A small molecule compound and its application in preparing anti-tumor composition
By developing small molecule compound 8914689 as a specific HK2 inhibitor and administering it in conjunction with PARP inhibitors, the problem of high cost of existing anti-tumor drugs has been solved and efficient and low-cost anti-tumor effects have been achieved.
Patent Information
- Application Number
- CN202411651426.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-19
- Publication Date
- 2025-04-04
AI Technical Summary
Existing anti-tumor drugs target HK2 expression through genomic means, which is costly and difficult to be widely used in clinical practice.
A small molecule compound 8914689 was developed as a specific HK2 inhibitor, obtained by chemical synthesis, at a low cost, and administered in conjunction with polyadenylasine diphosphate polymerase inhibitors to enhance antitumor effects.
Small molecule compound 8914689 can specifically inhibit HK2 expression, inhibit the proliferation and survival of tumor cells, enhance the sensitivity of tumor cells to apoptosis induced by PARP inhibitors, thereby improving the anti-tumor effect.
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Figure CN119745888A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of anti-tumor, and in particular to a small molecule compound and application thereof in the preparation of an anti-tumor composition. Background Art
[0002] Cancer is an extremely lethal disease and is rapidly becoming the number one cause of death worldwide. Therefore, the research and development of anti-tumor drugs is an important topic in the current medical field. Studies have shown that hexokinase 2 (HK2) is overexpressed in many types of tumors and is closely related to the occurrence and development of tumors. In tumor cells, HK2 is not only involved in the regulation of energy metabolism, but also related to multiple cellular processes such as cell apoptosis, autophagy, and oxidative stress. By promoting the glycolysis pathway, it provides tumor cells with energy and raw materials required for biosynthesis, supporting their rapid proliferation. In addition, HK2 can also inhibit cell apoptosis and increase the survival ability of tumor cells by binding to the voltage-dependent anion channel 1 (VDAC1) of the mitochondrial outer membrane. Therefore, inhibiting the expression of HK2 can play an anti-tumor role.
[0003] Currently, genetic methods are usually used to compound targeted gene sites in drugs to specifically inhibit the expression of HK2. However, this method is costly and difficult to be widely used in clinical practice. Summary of the invention
[0004] The object of the present invention is to provide a low-cost specific HK2 inhibitor and its application in the preparation of a composition for treating tumors.
[0005] In order to achieve the above object, the present invention provides the following technical solutions:
[0006] A specific hexokinase 2 inhibitor, wherein the inhibitor is a small molecule compound 8914689.
[0007] In a second aspect, the present invention also provides the use of the above-mentioned specific hexokinase 2 inhibitor in the preparation of an anti-tumor composition.
[0008] Optionally, the small molecule compound 8914689 inhibits the proliferation of tumor cells.
[0009] Optionally, the small molecule compound 8914689 is administered in combination with a poly(ADP-ribose) polymerase inhibitor, and the small molecule compound 8914689 enhances the promoting effect of the poly(ADP-ribose) polymerase inhibitor on tumor cell apoptosis.
[0010] Optionally, the poly(ADP-ribose) polymerase inhibitor is olaparib, and the small molecule compound 8914689 increases the patient's sensitivity to the poly(ADP-ribose) polymerase.
[0011] Optionally, the composition is an oral dosage form.
[0012] Optionally, the composition has a dosage form selected from the following:
[0013] Tablets, capsules, powders, granules and powders.
[0014] Optionally, the administration period of the composition is any value between 5 weeks and 10 weeks.
[0015] Optionally, the dosage of the small molecule compound 8914689 is any value between 30 mg / kg and 60 mg / kg.
[0016] The beneficial effect of the present invention is to provide an HK2 inhibitor with high specificity. Since the HK2 inhibitor is a small molecule compound, it can be obtained by chemical synthesis at a low cost.
[0017] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 This is a band diagram of HK2 detected by immunoblotting as shown in Example 1 of the present invention;
[0019] Figure 2 The figure is a band diagram of HK1, HK3 and HK4 in the immunoblotting detection shown in Example 1 of the present invention;
[0020] Figure 3 This is a curve diagram showing the changes in cell viability with component concentrations in the in vitro cell experiment shown in Example 1 of the present invention;
[0021] Figure 4 This is a schematic diagram of tumor size in the in vivo experiment shown in Example 1 of the present invention. DETAILED DESCRIPTION
[0022] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0023] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.
