Application of ginkgolic acid compound, inhibitor and medicine
The development of ATP citrate lyase inhibitors using ginkgolic acid compounds has solved the problem of the lack of effective treatments for triple-negative breast cancer in existing technologies. It has achieved significant inhibition of ACLY and anti-cancer effects at the cellular level, providing a new drug option for the treatment of triple-negative breast cancer.
Patent Information
- Application Number
- CN202410581623.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-11
- Publication Date
- 2025-11-11
AI Technical Summary
Current technologies lack effective drugs for treating triple-negative breast cancer, especially inhibitors of ATP citrate lyase, resulting in a lack of effective therapeutic targets for this difficult-to-treat and rapidly progressing disease.
Using ginkgolic acid compounds as active ingredients, we developed an ATP citrate lyase inhibitor for the preparation of drugs to prevent or treat triple-negative breast cancer, including dosage forms such as tablets, pills, injections, capsules, or granules. In vitro experiments showed that it has a significant inhibitory effect on ACLY and exhibits anticancer effects at the cellular level.
Ginkgolide compounds showed significant inhibitory effects on ACLY in vitro, with an IC50 of approximately 5 μM. In in vitro cell experiments, they also exhibited significant inhibitory activity against triple-negative breast cancer cells HS578T, demonstrating good anti-cancer effects.
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Figure CN120919142A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of natural medicine development, specifically relating to the use of natural ginkgolic acid compounds in the preparation of ATP citrate lyase inhibitors and in the treatment of triple-negative breast cancer. Background Technology
[0002] ATP citrate lyase (ACLY) is located in the cytosol and catalyzes the cleavage of citrate into acetyl-CoA and oxaloacetate. Acetyl-CoA is an important precursor molecule in the body's metabolism, participating in the biosynthesis of fatty acids and cholesterol. The citrate in the reaction originates from the tricarboxylic acid cycle. ACLY connects glycolysis, the tricarboxylic acid cycle, and lipid metabolism, and is a key enzyme in glycolipid metabolism (European Journal of Medicinal Chemistry, 2018, 157:1276-91.).
[0003] Triple-negative breast cancer is the most aggressive type of breast cancer, characterized by early recurrence, rapid progression, and a lack of effective therapeutic targets (Cancer Communications, 2018, 38(1):27). There is an urgent market demand for developing related therapeutic drugs. ACLY is a potential anti-cancer target (Cancer Communications, 2018, 38(1):27). Inhibiting ACLY activity can suppress the vigorous lipid synthesis in cancer cells, thereby inhibiting cancer cell proliferation and exerting an anti-cancer effect.
[0004] Natural products are numerous, diverse in activity, and rich in structure, making them an important source of drug molecules. This invention discovers ACLY inhibitors from natural products and explores their application in the treatment of triple-negative breast cancer. Summary of the Invention
[0005] The application of ginkgolic acid compounds in the preparation of ATP citrate lyase inhibitors, wherein the ginkgolic acid compounds have any one or more of the following structures:
[0006]
[0007] The use of ginkgolic acid compounds in the preparation of drugs for the prevention or treatment of triple-negative breast cancer, wherein the ginkgolic acid compounds have any one or more of the following structures:
[0008]
[0009] An ATP citrate lyase inhibitor, wherein at least one or more compounds of formula (1) to formula (4) are active ingredients.
[0010] A drug for the prevention or treatment of triple-negative breast cancer, comprising at least one or more compounds of formulas (1) to (4) as active ingredients.
[0011] The inhibitors or drugs mentioned above may also contain pharmaceutically acceptable excipients or carriers, and their dosage forms may be tablets, pills, injections, capsules or granules.
[0012] At least one or more compounds from formulas (1) to (4) can be used as active ingredients to prepare ATP citrate lyase inhibitors or to prepare ATP citrate lyase-related anti-triple-negative breast cancer drugs.
[0013] In the activity test, the above ginkgolic acid compounds showed significant inhibitory effects on ACLY, with a half-maximal inhibitory concentration (IC50) of 1,000%. 50 The concentrations were all around 5 μM, and they showed inhibitory effects on triple-negative breast cancer in in vitro cell experiments. Attached Figure Description
[0014] Figure 1 Inhibitory activity of compounds (1)-(4) against ACLY
[0015] Figure 2 Anticancer activity of compounds (1)-(4) in HS578T cells Detailed Implementation
[0016] The present invention will now be further illustrated with examples. These examples are merely illustrative and not intended to limit the scope of the invention.
[0017] The instruments and reagents used in the experiment mainly included: a microplate reader (PerkinElmer); ACLY (Promega); and ADP-Globe. TM Kinase Assay kit (Promega); CulturPlate 384-well plate (PerkinElmer); Ginkgo biloba compounds (1) to (4) (Shanghai Bid Pharmaceutical Technology Co., Ltd.), namely ginkgolic acid C13:0, ginkgolic acid C15:0, ginkgolic acid C15:1, and ginkgolic acid C17:1; Tris(hydroxymethyl)aminomethane, dithiothreitol, citric acid, coenzyme A, dimethyl sulfoxide, and bovine insulin (Shanghai Yuanye Biotechnology Co., Ltd.); HS578T cells (Chinese Academy of Sciences Cell Bank); DMEM medium (Gibco); CCK8 assay kit (Shanghai Beyotime Biotechnology Co., Ltd.).
