A small molecule inhibitor targeting DNA methyltransferase 3A and preparation method and application thereof

Abstract

This invention discloses a small molecule inhibitor targeting DNA methyltransferase 3A, its preparation method, and its applications, specifically relating to the field of biomedical technology. The application of Dieckol in the preparation of DNMT3A methyltransferase inhibitors is highlighted. This invention is the first to demonstrate that the marine natural polyphenol Dieckol can directly bind to the active pocket of the DNMT3A catalytic domain and efficiently inhibit the catalytic activity of DNMT3A methyltransferase, filling the gap in existing technologies for naturally sourced DNMT3A-biased inhibitors and providing a novel lead compound framework for the development of DNMT3A-targeted drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a small molecule inhibitor targeting DNA methyltransferase 3A, its preparation method, and its application. Background Technology

[0002] DNA methylation is a key epigenetic modification in eukaryotes, primarily occurring at the 5-carbon position of the CpG dinucleotide cytosine. This modification process is catalyzed by the DNA methyltransferase (DNMT) family. Functionally defined DNMT subtypes in mammals mainly include DNMT1, DNMT3A, and DNMT3B. DNMT1 is responsible for maintenance methylation after replication, while DNMT3A and DNMT3B mediate de novo methylation. Numerous clinical and basic studies have confirmed that abnormally high expression, functional mutations, or persistent activation of DNMT3A significantly increase the methylation levels of CpG islands across the entire genome and in tumor suppressor gene promoters, which is highly correlated with the occurrence and progression of hematologic malignancies such as acute myeloid leukemia and myelodysplastic syndromes. Therefore, developing small-molecule inhibitors that can precisely target DNMT3A has significant application value in anti-tumor drug development and epigenetic mechanism research.

[0003] Currently reported DNMT inhibitors are mainly divided into two categories: nucleoside analogs and non-nucleoside synthetic small molecule compounds. Representative nucleoside analogs include 5-azacytidine and decitabine. These compounds achieve their inhibitory effect by covalently capturing the DNMT protein through incorporation into the DNA / RNA chain. However, they have several significant drawbacks: they lack DNMT subtype selectivity, simultaneously inhibiting DNMT1, DNMT3A, and DNMT3B; they have poor in vivo stability, requiring intravenous administration; long-term administration can easily lead to bone marrow suppression, gastrointestinal adverse reactions, and tumor cells are prone to developing drug resistance. Non-nucleoside analogs, represented by compounds RG108 and SGI-1027, do not require embedding in nucleic acid molecules and rely on competitive binding to the DNMT active pocket to exert their inhibitory effect. However, they still suffer from broad-spectrum inhibition and struggle to achieve biased regulation targeting DNMT3A.

[0004] In recent years, screening low-toxicity DNMT inhibitors from natural products has become an important research direction in this field. Existing literature reports that EGCG and Withaferin A possess DNMT inhibitory activity; caffeic acid phenethyl ester has a certain binding affinity for DNMT3A and can induce demethylation of tumor suppressor gene promoters; the marine natural product Psammaplin A has an inhibitory effect on DNMT1; and some flavonoid derivatives can inhibit DNMT3A activity at micromolar concentrations. These results indicate that the polyphenolic chemical skeleton possesses the structural basis for binding to the DNMT3A active pocket, but none of the currently disclosed natural products have achieved highly efficient and selective targeted inhibition of DNMT3A.

[0005] Among the patents related to DNMT3A regulatory reagents derived from natural products, Chinese patent CN107875160A discloses that stilbene glycosides extracted from Polygonum multiflorum can downregulate the expression levels of the DNMT3A gene and protein in nerve cells, providing a strategy for intervention in related diseases. However, this patent only reveals the role of stilbene glycosides in gene expression regulation, that is, producing an effect by downregulating the expression level of DNMT3A protein. For pathological states where DNMT3A only exhibits abnormally hypercatalytic function without significant changes in protein expression levels, intervention strategies relying solely on downregulating protein expression may have limitations such as slow onset of action and insufficient precision in targeted intervention.

[0006] Existing DNMT inhibitors face significant technical bottlenecks: nucleoside analogs have obvious toxic side effects, poor in vivo stability, and lack subtype selectivity; synthetic non-nucleoside small molecules lack sufficient targeting bias towards DNMT3A. Therefore, there is an urgent need in this field to develop a class of natural small molecule inhibitors that can directly bind to the catalytic domain of DNMT3A, efficiently block its methyltransferase activity, and exhibit significant subtype bias towards DNMT3A, providing novel drug candidates and epigenetic research tools for DNMT3A-related diseases. Summary of the Invention

[0007] Therefore, this invention provides a small molecule inhibitor targeting DNA methyltransferase 3A, its preparation method, and its application, in order to solve the problems in the prior art.

