CCK-8 reagent, Flavokawain C (Shanghai Taoshu Biotechnology), and microplate reader (Biotek).

[0050] (2) Experimental methods Sh_IDH1 TS603 cells were seeded in 96-well plates at 8000 cells per well and divided into a blank group and a series of gradient concentrations of Flavokawain C administration groups. After the cells adhered to the wall, the culture medium was replaced. The administration group was added with 200 μL of Flavokawain C culture 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, and the blank group was given the same volume of 1640 culture medium containing 10% serum and treated for 48 hours. After 48 hours, it was replaced with 10% cck8 culture medium solution, and the absorbance was detected at a wavelength of 480 nM after 2 hours.

[0051] The results are as follows Figure 2 As shown, the IC of Flavokawain C against Sh_IDH1 TS603 cells at 48 hours 50 It is 36.07 μM.

[0052] Example 4 Evaluation of the cytotoxicity of Flavokawain C (1) Experimental materials BV2 cells, CCK-8 reagent, Flavokawain C (Shanghai Taoshu Biological), and microplate reader (Biotek).

[0053] (2) Experimental methods The cells were seeded in 96-well plates at 8000 cells per well and divided into a blank group and a group treated with a series of gradient concentrations of Flavokawain C. After the cells adhered to the wall, the culture medium was replaced. The treatment group was added with 200 μL of Flavokawain C culture medium solution 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, and the blank group was given an equal volume of 1640 culture medium containing 10% serum and treated for 48 hours. After 48 hours, it was replaced with 10% cck8 culture medium solution, and the absorbance was detected at a wavelength of 480 nM after 2 hours.

[0054] The results are as follows Figure 3 As shown, the IC of Flavokawain C on BV2 cells at 48 h 50 The value was greater than 200 μM, indicating good safety.

[0055] Example 5 IDH1-R132H recombinant protein expression (1) Experimental materials BL21(DE3) competent Escherichia coli strain (Nanjing Novozyme Biotechnology Co., Ltd.), BCA kit (Beijing Solebow Technology Co., Ltd.), IPTG (Shanghai Biotech Co., Ltd.), kanamycin (Shanghai Biotech Co., Ltd.), His-tag purification resin (Shanghai Biotech Co., Ltd.), tryptone (Thermo Fisher Scientific, USA), yeast extract (Thermo Fisher Scientific, USA), agar powder (Hubei Baiji Biotechnology Co., Ltd.), imidazole (Shanghai Aladdin Biochemical Technology Co., Ltd.), pET-28a+ vector (Beijing Qingke Biotechnology Co., Ltd.).

[0056] (2) Experimental methods The 132-arginine (R) of human IDH1 (NM_001282386.1, CDS region) was mutated to histidine (R132H) and then cloned into the NdeI / Xho I site of the pET-28a+ vector to obtain a recombinant expression vector.

[0057] The recombinant expression vector was transformed into Escherichia coli BL21 (DE3) and monoclonal colonies were formed in Luria-Bertani (LB) solid medium containing kanamycin. Culture and induction: Pick a monoclonal colony in 300 mL LB medium, place it in a shaker, and expand it at 37°C for 12 h. 0.4 mmol / L isopropyl-D-thiogalactose (IPTG) was induced at 16°C for 6 h. The cell debris in the supernatant was removed by centrifugation at 4000 rpm for 20 min. After resuspending in PBS, the bacteria were ultrasonically disrupted on ice, and the supernatant was loaded onto the pre-equilibrated His-tag purification resin after centrifugation at 4000 rpm for 30 min. The impurities were washed with 20 mM imidazole, and then the protein was eluted with 200 mM imidazole. The protein concentration was determined by BCA kit, and the protein purity was detected by SDS-PAGE combined with Coomassie Brilliant Blue. The imidazole solution was replaced by ultrafiltration with PBS solution, and the IDH1-R132H recombinant protein was concentrated to 10 mg / ml. Store at 4°C before use.

[0058] Example 6 Detection of the binding ability of Flavokawain C to IDH1-R132H protein (1) Experimental materials SMAP probe (Gator Company, USA), Biotin (Beijing Biolab Technology Co., Ltd.), FlavokawainC (Shanghai Taoshu Biological), Gator plus biomembrane interferometer (Gator Company, USA).