[0024] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.
[0025] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.
[0026] In the present invention, compound screening is performed by a computer-assisted virtual selection method. The structure of HK2 is obtained from the protein structure database (RCSB PDB), its binding mode is analyzed, and the surface shape and hydrophilic and hydrophobic properties of the receptor are calculated using the site finder (SiteFinder) of the computer-aided drug design software Molecular Operating Environment (MOE), and the active site suitable for small molecule inhibitor binding is determined. 200,000 compounds in the compound library of ChemBridge, USA, are screened, and the absorption, distribution, metabolism, excretion and toxicity of the drug of the compound are filtered using the drug property filter Oprea's leadlikeness filter, and candidate compounds suitable as lead compounds are retained. Each candidate compound is energy minimized, and then rigid and flexible docking methods are used in turn to dock each candidate compound to the aforementioned active site of the receptor, and the binding free energy is calculated. Candidate compounds with binding energy lower than -10kcal / mol are taken, the topological structure fingerprint of the molecule is calculated, and cluster analysis is performed according to the Tanimoto coefficient between the fingerprints, and the small molecule compound 8914689 is selected in combination with the visual judgment of the receptor-ligand action mode. Among them, 8914689 is the ID of the compound in the compound library of ChemBridge. The Chinese name of the small molecule compound 8914689 is: 4-[3-(5-methyl-1,2,3,4-tetrazolyl-2-yl)adamantan-1-yl]-13-(2-methylbutan-2-yl)-10-thia-3,5,6,8-tetraazatetracyclo[7.7.0.0 「2,6」 .0 「11,16」 】 Hexadecane-1(9),2,4,7,11(16)-pentaene, its structural formula is shown in the figure below:
[0027]
[0028] The ability of the small molecule compound 8914689 to specifically inhibit HK2 expression was verified through experiments, proving that the small molecule compound 8914689 can act as an HK2 inhibitor with strong specificity. It was also unexpectedly found that the small molecule compound 8914689 has a synergistic effect with the poly(ADP-ribose) polymerase inhibitor (PARP).
[0029] The present invention applies for protection of a specific HK2 inhibitor, namely the small molecule compound 8914689.
[0030] The present invention also claims the use of the specific hexokinase 2 inhibitor in the preparation of an anti-tumor composition. Since it can specifically inhibit the expression of HK2 but not HK1, HK3 and HK4, the composition prepared with the small molecule compound as an effective ingredient can play an anti-tumor role with less side effects.
[0031] In some embodiments, small molecule compound 8914689 inhibits the proliferation of tumor cells.
[0032] In some embodiments, the small molecule compound 8914689 is administered in combination with a poly(ADP-ribose) polymerase inhibitor (PARP), and the small molecule compound 8914689 enhances the effect of the poly(ADP-ribose) polymerase inhibitor on tumor cell apoptosis. HK2 inhibitors work synergistically with PARP to simultaneously inhibit glycolysis and PARP activity, thereby depriving tumor cells of their energy supply and exacerbating their DNA damage, leading to tumor cell death, and gradually reducing the activity of cells in tumor tissue, thereby improving the anti-tumor effect.
[0033] In some embodiments, the poly(ADP-ribose) polymerase inhibitor is olaparib, and the small molecule compound 8914689 increases the patient's sensitivity to poly(ADP-ribose) polymerase, helping to ensure the long-term effectiveness of the drug, thereby improving the anti-tumor effect.
[0034] In some embodiments, the content ratio of the poly(ADP-ribose) polymerase inhibitor to the small molecule compound 8914689 in the composition is any value between 2.5 and 25, for example, any value between 2.5, 5, 10, 15, 20 and 25.
[0035] In some embodiments, the composition is an oral dosage form.
[0036] In some embodiments, the composition has a dosage form selected from the following:
[0037] Tablets, capsules, powders, granules and powders.
