Application of geraniin in preparation of medicine for treating LDHA related diseases or medicine for inhibiting LDHA activity and medicine composition

Abstract

The invention discloses an application of geraniin in preparation of a lactic dehydrogenase A (LDHA) inhibitor. In an enzyme activity test, the geraniin has a very significant inhibition effect on LDHA, and the median inhibitory concentration (IC50) is 26.7 + / -7.3 nM; a substrate competition experiment and molecular simulation show that the geraniin is allosterally combined with LDHA; cell experiments show that geraniin has in-vitro triple negative breast cancer resistance activity, and geraniin treatment causes accumulation of reactive oxygen species (ROS) in cells, mitochondrial membrane potential disorder, inhibition of cell migration and retardation of a cell cycle in an S period. Therefore, the geraniin has a potential treatment value on LDHA related diseases, such as triple negative breast cancer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of drug development, and particularly relates to the use of a natural product, oroxin, in the preparation of lactate dehydrogenase A (LDHA) inhibitors and LDHA-related diseases such as triple-negative breast cancer. BACKGROUND

[0002] LDHA, as a key enzyme in aerobic glycolysis, mediates the interconversion between lactic acid and pyruvic acid in vivo. LDHA high expression is related to various types of cancers including triple-negative breast cancer (J Exp Clin Cancer Res, 2017, 36(2): 129). In addition, knocking down LDHA can inhibit tumor cell proliferation, inhibit invasion ability, reduce mitochondrial membrane potential and induce oxidative stress (Proc Natl Acad Sci USA, 2010, 107(5): 2037-2042). Therefore, LDHA is considered a promising potential target for TNBC.

[0003] Currently, there are many synthetic LDHA inhibitors reported, such as FX11 (Proc Natl Acad Sci USA, 2010, 107(5): 2037-2042), GEN-140 (Nat Cell Biol, 2016, 12(10): 779-786), GSK2837808A (Cancer Metab, 2013, 1(1): 19), NCATS-SM1440 (J Med Chem, 2020, 63(19): 10984-11011) and compound 7 (ACS Omega, 2020, 5(22): 13034-13041). Among them, GSK2837808A shows the greatest inhibitory potency on LDHA, with a half-inhibitory concentration (IC 50 ) value of 2.6 nM. Except for compound 7, all these synthetic LDHA inhibitors show antitumor activity in in vitro cell experiments. In addition, NCATS-SM1440 inhibits LDH activity in vivo, however, whether NCATS-SM1440 inhibits tumor growth in vivo is not mentioned. It is worth noting that compound 7 binds to LDHA in an allosteric manner. For natural LDHA inhibitors, only Machilin A (Cancers, 2019, 11(7): 963) and berberine (Clin Transl Med, 2021, 11(6): e467) have been identified as LDHA inhibitors and their in vitro activities are not high. The existing LDHA inhibitors have the problems of low cell level activity, few structural types and limited efficacy, and the development of new inhibitors will lay a foundation for the research of drugs targeting LDHA.

[0004] For a long time, traditional Chinese medicine has been used for anti-cancer treatment, which can alleviate pain, inhibit tumor growth and prolong the survival of patients. Natural products derived from traditional Chinese medicine are an important resource for screening anti-tumor candidate drugs.

[0005] The inventors' research group found that geraniin derived from Geranium wilfordii of Geraniaceae has good LDHA inhibitory activity, IC 50 = 26.7 ± 7.3 nM, and no report has been made on the medicinal potential of geraniin in LDHA-related disease triple-negative breast cancer.

[0006] Geraniin: Geraniin, also known as beta-D-glucopyranose, cyclic 3,6-(4,4',5,5',6,6'-hexahydroxy(1,1'-biphenyl)-2,2'-dicarboxylate)1-(3,4,5-trihydroxybenzoate), 2-(2-carboxy-4,5,6-trioxo-2-cyclohexen-1-yl)-3,4,5-trihydroxybenzoic acid; CAS Registry Number: 60976-49-0; Molecular Formula: C41H28O27; Molecular Weight: 952.64. SUMMARY

[0007] One of the purposes of the present application is to provide geraniin as a high-activity natural inhibitor of LDHA, and the second purpose of the present application is to provide a new medical use of geraniin.

[0008]

[0009] Specifically, the new medical use of the present application provides the use of amentoflavone in the preparation of a drug for treating LDHA-related diseases including triple-negative breast cancer.