[0018] Example 1: Determination of ACLY inhibitory activity of compound (1)
[0019] The stock solution of compound (1) has a concentration of 50 mM and uses dimethyl sulfoxide as the solvent. Take 1 μL of the stock solution and add it to 61.5 μL of aqueous buffer to obtain a 0.8 mM solution of compound (1). The aqueous buffer consists of: 40 mM tris(hydroxymethyl)aminomethane (tris), 4 mM dithiothreitol, 0.01% lauryl polyoxyethylene ether, 10 mM MgCl2, 1% dimethyl sulfoxide, and pH = 8.0. Dilute the ACLY stock solution to 30 nM with the aqueous buffer to obtain a diluted ACLY solution. Take 2.5 μL of the diluted ACLY solution and 2.5 μL of the compound (1) solution and place them in the same well of a 384-well plate. The aqueous stock solution of the enzyme (ACLY) consists of 50 mM tris, 400 mM NaCl, and 10% glycerol.
[0020] Gently tap the well plate to mix the liquid system thoroughly, and incubate at room temperature for 15 min. Then add 5 μL of substrate mixture solution to the well; the substrate mixture solution is: 600 μM citrate, 150 μM adenosine triphosphate (Promega), 150 μM coenzyme A, and water as solvent; centrifuge at low speed (1200 r, 1 min, 20 °C) to mix thoroughly. The above preparation operation yields an incubation solution with an initial concentration of 200 μM for compound (1). Following the same operation, the 0.8 mM compound (1) solution was serially diluted with aqueous buffer, and then mixed with ACLY solution and substrate mixture solution respectively, to prepare incubation solutions of compound (1) at concentrations of 200, 66.7, 22.2, 7.41, 2.47, 0.82, 0.27 and 0.09 μM in different wells of a 384-well plate.
[0021] The 384-well plate was incubated at 37°C for 55 min, then removed and allowed to stand at room temperature for 5 min. After reaching room temperature, ADP-Glo was added to each well. TM 10 μL of ADP-Glo reagent from the Kinase Assay kit was added and reacted at room temperature for 40 min. Then, ADP-Glo was added to each well. TM 20 μL of the kinase assay reagent in the Kinase Assay kit was used to convert ADP into ATP and emit fluorescence, a process that lasted 40 min. The fluorescence intensity was detected on a PE Ensight microplate reader. Data processing was performed using GraphPadPrism 6.0 to obtain the dose-response curve of compound (1). Figure 1 The x-axis represents the logarithm of the molar concentration of compound (1), and the y-axis represents the fluorescence intensity measured by the instrument. The inhibitory activity of compound (1) targeting ACLY is IC50. 50 =5.23±0.50μM, indicating that compound (1) is an inhibitor of ACLY.
[0022] Example 2: Determination of the anticancer activity of compound (4) in HS578T cells
[0023] HS578T cells were seeded at a density of 4000 / well in 96-well cell culture plates. The culture medium was DMEM + 10% FBS (fetal bovine serum, volume fraction) + 0.01 mg / mL bovine insulin, with a culture volume of 100 μL / well. The cells were cultured at 37°C in a carbon dioxide (clean air with a volume concentration of 5% carbon dioxide) incubator for 24 h. Subsequently, different concentrations of compound (4) were added to the cells by changing the medium. The 2‰ DMSO treatment group served as a control. The compound concentration gradient was prepared as follows: the stock solution of the compound was 50 mM, and the solvent was dimethyl sulfoxide. 1 μL of the stock solution was added to 499 μL of the above culture medium to obtain a final concentration of 100 μM. Based on this concentration, the cells were serially diluted with the above culture medium to obtain eight concentrations of compound (4): 100, 50, 25, 12.5, 6.25, 3.13, 1.56, and 0.78 μM. After treatment with compound (4) for 72 h, the culture medium was discarded, and 100 μL of CCK8 detection reagent was added. The mixture was incubated at 37 °C for 1 h. The absorbance was measured at 450 nm using an Ensight microplate reader. Data processing was performed using GraphPad Prism 6.0 to obtain the dose-response curve of the anticancer effect of compound (4). Figure 2 The horizontal axis represents the logarithm of the molar concentration of compound (4), and the vertical axis represents the inhibition rate. The inhibition rate is calculated using the formula: Inhibition rate (%) = (1 - OD of experimental group) 450 / Control group OD 450 The inhibitory activity of compound (4) on HS578T cells was 5.57±0.12μM, which indicates that compound (4) has a good anti-cancer effect.
[0024] The assay procedures for the ACLY inhibitory activity of compounds (2), (3), and (4) were the same as in Example 1, except that the compounds were changed. Results are available in [link to example]. Figure 1 The assays for the anticancer activity of compounds (1), (2), and (3) on HS578T cells were performed in the same manner as in Example 2, except for the changes in the compounds. Results are shown in Table 1. Figure 2 And Table 2.
[0025] Table 1. Inhibitory activity of compounds (1) to (4) against ACLY
[0026]
[0027] Table 2. Anticancer activity of compounds (1) to (4) in triple-negative breast cancer cells HS578T
[0028]
Claims
1. The application of ginkgolic acid compounds in the preparation of ATP citrate lyase inhibitors, characterized in that, The ginkgolic acid compound has one or more of the following structures:
2. The application of ginkgolic acid compounds in the preparation of drugs for the prevention or treatment of triple-negative breast cancer, characterized in that, The ginkgolic acid compound has one or more of the following structures:
3. An ATP citrate lyase inhibitor, characterized in that, The active ingredient comprises at least one or more compounds of formulas (1) to (4) of claim 1.
4. The drug according to claim 3, characterized in that, It also includes pharmaceutically acceptable excipients or carriers, and its dosage form may be tablets, pills, injections, capsules or granules.
5. A drug for the prevention or treatment of triple-negative breast cancer, characterized in that, The active ingredient comprises at least one or more compounds of formulas (1) to (4) of claims 2.
6. The drug according to claim 5, characterized in that, It also includes pharmaceutically acceptable excipients or carriers, and its dosage form may be tablets, pills, injections, capsules or granules.