[0008] To achieve the above objectives, the present invention provides the following technical solution: The application of Dieckol, provided in the first aspect of the present invention, in the preparation of DNMT3A methyltransferase inhibitors. The CAS number of Dieckol is 88095-77-6, and its molecular formula is C1. 36 H 22 O 18 The molecular weight is 742.55. The Dieckol can bind to the active pocket of the catalytic domain of DNMT3A and directly inhibit the catalytic activity of DNMT3A methyltransferase. The inhibitory activity of Dieckol on DNMT3A is stronger than that of DNMT1 and DNMT3B, and it has a DNMT3A subtype bias.

[0009] Furthermore, the Dieckol binds to the catalytic domain or its nearby active pocket of the DNMT3A protein; the active pocket is composed of one or more amino acid residues from Arg887, Arg788, Asn707, Val755, Ala756, Ser704, Glu752, Gly886, Trp889, Phe636, Ala683, and Ser659; the source is a mouse and human common PDB number 3SWC.

[0010] Furthermore, the Dieckol binds to DNMT3A through one or more of the following mechanisms: hydrogen bonding, hydrophobic interaction, van der Waals forces, π-π interactions, or polar interactions; the half-maximal inhibitory concentration (IC50) of the Dieckol on the DNMT3A enzyme activity is... 50 It is 7.196 μM.

[0011] Furthermore, the Dieckol can reduce the expression level of DNMT3A protein in cells.

[0012] Furthermore, Dieckol can reduce the overall DNA methylation level in cells and / or reduce the methylation level of CpG islands in the promoter regions of tumor suppressor genes.

[0013] Furthermore, the tumor suppressor gene is p16.

[0014] The use of Dieckol according to a second aspect of the present invention in the preparation of a medicament for treating diseases related to abnormal expression or function of DNMT3A, characterized in that the disease is acute myeloid leukemia or myelodysplastic syndrome.

[0015] The use of Dieckol according to a third aspect of the present invention in the preparation of a drug for inhibiting the proliferation of DNMT3A-related tumor cells, characterized in that the tumor cells are HL-60 cells.

[0016] The use of Dieckol as a lead compound according to the fourth aspect of the present invention in screening, designing or preparing small molecule inhibitors of DNMT3A or DNMT3A biased inhibitors.

[0017] According to a fifth aspect of the present invention, a DNMT3A-biased methyltransferase inhibitory composition comprises an effective amount of Dieckol and a pharmaceutically acceptable carrier or excipient thereof. The composition is available in any one of the following dosage forms: injection, tablet, capsule, suspension, or powder. The composition is used to reduce cellular DNA methylation levels and inhibit the proliferation of DNMT3A-overexpressing hematologic malignancies.

[0018] The present invention has the following advantages: This invention is the first to demonstrate that the marine natural polyphenol Dieckol can directly bind to the active pocket of the DNMT3A catalytic domain and efficiently inhibit the catalytic activity of DNMT3A methyltransferase, filling the gap in the existing technology for the lack of naturally sourced DNMT3A bias inhibitors and providing a novel lead compound skeleton for the development of DNMT3A targeted drugs.

[0019] This invention integrates multiple experimental systems including molecular docking, molecular dynamics simulation, and in vitro DNMT enzyme activity detection, to fully elucidate the binding conformation of Dieckol and DNMT3A, the key interacting amino acid residues, and to quantitatively determine its inhibitory activity IC50. 50 The concentration is 7.196 μM; the target action mode is clear and the experimental evidence chain is complete, which solves the technical shortcoming of most existing natural DNMT inhibitors that lack systematic verification of the target binding mechanism.

[0020] Dieckol exhibits significantly stronger inhibitory activity against DNMT3A than against DNMT1 and DNMT3B, demonstrating a clear DNMT3A bias. Unlike clinical nucleoside and synthetic non-nucleoside broad-spectrum DNMT inhibitors, it does not indiscriminately inhibit the normal physiological functions of intracellular DNMT1 / DNMT3B, thus significantly reducing the risk of toxicity to the body caused by non-selective inhibition.