[0038] In some embodiments, the administration cycle of the composition is any value between 5 weeks and 10 weeks, for example, it can be any value between 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks and 10 weeks, so that within one cycle of administration, the patient's condition is controlled and the drug resistance produced is low.
[0039] In some embodiments, the dosage of the small molecule compound 8914689 is any value between 30 mg / kg and 60 mg / kg, for example, any value between 30 mg / kg, 40 mg / kg, 50 mg / kg and 60 mg / kg.
[0040] Please refer to the following examples for details.
[0041] Embodiment 1:
[0042] In this embodiment, the ability of small molecule compound 8914689 to inhibit HK2 protein expression in cells was detected by immunoblotting. In this embodiment, two identical human ovarian cancer cells (HEY) were taken, one of which was directly cultured as the Ctrl group, and the other was cultured for the same time after adding small molecule compound 8914689 as the HIC-29 group. Protein extraction was performed on the two groups of samples, and the extracted protein samples were electrophoretically separated by polyacrylamide gel electrophoresis (SDS-PAGE). The protein on the gel after electrophoresis was transferred to a polyvinylidene fluoride (PVDF) membrane by an electrotransfer method. The PVDF membrane was treated with a skim milk powder solution, and after the hydrophobic binding sites on the membrane were blocked, antibody incubation was performed. First, the PVDF membrane was incubated with a primary antibody for HK2, so that the primary antibody specifically binds to the HK2 protein to form an antigen-antibody complex. After washing and removing the unbound primary antibody, it was incubated with a secondary antibody with a colorimetric marker and coordinated to the primary antibody, so that the secondary antibody binds to the primary antibody to form an antibody complex. At the same time, in order to verify the integrity and accuracy of the experimental system, it is necessary to incubate the same membrane with an antibody against tubulin in the same way as an internal reference. The PVDF membrane is treated with a colorimetric solution that matches the colorimetric marker for color development. The results are shown in Figure 1 , where the blank group is marked as Ctrl and the experimental group is marked as HIC-29. As can be seen from the figure, there is no significant change in the content of tubulin in the Ctrl group and the HIC-29 group, while the content of HK2 in the HIC-29 group is significantly reduced compared with that in the Ctrl group. This shows that the small molecule compound 8914689 has the ability to inhibit the expression of HK2 protein in tumor cells.
[0043] The ability of small molecule compound 8914689 to inhibit the expression of HK1, HK3 and HK4 proteins in cells was tested by the same method as above. The results are shown in Figure 2 As can be seen from the figure, the small molecule compound 8914689 has no significant effect on the expression of HK1, HK3 and HK4 proteins in tumor cells. Combined with the above conclusions, it can be seen that the small molecule compound 8914689 has a strong specificity in inhibiting the expression of HK2 protein in tumor cells, which helps to reduce the side effects of the composition on the body after administration.
[0044] Different masses of poly ADP-ribose polymerase inhibitor (PARP) were dissolved in dimethyl sulfoxide (DMSO) to obtain five PARP samples with concentrations of 0.1 μmol / L, 12.5 μmol / L, 25 μmol / L, 50 μmol / L and 100 μmol / L, respectively. Parts of the five PARP samples were taken, and appropriate amounts of small molecule compound 8914689 were added and dissolved to obtain five composite samples, so that the concentration of small molecule compound 8914689 in each composite sample was 10 μmol / L. In this embodiment, PARP is Olaparib.
[0045] In this embodiment, an in vitro cell experiment was performed by cell counting method to detect the tumor killing ability of the above PARP samples and composition samples. The specific method includes:
[0046] S110. Take HEY cells with good growth status, digest them and wash them with cell culture medium, count and resuspend them to make the cell concentration 5×10 4 Pieces / L.
[0047] S120. Place cells on a 96-well plate and add 100 μL of cell suspension to each well. The cell density is 5000 cells / well. Place the 96-well plate in a 37°C, 5% CO 2 The cells were cultured overnight in a constant temperature incubator for 20 h to obtain adherent cells.