[0010] The present application research shows that geraniin has a very significant inhibitory effect on LDHA, can inhibit the proliferation of triple-negative breast cancer cells, cause accumulation of reactive oxygen species (ROS) in cells, mitochondrial membrane potential disorder, inhibit cell migration and block cell cycle at S phase. Therefore, geraniin can be used for preparing a drug for treating LDHA-related diseases including triple-negative breast cancer.

[0011] According to another aspect of the present application, the use of a pharmaceutical combination of geraniin in the preparation of a drug for treating LDHA-related diseases including triple-negative breast cancer is provided

[0012] The pharmaceutical composition is geraniin as an active ingredient and a pharmaceutical carrier.

[0013] The carrier can be any inert material, organic or inorganic, suitable for enteral, transdermal or parenteral administration, such as water, gelatin, acacia, lactose, microcrystalline cellulose starch, starch, sodium starch glycolate, calcium hydrogen phosphate, magnesium stearate, talc, colloidal silicon dioxide, etc. The composition can also contain other pharmaceutically active agents, and conventional additives such as stabilizers, wetting agents, emulsifiers, flavoring agents, buffers, etc.

[0014] The pharmaceutical composition is a tablet, capsule, granule, suspension, drop pill, injection or aerosol, etc.

[0015] The present application discovers a new medical use of the known compound geranin, opening up a new application field. Geranin has strong pharmacological effects and can be used for preparing a drug for treating LDHA related diseases including triple-negative breast cancer.

[0016] Advantages of the present application

[0017] The present application discloses a use of geranin in preparation of a drug for LDHA related diseases. The compound has a significant anti-triple-negative breast cancer cell proliferation effect and a clear high activity of LDHA inhibition. The compound has a good development prospect in the treatment of LDHA related diseases.

[0018] In the enzyme activity test, geranin has a very significant inhibitory effect on LDHA, and the half inhibitory concentration (IC 50 ) is IC 50 = 26.7 ± 7.3 nM; substrate competition experiment and molecular simulation show that geranin binds to LDHA allosterically; cell experiments show that geranin has in vitro anti-triple-negative breast cancer activity, and geranin can cause intracellular ROS accumulation, mitochondrial membrane potential disorder, inhibit cell migration and block cell cycle at S phase. Therefore, geranin has potential therapeutic value for LDHA related diseases such as triple-negative breast cancer. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 : Inhibitory activity of geranin on LDHA

[0020] Figure 2 : Effect of geranin on triple-negative breast cancer cell proliferation

[0021] Figure 3 : Effect of geranin on ROS of triple-negative breast cancer cell SUM159

[0022] Figure 4 : Effect of geranin on mitochondrial membrane potential of triple-negative breast cancer cell SUM159

[0023] Figure 5Effect of geranin on migration of triple-negative breast cancer cell SUM159

[0024] Figure 6 Effect of geranin on cell cycle of triple-negative breast cancer cell SUM159 DETAILED DESCRIPTION

[0025] The present application will be further described in conjunction with examples. The examples are only limited to illustrate the present application, but not limit the present application.

[0026] The instruments and reagent materials used in the experiment mainly include: LDHA, PE Ensight enzyme label instrument (Perkinelmer), flow cytometer Sony SH800 (Sony), Corning 6-hole plate (Corning), Corning 96-hole plate (Corning), lactic acid (Shanghai Yuan Ye Biotechnology Co., Ltd.), dimethyl sulfoxide (Shanghai Yuan Ye Biotechnology Co., Ltd.), water-soluble tetrazole-8 (wst-8) (Shanghai Yuan Ye Biotechnology Co., Ltd.), oxidized coenzyme I (NAD + ) (Shanghai Yuan Ye Biotechnology Co., Ltd.), geranin (Shanghai Yuan Ye Biotechnology Co., Ltd.), 1-methoxyphenazine methanesulfonate (pms) (MedChemExpress), DCFH-DA active oxygen detection kit (Shanghai Biyun Tian Biotechnology Co., Ltd.), JC-1 mitochondrial membrane potential detection kit (Shanghai Biyun Tian Biotechnology Co., Ltd.), PI cell cycle detection kit (Shanghai Biyun Tian Biotechnology Co., Ltd.).

[0027] Triple-negative breast cancer cell strains HCC1937, MDA-MB-231 and MDA-MB-468 are purchased from the Chinese Academy of Sciences Cell Library (Shanghai), and SUM159 is derived from the Second Affiliated Hospital of Dalian Medical University.