[0021] Dieckol can achieve two major regulatory effects simultaneously: on the one hand, it directly binds to DNMT3A to block its catalytic activity, and on the other hand, it downregulates the intracellular DNMT3A protein expression level. Compared with existing natural products such as stilbene glycosides, which only exert their effects by downregulating protein expression, it can intervene in DNMT3A at both the protein function and protein content levels, with faster onset and more significant regulatory effects. It is suitable for pathological scenarios where DNMT3A only has abnormal enzyme activity but no significant increase in expression.

[0022] Dieckol can reduce the whole genome 5mC methylation level of HL-60 cells in a concentration-dependent manner, while downregulating the CpG island hypermethylation modification of the tumor suppressor gene p16 promoter and upregulating p16 mRNA expression; it can unsilence the tumor suppressor gene through epigenetic pathways and reverse the malignant epigenetic characteristics of tumor cells from the root.

[0023] Dieckol can continuously inhibit the proliferation of DNMT3A-overexpressing HL-60 leukemia cells and has direct anti-tumor activity, providing a novel candidate drug for the treatment of hematologic malignancies related to DNMT3A abnormalities, such as acute myeloid leukemia and myelodysplastic syndrome.

[0024] Dieckol is extracted from natural marine resources such as brown algae, with a wide range of raw material sources and a polyphenol core structure that differs from existing synthetic inhibitors. It can be used as a lead compound for the structural modification, screening and optimization of next-generation highly selective DNMT3A inhibitors, and as a specific tool reagent for basic research on the functional mechanism of epigenetic DNMT3A, with a wide range of applications. Attached Figure Description

[0025] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0026] The structures, proportions, sizes, etc. illustrated in this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed herein, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0027] Figure 1 The diagram illustrates the docking and binding pattern of Dieckol with DNMT3A protein, provided by this invention. A- shows the molecular docking result between Dieckol and DNMT3A protein; B- shows the two-dimensional interaction between Dieckol and multiple amino acid residues within the catalytic domain or its vicinity in the active pocket of DNMT3A. Figure 2 The molecular dynamics simulation analysis results of the Dieckol-DNMT3A complex provided for this invention include: A - RMSD analysis results; B - Radius of gyration (Rg) analysis results; C - RMSF analysis results; D - Hydrogen bond quantity analysis results; E - Solvent and surface area (SASA) analysis results; F - Secondary structure analysis results; G - Free energy landscape analysis results; and H - Three-dimensional free energy volcano plot. Figure 3 A bar chart showing the inhibitory effect of Dieckol on DNMT3A methyltransferase activity provided by this invention; Figure 4 The IC50 fitting curve for Dieckol inhibiting DNMT3A enzyme activity provided by this invention; Figure 5 The present invention provides the results of Western blot detection of DNMT3A protein expression level after Dieckol treatment; wherein, A-Western blot detection of DNMT3A protein expression level after Dieckol treatment at different concentrations; B-Statistical graph of DNMT3A protein expression level after Dieckol treatment at different concentrations; Figure 6 The effect of Dieckol provided by this invention on the proliferation of HL-60 cells; Figure 7This is a diagram showing the changes in overall DNA methylation levels in cells after Dieckol treatment, provided by the present invention. Figure 8 This is a graph showing the change in CpG island methylation level of the p16 promoter after Dieckol treatment, as provided by the present invention. Detailed Implementation

[0028] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Unless otherwise specified in the embodiments of this invention, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products; different manufacturers and models of raw materials do not affect the implementation of the technical solution or the achievement of the technical effect of this invention.

[0030] 1. Dieckol: CAS 88095-77-6, purity ≥98%, prepare 10mM stock solution with DMSO, store at -20℃ protected from light; 2. Comparative compounds: decitabine, SGI-1027, stilbene glycoside, EGCG, phenethyl caffeate, Psammaplin A, purity ≥98%, prepared as 10 mM stock solution in DMSO; 3. Recombinant human proteins: DNMT1 (AntibodySystem, catalog number: YHJ72201), DNMT3A (AntibodySystem, catalog number: YHJ91401), DNMT3B (AntibodySystem, catalog number: YHD69901) (active purified proteins). 4. Reagent kits: EpiQuik™ DNMT Activity / Inhibition ELISA Easy Kit (P-3139), 5mC DNA ELISA Kit (P-1030), CCK-8 Cell Proliferation Kit; 5. Cells: Human acute myeloid leukemia HL-60 cells, normal human bone marrow hematopoietic cells; 6. Cell culture medium: RPMI 1640 complete medium (10% fetal bovine serum, 1% penicillin and antibiotics); 7. Molecular simulation software: AutoDock Vina, GROMACS 2021, PyMOL; 8. Others: SDS-PAGE gel preparation reagent, DNMT3A primary antibody, β-Tubulin internal control primary antibody, HRP secondary antibody, bisulfite conversion kit, qRT-PCR kit.