[0059] (2) Experimental methods A 2-fold molar amount of biotin reagent was mixed with the IDH1-R132H recombinant protein and incubated at room temperature in the dark for 2 h. Biotin not bound to the protein was removed by ultrafiltration.

[0060] The SMAP probe was pre-wetted in PBS buffer (0.05% BSA, 0.01% Tween-20) for 10 min. A protein sample plate was taken, and 250 μL of biotinylated protein solution and control solution were added to each well, for a total of 5 wells, with one probe placed in each well. The probe was fixed with biotinylated protein (10 μg / mL), and the analyte was a gradient diluted Flavokawain C solution. The binding affinity of Flavokawain C to recombinant IDH1 was determined using a Gatorplus biomembrane interferometer.

[0061] The parameters were set as follows: temperature 25°C; baseline 120 sec, 1000 rpm; load 160 sec, 400 rpm; baseline 120sec, 1000 rpm; binding 300 sec, 400 rpm; dissociation 600 sec, 400 rpm; binding mode was 1:1.

[0062] When loading biotin-labeled IDH1 protein, stop loading protein when the signal is greater than 0.6 nm and non-saturated. After the baseline is stable, place the probe in the drug solution for binding force detection. The background control is a set of repeated probes without protein loading, which is used to deduct small molecule background signals. All data were statistically analyzed using Gator Bio data analysis software. According to K off With K on The equilibrium dissociation constant (K d value).

[0063] The results are as follows Figure 4 As shown, the binding affinity of Flavokawain C to IDH1-R132H recombinant protein is 8.16×10 -7 M, has strong binding ability.

[0064] Example 7 Effect of Flavokawain C on the Level of 2-HG in TS603 Cells (1) Experimental materials D-2-Hydroxyglutaric acid (D2HG) ELISA kit (Shanghai Hengyuan Biotechnology), Flavokawain C (Shanghai Taoshu Biotechnology), and microplate reader (Biotek).

[0065] (2) Experimental methods 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.

[0066] The results are as follows Figure 5 As shown, Flavokawain can inhibit 2-HG production in TS603 cells.

[0067] Example 8 Flavokawain C downregulates the methylation levels of H3K9me3 and H3K9me2 proteins in TS603 cells (1) Experimental materials 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.).

[0068] (2) Experimental methods TS603 cells were cultured to an appropriate density, and Flavokawain C was added and incubated for 24 hours. The culture medium was discarded, and the cells were washed with PBS for 2-3 times, and then digested with trypsin and collected. RIPA cell lysis buffer and protease inhibitors were added and ultrasonically disrupted on ice. The cells were centrifuged at 20000×g for 5 min at 4℃, and the supernatant was collected. The loading buffer was added and the cells were incubated at 95℃ for 10 min. The prepared loading solution was separated by SDS-PAGE electrophoresis and then transferred to a PVDF membrane. The PVDF membrane was blocked in 5% skim milk powder for half an hour at room temperature, and then placed in the primary antibody incubation solution at 4℃ overnight. It was washed in PBST for 5 minutes and repeated 3 times. It was then placed in the secondary antibody incubation solution at room temperature for 30 min. It was washed in PBST for 5 minutes and repeated 3 times. The target protein was detected using a chemiluminescence kit, and the PVDF membrane was analyzed by a multifunctional imager and quantified by ImageJ software.

[0069] The results are as follows Figure 6 As shown, Flavokawain C can inhibit the methylation levels of H3K9me3 and H3K9me2 in TS603 cells.

[0070] Example 9: Flavokawain C inhibits proliferation of TS603 cell transplanted tumors in nude mice (1) Experimental materials TS603 cells, BALB / c mice (Speifo), Flavokawain C (Shanghai Taoshu Biotechnology), AG120 (Shanghai MacLean), sodium carboxymethylcellulose (Solarbio).

[0071] (2) Experimental methods TS603 cells in good condition were collected and placed in saline at 2×10 6 The cells were injected into the right axilla of 5-week-old BALB / c mice to form xenograft tumors. Dosing began one week after tumor inoculation. After preparing 0.5% sodium carboxymethylcellulose with normal saline, 30mg / kg and 20mg / kg Flavokawain C suspensions, 50mg / kg AG120 suspensions of the positive drug, and solvent control suspensions were prepared. The drugs were administered by intraperitoneal injection every day for 2 consecutive weeks.