[0028] The LDHA gene described in the present application is purchased from Jin Sui Company, connected into pET28a(+) vector to construct a recombinant plasmid, and the successful construction of the plasmid is confirmed by Nde I and BamH I double enzyme digestion and DNA sequencing. The correct recombinant plasmid is transformed into Escherichia coli BL21 (DE3) strain to obtain an expression strain of LDHA. Further, LDHA is expressed by IPTG induction, the bacterial body is collected and ultrasonic broken, the broken supernatant is enriched by nickel column affinity chromatography, and the collected fraction is replaced and concentrated by 3KDa ultrafiltration tube, so that the target protein with high purity is finally obtained.

[0029] Example 1

[0030] LDHA inhibitory activity determination of geranin

[0031] The composition of the aqueous buffer solution is as follows: final concentration of 50 mM TrisCl, final mass concentration of 0.01% BSA, final mass concentration of 0.003% Brij-35, final volume concentration of 0.001% Tween 20 in water, pH 9.2.

[0032] The stock solution of geranyl is 80 mM in dimethyl sulfoxide (DMSO). Add 1 μL of the stock solution to 399 μL of DMSO to obtain a 200 μM geranyl solution. This 200 μM geranyl solution is then diluted 2-fold with DMSO to prepare a total of eight geranyl solutions.

[0033] 1 μL of geranyl solution with different concentrations (the concentrations are 200, 66.67, 22.22, 7.41, 2.47, 0.82, 0.27, 0.09 μM) was placed in different wells of a 96-well plate, and 1 μL of DMSO was placed in 6 wells of the 96-well plate as control wells and blank wells. 49 μL of aqueous buffer containing 20 nM LDHA was added to each well. Incubate at room temperature for 30 minutes. Then 50 μL of substrate mixture solution was added to each well, and the total volume of the solution in each well was 100 μL; the substrate mixture solution (final concentration) was: lactic acid final concentration 30 mM, NAD + The final concentration was 200 μM, the final concentration of wst-8 was 0.5 mM, and the final concentration of pms was 10 μM. The final concentration of LDHA was 10 nM, and the final concentrations of geranyl were 2000, 666.67, 222.22, 74.07, 24.69, 8.23, 2.74, and 0.91 nM, respectively. The mixture was incubated at room temperature for 30 minutes, and then the absorbance at 450 nm was detected on a PE Ensight microplate reader. The relationship between the concentration and absorbance of geranyl was fitted using Origin 8.5 to obtain the dose-effect curve of the compound geranyl ( Figure 1 ), the horizontal axis is the molar concentration of the compound geranyl, and the vertical axis is the absorbance value at 450nm measured by the instrument. The results show that the inhibitory activity of geranyl targeting LDHA is IC 50 =26.7±7.3 nM, indicating that geranyl is a highly active inhibitor of ACLY.

[0034] Example 2

[0035] Testing the anti-triple-negative breast cancer activity of geranyl

[0036] MDA-MB-231, MDA-MB-468, SUM159, HCC1937 cells were seeded in 96-well cell culture plates at a density of 4000 cells per well, and the culture medium was LEIBOVITZ'S L15 (L-15 medium), L-15, DMEM: F12K = 1:1 (volume ratio), RPMI 1640, respectively, and 10% fetal bovine serum (FBS) was added to the medium at a final volume concentration of 10% (complete medium). The volume of the culture medium was 100 μL per well, and SUM159 and HCC1937 were cultured in a constant temperature carbon dioxide (5% volume carbon dioxide in air) incubator at 37°C for 24 hours, and MDA-MB-231 and MDA-MB-468 were cultured in a constant temperature incubator at 37°C for 24 hours. Then 100 μL of different concentrations of oldenlandin medium was added by replacing the medium, and the DMSO treatment group was used as a control. The oldenlandin medium was prepared as follows: 1 μL of different concentrations (concentrations were 80, 40, 20, 10, 5, and 2.5 mM, respectively) of oldenlandin solution was placed in a 1.5 mL sterile centrifuge tube, 1 μL of DMSO was placed in a 1.5 mL sterile centrifuge tube as a control well, and 399 μL of complete medium was added to each centrifuge tube and mixed. After incubation in a constant temperature carbon dioxide (containing 5% volume concentration of air) incubator at 37°C for 72 hours, the culture medium was discarded, and 100 μL of serum-free medium containing 0.5 mM wst-8 and 10 μM pms at a final concentration was added to each well, respectively (corresponding to the above-mentioned medium, respectively), and incubated in a constant temperature incubator at 37°C for 1 hour. The absorbance was measured at 450 nm by an Ensight enzyme label instrument, and the data was processed by Origin 8.5 software to obtain the dose-effect curve of oldenlandin anticancer effect (Figure 1). Figure 2 450 450 The inhibition activity of oldenlandin on MDA-MB-231, MDA-MB-468, SUM159, and HCC1937 cells was 38.1 ± 1.3, 92.0 ± 32.1, 52.2 ± 4.2, and 111.4 ± 31.4 μM, respectively, indicating that oldenlandin had good in vitro anti-triple negative breast cancer effect.