[0031] All experimental groups had a final DMSO concentration of ≤1%; each group had 3 technical replicates, and all experiments were independently repeated 3 times; background absorbance was subtracted from the blank group, and the data were calculated using GraphPad Prism nonlinear regression to calculate IC50. 50 Statistical analysis was performed using the t-test, with P < 0.05 considered statistically significant.

[0032] Example 1

[0033] Dieckol docking with DNMT3A molecules: 1) DNMT3A protein pretreatment: Download the DNMT3A crystal structure from the PDB database, remove bound water molecules and irrelevant ligands using PyMOL; add missing side chains, add hydrogen, calculate Gasteiger charge; minimize energy to eliminate spatial conflicts, and save the protein receptor file.

[0034] 2) Dieckol ligand preprocessing: Draw the 2D structure of Dieckol, optimize the 3D conformation with Chem3D, minimize the force field energy of MMFF94, assign charges, and export the ligand docking file.

[0035] 3) Docking grid settings: The active pocket of the DNMT3A catalytic domain is located at the center of the grid, the grid size covers key residue regions such as Arg887 and Arg788, and the grid accuracy is 0.375 Å.

[0036] 4) Molecular docking operation: AutoDock Vina 1.1.2 performs global flexible docking, sets ligand flexibility and protein pocket residue local flexibility, and generates 20 binding conformations.

[0037] 5) Conformation screening and analysis: The conformation with the lowest binding energy (-10.4 kcal / mol) was selected as the optimal conformation; PyMOL 2.5 was used to draw a 3D binding map; Discovery Studio 4.5 was used to analyze the two-dimensional interaction between ligand and protein, and to count hydrogen bonds, hydrophobicity, π-π interaction residues and interaction distances.

[0038] The results are as follows Figure 1As shown, in the optimal conformation, Dieckol is stably embedded in the DNMT3A catalytic pocket. The magnified conformation of Dieckol in the DNMT3A binding pocket shows that Dieckol forms hydrogen bonds or polar interactions with residues such as Arg887, Arg788, Asn707, Val755, and Ala756, with interaction distances of approximately 2.60 Å, 2.80 Å, 3.43 Å, 3.21 Å, and 3.08 Å, respectively, with the interaction distance ranging from 2.60 to 3.43 Å. The two-dimensional interactions involve 12 amino acid residues, including Arg887, Arg788, Asn707, Ser704, Glu752, Gly886, Trp889, Phe636, Ala683, and Ser659, and also involve hydrophobic, van der Waals, and π-π stacking interactions.

[0039] Example 2

[0040] 100ns molecular dynamics simulation of the Dieckol-DNMT3A complex: 1) Initial system construction: The optimal docking conformation of Example 1 was taken and placed in a cubic TIP3P water box, with the protein ≥10 Å from the box wall; Na was added. + Neutralize the negative charge of the system to construct a solvated complex system.

[0041] 2) Force field allocation: The protein was subjected to the AMBER14SB force field, the Dieckol ligand GAFF2 force field, and the water molecule TIP3P model.

[0042] 3) Three-step energy minimization: ① Fix protein ligands and optimize only water molecules; ② Fix protein backbone and optimize side chains and ligands; ③ Minimize the energy of the entire system without constraints and eliminate steric hindrance.

[0043] 4) Temperature rise equilibrium simulation: NVT ensemble, linear temperature rise from 0-300K, duration 1ns; NPT ensemble, 1 bar pressure equilibrium 2ns, stabilizing system temperature, pressure, and density.

[0044] 5) Production simulation: Unconstrained 100ns molecular dynamics simulation, step size 2fs, one frame of trajectory is saved every 10ps.

[0045] 6) Trajectory analysis: The GROMACS tool was used to calculate RMSD (protein, ligand), Rg radius of gyration, residue RMSF, number of transient hydrogen bonds, SASA solvent accessible surface area, and protein secondary structure; MM / GBSA was used to calculate binding free energy; and FEL and 3D volcano maps were plotted.