[0048] S130. Add five PARP samples and five combination samples to different wells of a 96-well plate and continue to incubate at 37°C and 5% CO. 2 Incubate in a constant temperature incubator for 72 h. After incubation, discard the culture medium and add 90 μL of cell culture medium to each well.
[0049] S140. Add 10 μL of colorimetric reagent CCK8 to each well in a dark environment and place in a 37°C, 5% CO 2 The cells were incubated in a constant temperature incubator for 1.5 h, and the absorbance of each group was measured at 490 nm using an ELISA reader (Bio-Rad).
[0050] See also Figure 3 , the cell viability was obtained by converting the absorbance value, and a curve was drawn by combining the concentration of Olaparib and the concentration of the small molecule compound 8914689. As can be seen from the figure, the small molecule compound 8914689 can enhance the sensitivity of tumor cells to apoptosis induced by PARP inhibitors, inhibit the cloning ability of tumor cells and the migration ability of tumor cells, thereby significantly improving the ability of PARP to kill tumor cells, which is helpful for the treatment of cancer.
[0051] In this example, the anti-tumor effect of the small molecule compound 8914689 was also verified by in vivo experiments in mice.
[0052] Six immunodeficient (NOD / SCID) mice were randomly divided into two groups: the control group and the HIC-29 group. The cultured HEY cells were sampled and counted, and then quantitatively injected into the two groups of mice. The injected cell amount was 5×10 6 One week after cell inoculation, mice in the control group were treated with solvent by gavage, while HIC-29 mice were treated with small molecule compound 8914689 dissolved in solvent by gavage. The solvent was DMSO, and the dosage of small molecule compound 8914689 was 50 mg / kg. After 8 weeks of continuous administration 5 times a week, mice were killed and tumor tissues were isolated. The size of tumor tissue is shown in Figure 4 It can be seen that after continuous administration of the drug, the tumor tissue in the mice has been significantly reduced, which proves that oral administration of the small molecule compound 8914689 can kill tumor cells in the body, alleviate or even treat cancer.
[0053] Since the small molecule compound 8914689 is administered orally, it can be prepared into any one of tablets, capsules, powders, pulverulent preparations and granules.
[0054] The beneficial effects of the present invention are: providing a HK2 inhibitor with strong specificity and obtainable by chemical synthesis, namely, small molecule compound 8914689. This HK2 inhibitor can inhibit the clone formation ability of tumor cells and the migration ability of tumor cells by inhibiting the expression of HK2 in tumor cells, thereby achieving an anti-tumor effect. This inhibitor can increase the sensitivity of tumor cells to apoptosis induced by PARP inhibitors. A composition with this HK2 inhibitor as an effective ingredient can inhibit the growth and spread of tumors and kill tumor cells. And this composition has a good therapeutic effect when combined with existing treatment methods for cancer treatment after administration. A composition with this HK2 inhibitor as an effective cost and added with PARP can also effectively treat cancer.
[0055] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0056] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.
Claims
1. A specific hexokinase 2 inhibitor, characterized in that The inhibitor is a small molecule compound 8914689.
2. Use of the specific hexokinase 2 inhibitor as claimed in claim 1 in the preparation of an anti-tumor composition.
3. The use according to claim 2, characterized in that The small molecule compound 8914689 inhibits the proliferation of tumor cells.
4. The use according to claim 3, characterized in that The small molecule compound 8914689 is administered in combination with a poly(ADP-ribose) polymerase inhibitor, and the small molecule compound 8914689 enhances the promoting effect of the poly(ADP-ribose) polymerase inhibitor on tumor cell apoptosis.
5. The use according to claim 4, characterized in that The poly(ADP-ribose) polymerase inhibitor is olaparib, and the small molecule compound 8914689 increases the patient's sensitivity to the poly(ADP-ribose) polymerase.
6. The use according to claim 2, characterized in that The composition is an oral preparation.
7. The use according to claim 6, characterized in that The composition has a dosage form selected from the following: Tablets, capsules, powders, granules and powders.
8. The use according to claim 6, characterized in that The administration period of the composition is any value between 5 weeks and 10 weeks.
9. The use according to claim 8, characterized in that The dosage of the small molecule compound 8914689 is any value between 30 mg / kg and 60 mg / kg.