[0037] Example 3

[0038] Determination of the effect of oldenlandin on SUM159 ROS

[0039] ​​SUM159 cells were seeded in 6-well cell culture plates at a density of 200,000 cells per well in DMEM:F12K=1:1 + 10% FBS (final volume concentration). The volume of culture medium per well was 2 mL, and the cells were incubated in a 37 °C constant temperature carbon dioxide (5% volume carbon dioxide in air) incubator for 24 h. Then 2 mL of different concentrations of geraniin was added by changing the culture medium, and the DMSO treatment group was used as a control. The final concentrations of geraniin were 100 and 200 μM, respectively. The culture medium containing geraniin was prepared as follows: 16 μL of different concentrations (concentrations were 80, 40 mM, respectively) of geraniin solution was placed in a 15 mL sterile centrifuge tube, and 16 μL of DMSO was placed in a 1.5 mL sterile centrifuge tube as a control well. Then 6384 μL of complete culture medium was added to each centrifuge tube and mixed well. After incubation in a 37 °C constant temperature carbon dioxide (containing 5% volume concentration of air) incubator for 48 h, 10 μL of ROS UP was added to each of the three replicate wells in the 6-well plate as a positive control group. The old culture medium was discarded, and 1 mL of trypsin was used to digest the cells at room temperature for 5 min. The cells were collected for DCFH-DA staining. The staining steps were as follows: the cells were washed with culture medium without FBS (DMEM:F12K=1:1, volume ratio), incubated with DCFH-DA probe at room temperature for 20 min in the dark, and washed twice with culture medium without FBS. The cells were placed on ice and detected by SH800 flow cytometry. The flow cytometry data were analyzed by Cell Sorter software (Sony), and the data were plotted using GraphPad Prism 6.0 (x-axis: different groups, y-axis: mean fluorescence intensity). Figure 3 The results showed that the mean fluorescence intensity of SUM159 cells treated with 100 μM and 200 μM geraniin and the positive control group was significantly higher than that of the control group, i.e., geraniin treatment led to a significant increase in the content of reactive oxygen species in SUM159 cells.

[0040] Example 4

[0041] Determination of the effect of geraniin on the mitochondrial membrane potential of SUM159

[0042] SUM159 cells were seeded in 6-well plates at a density of 200,000 cells per well in DMEM:F12K = 1 : 1 + 10% FBS (final concentration) and incubated for 24 h at 37 °C in a CO2 incubator (5% CO2 in air). Then, 2 mL of different concentrations of geraniin were added by replacing the medium, and DMSO was used as a control. The final concentrations of geraniin were 100 and 200 mM, respectively. The medium containing geraniin was prepared as follows: 16 mL of different concentrations (80, 40 mM, respectively) of geraniin solution was placed in a 15 mL sterile centrifuge tube, and 16 mL of DMSO was placed in a 1.5 mL sterile centrifuge tube as a control well. Then, 6384 mL of complete medium was added to each centrifuge tube and mixed well. After incubation in a CO2 incubator (5% CO2 in air) at 37 °C for 48 h, the old medium was discarded, and 1 mL of trypsin was used to digest the cells at room temperature for 5 min. The cells were collected for JC-1 staining. The staining procedure was as follows: the cells were incubated with JC-1 probe in a CO2 incubator (5% CO2 in air) at 37 °C for 20 min, washed twice with pre-cooled staining buffer, and then 500 mL of complete medium was added. The cells were placed on ice and detected by SH800 flow cytometry. The flow cytometry data were analyzed by Cell Sorter software (Sony), and the data were plotted using GraphPad Prism 6.0 (x-axis: different groups, y-axis: red / green median fluorescence intensity ratio). Figure 4 The results showed that the median average red / green fluorescence ratio of SUM159 cells treated with 200 mM geraniin was significantly lower than that of the control group, indicating that geraniin treatment induced a decrease in mitochondrial membrane potential in SUM159 cells.