[0046] The results are as follows Figure 2As shown, after 25 ns of simulation, the RMSD of the protein and ligand tends to stabilize; Rg is stable at 3.10-3.20 nm; the RMSF of the catalytic pocket residues fluctuates very little; stable hydrogen bonds are maintained throughout; the protein secondary structure is not disrupted; the complex has a low free energy dominant conformation region, and thermodynamic binding is stable. Under physiological conditions, Dieckol can form a long-term stable complex with DNMT3A.

[0047] Example 3

[0048] In vitro ELISA detection of Dieckol's direct inhibition of DNMT3A enzyme activity: 1. Experimental Grouping The control group consisted of three replicates: blank control group, solvent control group (0 μM MDieckol, equal volume of DMSO), 1.5625 / 3.125 / 6.25 / 12.5 / 25 / 50 / 100 μM MDieckol groups, and enzyme-free background group.

[0049] 2. Reagent preparation 1) 10×Dieckol working solution: 10mM stock solution serially diluted to a final concentration corresponding to 10× concentrate; 2) 1× Wash Buffer: 26mL 10× Wash Buffer + 234mL pure water, pH adjusted to 7.2-7.5; 3) Prepare working DAB fresh: 96 μL DAB base solution + 2 μL SAM methyl donor, store on ice.

[0050] 3. Sample loading system (total 50 μL / well) 1) Solvent control wells: 43 μL Working DAB + 2 μL DNMT3A enzyme solution + 5 μL DMSO solvent; 2) Dosage well: 43 μL Working DAB + 2 μL DNMT3A enzyme solution + 5 μL 10×Dieckol working solution; 3) Blank well: 45μL Working DAB + 5μL DMSO, without DNMT3A; 4) Base well: 45μL Working DAB + 5μL corresponding concentration of Dieckol, without DNMT3A.

[0051] 4. Steps 1) Seal the 96-well plate with a sealing film and incubate at 37°C for 75 minutes to complete the methylation reaction; 2) Discard the reaction solution, wash each well three times with 150 μL of 1× washing solution, and let it stand for 1 min each time; 3) Prepare the detection mixture: Add 1 μL of capture antibody + 1 μL of signal indicator + 0.5 μL of enhancement solution to each 1 mL of washing buffer; add 50 μL of detection mixture to each well and incubate at room temperature in the dark for 50 min. 4) Discard the liquid and wash the plate 5 times with 150 μL of washing solution; 5) Add 100 μL of colorimetric solution to each well, incubate at room temperature in the dark for 3 min, and add 100 μL of stop solution after the positive control turns blue; 6) Read the absorbance OD value at 450nm using an ELISA reader.

[0052] 5. Data Calculation Corrected OD = Sample OD - Background OD at the same concentration without enzyme; Relative enzyme activity % = (Drug-corrected OD / Solvent-corrected OD) × 100%; Inhibition rate % = (1 - relative enzyme activity) × 100%; GraphPad Prism uses four parameters to fit the concentration-inhibition rate curve and calculates the IC50. 50 .

[0053] Table 1. Inhibitory effect of Dieckol on DNMT3A enzyme activity

[0054] The results are as follows Figure 3 As shown in Table 1, the inhibition rate at a concentration of 100 μM exceeded 100%; Figure 4 As shown, the inhibition rate gradually increases with increasing Dieckol concentration, IC50... 50 =7.196 μM. This demonstrates that Dieckol can directly and in a concentration-dependent manner inhibit the in vitro DNMT3A methyltransferase activity.

[0055] Example 4

[0056] Dieckol's selective detection of DNMT1 / DNMT3A / DNMT3B subtypes: The overall system, reagents, incubation, and detection procedures are exactly the same as in Example 3, except that the recombinant enzyme protein is replaced: Group 1: DNMT1 enzyme system, gradient Dieckol 0-100μM; Group 2: DNMT3A enzyme system (same as Example 3); Group 3: DNMT3B enzyme system, gradient Dieckol 0-100μM; Fit three sets of IC respectively 50 It inhibits the activity of the bipolar type.

[0057] The results are shown in Table 2: DNMT3A IC 50=7.196μM, DNMT3B IC 50 =56.489μM, DNMT1 IC 50 >100μM. Dieckol exhibits a significant subtype bias towards DNMT3A.

[0058] Table 2 Comparison of the inhibitory activity of Dieckol against different DNMT isoforms

[0059] Example 5

[0060] Western blot analysis of Dieckol downregulating DNMT3A protein expression: 1. Cell treatment HL-60 cells were seeded in 6-well plates at a density of 5 × 10⁻⁶ cells / well. 5 Cells / well; three groups were set up: 0, 15, and 30 μM Dieckol, with a final DMSO concentration of 0.3% in the culture medium; cultured at 37℃ and 5% CO2 for 48 h.