[0072] After drug administration, the long and short diameters of the nude mouse transplanted tumors were measured every 3-4 days using a vernier caliper or other tool, and the data were recorded to calculate the tumor volume.

[0073] 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 animals were sacrificed, blood was collected, the tumors were dissected, weighed, and photographed.

[0074] The results are as follows Figure 7 As shown, 30mg / kg, 20mg / kg Flavokawain and 50mg / kg AG120 can significantly inhibit the growth of nude mouse transplanted tumors, and there is no significant difference in the inhibitory effect of 30mg / kg Flavokawain C and 50mg / kg AG120 on TS603 nude mouse transplanted tumors.

[0075] Example 10 Flavokawain C down-regulates 2-HG levels in TS603 cell transplanted tumors and blood in nude mice (1) Experimental materials D-2-Hydroxyglutaric acid (D2HG) ELISA kit (Shanghai Hengyuan Biotechnology), microplate reader (Biotek).

[0076] (2) Experimental methods The same position of the nude mouse transplanted tumor was cut and weighed, and ultrasonic extraction was performed using butanol:methanol:water (5:25:70 V:V:V). After centrifugation at 6000 rpm for 10 minutes, the supernatant was collected and the 2-HG level in the TS603 cell nude mouse transplanted tumor and blood was detected using the same method as before.

[0077] The results are as follows Figure 8 As shown, 30mg / kg, 20mg / kg Flavokawain and 50mg / kg AG120 can significantly inhibit the 2-HG level in nude mouse transplanted tumors.

[0078] Example 11 Covalent Binding of Flavokawain C to IDH1-R132H Protein Cysteine (1) Experimental materials 5-TAMRA azide (Xi'an Ruixi Biotechnology), IAA-yne (Xi'an Ruixi Biotechnology), CuSO4 (Shanghai Aladdin), TCEP (Shanghai McLean), TBTA (Shanghai McLean), acetone (Sinopharm Chemical Reagent), tert-butyl alcohol (Shanghai Aladdin), Flavokawain C (Shanghai Taoshu Biotechnology).

[0079] (2) Experimental methods A blank control group, IAA-yne probe control group, IAA+IAA-yne competition control group, and Flavokawain C+IAA-yne competition group were set up.

[0080] Probe labeling: Prepare 5 μg of IDH1-R132H recombinant protein for each group.

[0081] The blank group was only added with IDH1-R132H protein and control solvent, and the IAA-yne probe control group was added with IDH1 protein.

[0082] The IAA competition group was added with IDH1 protein and IAA.

[0083] IDH1-R132H protein and Flavokawain C were added to the Flavokawain C competition group.

[0084] After the above system is prepared, place it in an EP tube. Place the EP tube in a 37℃ shaking incubation for 2 h, then add the IAA-yne probe and continue shaking incubation at 37℃ for 1 h. Keep the total system at 40 μL.

[0085] Prepare click reagent: Prepare click reagent according to 72 μL tert-butyl alcohol, 18 μL 1.7 mM TBTA, 30 μL 50 mM CuSO4, 9 μL 10 mM 5-TAMRA azide, and 30 μL 50 mM TCEP. Prepare before use and mix thoroughly.

[0086] Click reaction: add 12 μL of click reagent to every 100 μL of system, quickly add to the sample, mix thoroughly, and incubate at 29℃ for 1 h. Acetone precipitation of protein: after the click reaction is completed, add 1 mL of cold acetone and let stand at -20℃ for more than 30 min. Centrifuge at 20,000×g for 5 min, discard the supernatant, and fully evaporate the acetone.

[0087] 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, stain with Coomassie brilliant blue and take photos for record.

[0088] The results are as follows Fig. 9 As shown, Flavokawain C was able to compete with IAA-YNE for binding to IDH1-R132H protein, indicating that Flavokawain C covalently binds to the IDH1-R132H cysteine ​​site.

[0089] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. Application of kava pepper C in the preparation of IDH1 inhibitors.

2. 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.

3. The use according to claim 2, 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.

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

5. 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.

6. A drug for treating IDH1 mutant tumors, characterized in that: The medicament comprises kava piperine C.

7. The drug according to claim 6, 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.

8. The drug according to claim 6, characterized in that The drug reduces the expression level of 2-HG in tumor cells.

9. The drug according to claim 6, 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.

10. The drug according to claim 6, characterized in that Kava C is used at a concentration of 1-300 mg / kg.

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