[0043] Example 5

[0044] Determination of the effect of geraniin on the migration of SUM159 cells

[0045] SUM159 cells were seeded into 6-well cell culture plates at a density of 600,000 cells per well in DMEM:F12K=1:1+10% FBS (final volume concentration). The culture volume was 2 mL per well, and the cells were incubated in a 37 °C constant temperature carbon dioxide (5% volume concentration of carbon dioxide in air) incubator for 24 h. The cells were treated with 1 mL of a pipette tip vertically drawn line, washed twice with serum-free medium, and then treated with medium containing different concentrations of strigolactone and 1% FBS. The DMSO treatment group was used as a control, and the final concentration of strigolactone was 100 and 200 μM, respectively. The strigolactone-containing medium was prepared as follows: 16 μL of strigolactone solution at different concentrations (concentrations were 80, 40 mM, respectively) was placed in a 15 mL sterile centrifuge tube, and 16 μL of DMSO was placed in a 1.5 mL sterile centrifuge tube as a control well. 6384 μL of complete medium was added to each centrifuge tube and mixed well. The width of the scratch at 0 h was recorded by taking a photograph under a microscope, and the width of the scratch was also recorded after incubation in a 37 °C constant temperature carbon dioxide (containing 5% volume concentration of air) incubator for 24 h. The blank area was measured using Image J software. The migration rate was calculated using the following formula: migration rate % = (0 h scratch area-24 h scratch area) / 0 h scratch area x 100%. The data were analyzed and plotted using GraphPad Prism 6.0 (x-axis: different groups, y-axis: cell migration rate). The results showed that the migration rate of SUM159 cells treated with 100 μM and 200 μM strigolactone was significantly lower than that of the control group, indicating that strigolactone treatment inhibited the migration ability of SUM159 cells. Figure 5

[0046] Example 6

[0047] Determination of the effect of strigolactone on the cell cycle of SUM159 cells

[0048] ​SUM159 cells were seeded at a density of 200,000 / well in a 6-well cell culture plate using a 1:1 ratio of DMEM:F12K plus 10% FBS (final concentration). The culture medium volume was 2 mL / well and the cells were incubated at 37°C in a CO2 incubator (5% CO2 in air) for 24 hours. The cells were then starved for 48 hours by adding FBS-free culture medium as a medium exchange. The cells were then treated with various concentrations of geranin as a medium exchange (DMSO-treated group served as a control). The final geranin concentrations were 100 and 200 μM, respectively. The geranin-containing culture medium was prepared as follows: 16 μL of a gradient of geranin solutions (80 and 40 mM, respectively) was placed in a 15 mL sterile centrifuge tube. 16 μL of DMSO was placed in a 1.5 mL sterile centrifuge tube as a control. 6384 μL of complete culture medium was added to each centrifuge tube and mixed thoroughly. After incubation at 37°C in a constant temperature carbon dioxide (5% volume concentration of air) incubator for 48 hours, the old culture medium was discarded and 1 mL of trypsin was added to digest the cells at room temperature for 5 minutes. The cells were collected for PI staining. The cells were fixed with pre-cooled 70% ethanol for 1 hour and incubated with PI probe working solution containing RNase at room temperature in the dark for 30 minutes. The cells were placed on ice and detected using an SH800 flow cytometer. The flow cytometry data were analyzed using Cell Sorter software (Sony). The data were analyzed and plotted using GraphPad Prism 6.0 ( Figure 6 ), with the horizontal axis representing the different groups and the vertical axis representing the percentage of nucleotides. The results showed that the proportion of SUM159 cells in the S phase was significantly increased in the 100μM and 200μM geranyl-treated groups compared to the control group, indicating that geranyl-treated SUM159 cells were arrested in the S phase and cell proliferation was affected.

Claims

1. Use of geranyl in the preparation of drugs for treating LDHA-related diseases or inhibiting LDHA activity.

2. The use according to claim 1, characterized in that The structure of the geranin is as follows: it has significant inhibitory activity against LDHA; 3. The use according to claim 1, characterized in that The LDHA-related disease is triple-negative breast cancer.

4. The use according to claim 1 or 3, characterized in that The drug that inhibits LDHA activity can inhibit the proliferation of triple-negative breast cancer by inhibiting LDHA activity.

5. A pharmaceutical composition, characterized in that The invention is a pharmaceutical composition prepared by taking geranium as an active ingredient and one or more pharmaceutical carriers or pharmaceutically acceptable carriers or auxiliary materials.

6. The pharmaceutical composition according to claim 5, characterized in that: The pharmaceutical composition is in the form of tablets, capsules, granules, suspensions, dripping pills, injections or aerosols.

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