[0061] 2. Protein extraction Discard the culture medium, wash the cells twice with PBS; lyse the cells on ice for 30 min with RIPA lysis buffer (with protease inhibitor); centrifuge at 12000 rpm and 4℃ for 15 min, and collect the supernatant; determine the protein concentration using the BCA kit, and balance the protein concentrations of each group.

[0062] 3. Electrophoretic transfer and blocking 1) SDS-PAGE separating gel 8%, stacking gel 5%; load 30μg total protein per well, electrophoresis at 80V to the separating gel, then adjust to 120V; 2) Wet transfer: Transfer protein to PVDF membrane at a constant current of 300mA for 90 min; 3) Seal a 5% skim milk in a plastic film at room temperature for 1 hour.

[0063] 4. Antibody incubation and development 1) Primary antibody dilution: DNMT3A antibody 1:1000, β-Tubulin internal control 1:5000; incubate overnight on a shaker at 4°C; 2) Wash the membrane 3 times with TBST, 10 min each time; dilute HRP-labeled secondary antibody 1:5000 and incubate at room temperature for 1 h; 3) Wash the membrane 3 times with TBST; uniformly cover the membrane with ECL luminescent solution, and collect the bands with a gel imaging system; quantify the gray value with ImageJ, and obtain the relative expression level by comparing the gray value of DNMT3A with the internal reference gray value.

[0064] The results are as follows Figure 5As shown in Table 3, the relative expression levels of DNMT3A protein in the 15μM and 30μM groups were significantly lower than those in the 0μM control group, and the expression was downregulated in a concentration-dependent manner. Dieckol can reduce the abundance of DNMT3A protein in HL-60 cells.

[0065] Table 3. Changes in DNMT3A protein expression levels after Dieckol treatment.

[0066] Example 6

[0067] CCK-8 assay showed that Dieckol inhibited HL-60 cell proliferation. 1. Plate laying and drug administration HL-60 cells were seeded at 1×10⁶ cells per well in a 96-well plate. 4 Each group was divided into three wells: a blank culture medium well, a blank cell control group, and a 30 μM Dieckol drug administration group; each group had six replicates.

[0068] 2. Culture and detection time points The assay was performed on days 1, 2, 3, 4, 5, 6 and 7 after administration. Before the assay, 10 μL of CCK-8 solution was added to each well and incubated at 37°C in the dark for 2 hours. The OD value was measured at 450 nm using an ELISA reader.

[0069] 3. Data Processing Cell proliferation OD = sample OD - blank culture medium OD; compare the absorbance difference between the control group and the drug treatment group at the same time point.

[0070] The results are as follows Figure 6 As shown in Table 4, there was no significant difference between the two groups after 1 day; from the 2nd day onwards, the OD of the Dieckol group was significantly lower than that of the control group, and it continued to inhibit proliferation for 7 days. Dieckol has a long-term inhibitory effect on the proliferation of DNMT3A-overexpressing leukemia cells.

[0071] Table 4. Effects of Dieckol on tumor cell proliferation

[0072] Example 7

[0073] 5mC ELISA was used to detect the overall DNA methylation level in cells. 1. Cell-based drug delivery HL-60 cells were cultured in 6-well plates with 0, 5, 10, 15, and 30 μM Dieckol for 72 h.

[0074] 2. Genomic DNA extraction After cell collection, the DNA extraction kit was used for lysis, precipitation, washing, and elution. The DNA concentration was quantified using Nanodrop and uniformly diluted to 50 ng / μL.

[0075] 3.5mC ELISA Detection Procedure 1) Each well of the ELISA plate was coated with 100 ng of genomic DNA and fixed by incubation at 37°C for 90 min; 2) Wash the plate 3 times with washing solution, and seal it at room temperature for 30 minutes with sealing solution; 3) Add the primary antibody against 5mC and incubate at room temperature for 60 min; after washing the plate, add the secondary antibody and incubate for 30 min. 4) Develop color and terminate the reaction; read the OD at 450nm; calculate the relative methylation ratio of each group with the control group's 5mC level as 100%.

[0076] The results are as follows Figure 7 As shown in Table 5, the total 5mC level in cells decreased continuously with increasing Dieckol concentration, and Dieckol concentration-dependently reduced whole-genome DNA methylation.

[0077] Table 5. Effects of Dieckol on whole-cell 5mC levels

[0078] Example 8

[0079] p16 promoter methylation and mRNA expression detection: 1. Cell treatment HL-60 cells were divided into two groups: a 0 μM control group and a 30 μM Ieckol treatment group, and treated for 48 h. Cells were collected for DNA extraction and RNA extraction, respectively.

[0080] 2. Methylation-specific PCR (MSP) detection of p16 promoter methylation 1) Genomic DNA bisulfite transformation: The kit was incubated for 16 hours according to the instructions, and the transformed DNA was purified and recovered; 2) Perform PCR amplification using methylation-specific primers and non-methylation-specific primers, respectively; 3) Agarose gel electrophoresis was used to image and quantify the grayscale of the bands, and the relative level of methylation of the p16 promoter was calculated.

[0081] 3. qRT-PCR detection of p16 mRNA 1) Total RNA was extracted from cells using Trizol, and cDNA was synthesized using a reverse transcription kit; 2) Real-time quantitative PCR using p16 gene primers and internal reference GAPDH primers, 2 -ΔCt Calculate the relative fold increase in mRNA expression.

[0082] The results are as follows Figure 8As shown in Table 6, the p16 promoter methylation grayscale was significantly reduced in the Dieckol group, and the p16 mRNA expression level was upregulated by 2.25-fold. Dieckol reduced the methylation of the CpG island in the promoter of the tumor suppressor gene p16 and restored p16 expression.

[0083] Table 6. Effects of Dieckol on p16 promoter methylation levels and mRNA.

[0084] Comparative Example 1 Comparative study of broad-spectrum inhibitor SGI-1027 DNMT subtype inhibition: 1) SGI-1027 concentration gradient settings: 0, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100 μM; 2) Three enzyme activation systems were used: DNMT1, DNMT3A, and DNMT3B. The operation procedures, incubation time, and detection methods were completely consistent with those in Example 3. 3) Fit the IC values ​​of the three systems respectively. 50 The inhibitory activity of the three subtypes was compared.

[0085] SGI-1027 for DNMT1, DNMT3A, DNMT3B IC 50 All values ​​are <20 μM, with no subtype selectivity, and simultaneously inhibit all DNMT subtypes. Existing synthetic non-nucleoside inhibitors offer broad-spectrum inhibition but are prone to causing off-target toxicity to normal cells; the Dieckol of this invention potently inhibits only DNMT3A, resulting in higher safety.

[0086] Comparative Example 2 Clinical comparative study of the cytotoxicity of the nucleoside drug decitabine: 1. Cells and Grouping Normal human bone marrow hematopoietic cells, seeded in 96-well plates at 1×10⁶ wells 4 cells / well; three groups: blank control group, equivalent inhibitory concentration decitabine group, and equivalent inhibitory concentration Dieckol group; 6 replicates per group.

[0087] 2. Matching of drug concentrations Concentrations with an inhibition rate of 50% for DNMT3A were selected: Dieckol 7.196 μM, decitabine 8.2 μM (as determined in this experiment); the final concentration of DMSO was uniformly 0.3%.

[0088] 3. Cultivation and Detection The cells were cultured at 37℃ for 72 hours; incubated with CCK-8 for 2 hours; and the OD was measured at 450 nm. The cell viability % was calculated as (OD of drug treatment / OD of control) × 100%.

[0089] Results: In the decitabine group, the survival rate of normal hematopoietic cells was only 52.3%, indicating significant cytotoxicity; in the Dieckol group, the cell survival rate was 94.7%, with almost no significant toxicity. Nucleoside analogues in clinical practice damage normal hematopoietic cells and have significant toxic side effects; Dieckol, a natural polyphenol, has significantly better safety.

[0090] Comparative Example 3 1. Grouping HL-60 cells were divided into three groups: blank control group, 30 μM stilbene glycoside group (prepared in Example 1 of CN107875160A), and 30 μM Dieckol group; treated for 48 h, and four indicators were detected simultaneously.

[0091] 2. Complete steps for four tests 1) In vitro enzyme activity of DNMT3A: Cell lysate supernatant was collected from each group, and the total catalytic activity of intracellular DNMT3A was detected using the ELISA system described in Example 3; 2) DNMT3A protein expression: quantified by Western blot as in Example 5; 3) Whole cell 5mC level: Same as Example 7 5mC ELISA; 4) Cell proliferation inhibition: Same as in Example 6, OD was detected by CCK-8 for 7 days.

[0092] Results: 1) Stilbene glycosides: only downregulated DNMT3A protein expression, with no significant decrease in intracellular DNMT3A enzyme activity; demethylation and anti-proliferation effects were weak; 2) Dieckol: Simultaneously inhibits DNMT3A enzyme activity and downregulates protein expression, with a significantly greater decrease in 5mC and a more effective inhibitory effect on cell proliferation than stilbene glycoside.

[0093] Existing natural products can only downregulate protein expression and cannot quickly block enzyme activity; this invention has dual regulation, which has higher intervention efficiency and is applicable to pathological scenarios where enzyme activity is hyperactive but protein levels are not elevated.

[0094] Comparative Example 4 1. Test compound EGCG, phenethyl caffeate, Psammaplin A, flavonoid derivatives, and Dieckol were all prepared in 10 mM DMSO stock solution.

[0095] 2. Experimental Procedure The in vitro DNMT3A ELISA enzyme activity system of Example 3 was completely reused, with a concentration gradient of 0-100 μM for each compound, and 3 replicates per group; the mixture was incubated for 75 min, and the color readings were taken. The IC50 values ​​of each compound were then fitted. 50 .

[0096] 3. Results EGCG IC 50 >100μM; Phenethyl caffeate I 50 =68.7 μM; Psammaplin A showed almost no DNMT3A inhibition; flavonoid derivatives IC 50 =54.2μM; Dieckol IC 50 =7.196 μM. Therefore, it is evident that the existing publicly available natural polyphenols exhibit far weaker inhibitory activity against DNMT3A than Dieckol.

[0097] Comparative Example 5 1. Grouping and Processing HL-60 cells were divided into three groups: blank control, 30 μM stilbene glycoside, and 30 μM Dieckol; and treated for two durations: short duration 4 h and long duration 48 h.

[0098] 2. Testing Indicators and Procedures 1) Short-term 4h treatment: Only the transient DNMT3A enzyme activity and genomic 5mC level of cells were detected (same as Examples 3 and 7); 2) Long-term 48h treatment: Simultaneous detection of DNMT3A enzyme activity, DNMT3A protein expression, and cell proliferation.

[0099] 3. Results 1) 4h short time: No significant changes in enzyme activity and 5mC in the stilbene glycoside group; Dieckol group directly inhibited enzyme activity, and 5mC decreased rapidly; 2) 48h long duration: Stilbene glycosides only downregulate protein expression; Dieckol simultaneously inhibits enzyme activity and downregulates protein, thus having a dual effect.

[0100] For the pathological state of acute hypercatalytic function of DNMT3A, natural products that only downregulate protein expression have a slow onset of action, while Dieckol can rapidly and directly block enzyme activity and has a wider intervention window.

[0101] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. Application of Dieckol in the preparation of DNMT3A methyltransferase inhibitors.

2. The application according to claim 1, characterized in that, The Dieckol binds to the catalytic domain of the DNMT3A protein or its nearby active pocket; the active pocket is composed of one or more amino acid residues selected from Arg887, Arg788, Asn707, Val755, Ala756, Ser704, Glu752, Gly886, Trp889, Phe636, Ala683, and Ser659.

3. The application according to claim 2, characterized in that, The Dieckol binds to DNMT3A through one or more of the following mechanisms: hydrogen bonding, hydrophobic interaction, van der Waals forces, π-π interactions, or polar interactions; the half-maximal inhibitory concentration (IC50) of the Dieckol on the DNMT3A enzyme activity is... 50 It is 7.196 μM.

4. The application according to claim 2, characterized in that, The Dieckol reduces the expression level of DNMT3A protein in cells.

5. The application according to claim 2, characterized in that, Dieckol can reduce the overall DNA methylation level in cells and / or reduce the methylation level of CpG islands in the promoter regions of tumor suppressor genes.

6. The application according to claim 5, characterized in that, The tumor suppressor gene is p16.

7. The use of Dieckol in the preparation of drugs for treating diseases related to abnormal expression or function of DNMT3A, characterized in that, The disease is acute myeloid leukemia or myelodysplastic syndrome.

8. The use of Dieckol in the preparation of drugs that inhibit the proliferation of DNMT3A-related tumor cells, characterized in that, The tumor cells were HL-60 cells.

9. Application of Dieckol as a lead compound in the screening, design, or preparation of small molecule inhibitors or DNMT3A biased inhibitors.

10. A DNMT3A-biased methyltransferase inhibitory composition, characterized in that, It contains an effective amount of Dieckol and its pharmaceutically acceptable carrier or excipient.

Images