New application of luteoloside

Abstract

The invention provides a new application of luteoloside, and luteoloside inhibits mutual combination of IGF2BP3 protein and RNA, so that protein expression of IGF2BP3 target genes c-Myc, c-Met, CDK6 and the like is reduced, cell growth is remarkably inhibited, growth of 4T1 mouse transplanted tumors is inhibited, and the luteoloside can be used as a good targeting inhibitor for treatment of IGF2BP3 high-expression tumors.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical biotechnology, and in particular to use of luteolin in preparing an inhibitor targeting IGF2BP3. Background Art

[0002] Triple-negative breast cancer (TNBC) accounts for about 15%-20% of all invasive breast cancers. Compared with other types of breast cancer, TNBC patients develop at an earlier age, are more likely to have distant metastasis, have a shorter survival time, and a mortality rate of 23% within 5 years after diagnosis. At the molecular level, it manifests as the loss of expression of estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2. Unfortunately, current hormone therapy and targeted therapy mainly rely on the expression of these receptors and are therefore ineffective in the treatment of triple-negative breast cancer, while the drug resistance and organ toxicity caused by traditional chemotherapy pose a huge obstacle to the treatment of triple-negative breast cancer. Therefore, how to improve the prognosis of TNBC patients has become an important problem in clinical treatment, and finding effective therapeutic targets is urgent.

[0003] IGF2BP3 is a member of a broad family of RNA-binding proteins, consisting of two RNA recognition domains (RRM) and four K homology domains (KH). IGF2BP3 recognizes N6 methylation (m6A) sites through the KH domain, thereby promoting RNA stability and translation efficiency in an m6A-dependent manner and regulating a variety of biological behaviors. IGF2BP3 expression increases in a variety of cancers, including lung cancer, gastric cancer, and colorectal cancer, and plays the role of a tumor-promoting factor. For breast cancer, IGF2BP3 expression is elevated only in one of its subtypes, triple-negative breast cancer. A large number of experimental studies at the tissue, cell, and animal levels have jointly confirmed that IGF2BP3 has a unique cancer-promoting effect in triple-negative breast cancer and is a good therapeutic target. Therefore, the development of an effective small molecule inhibitor of IGF2BP3 can provide a new direction for the targeted treatment of triple-negative breast cancer.

[0004] Luteolin is a flavonoid compound that can be isolated from several plants in the families of Poaceae, Labiatae, Solanaceae, Zingiberaceae, Asteraceae, etc. This flavonoid has anti-inflammatory, antibacterial, antifungal, anti-leishmanial, antioxidant, liver-protecting, anti-diabetic and anti-cancer effects, and is mainly used to treat chronic cough caused by COPD, bronchial asthma, chronic pharyngitis, allergic rhinitis, etc.

[0005] In recent years, studies have also reported its role in anti-tumor effects. For example, it can inhibit the proliferation of colorectal cancer cells by reducing the expression of CDC25A to induce cell cycle arrest in the G1 phase; at the same time, luteolin blocks the MET / AKT / mTOR axis by reducing the phosphorylation levels of AKT, mTOR and P70S6K, playing a key role in gastric cancer cell proliferation, apoptosis, migration and invasion. However, there are currently no reports on luteolin's direct target. Summary of the invention

[0006] The present invention aims to provide a new use of luteolin.

[0007] According to a first aspect of the present invention, there is provided a use of luteolin in preparing an inhibitor drug targeting IGF2BP3.

[0008] As a preferred embodiment, the structural formula of the luteolin is as shown in Formula I:

[0009]

[0010] As a preferred embodiment, the use of the luteolin is to inhibit the binding between the IGF2BP3 protein and RNA, thereby reducing the protein expression of the IGF2BP3 target gene.

[0011] As a preferred embodiment, the IGF2BP3 target genes include c-Myc, c-Met and CDK6.

[0012] According to a second aspect of the present invention, an inhibitor targeting IGF2BP3 is provided, comprising the aforementioned luteolin.

[0013] According to a third aspect of the present invention, a pharmaceutical composition is provided, comprising the aforementioned inhibitor targeting IGF2BP3.

[0014] As a preferred embodiment, the pharmaceutical composition is a drug for treating cancers with high expression of IGF2BP3.

[0015] As a preferred embodiment, the cancers with high expression of IGF2BP3 include: triple-negative breast cancer, gastric cancer, acute leukemia, lung cancer, colorectal cancer, oral cancer, esophageal cancer, hepatocellular carcinoma, Ewing sarcoma, meningioma, glioma, nasopharyngeal carcinoma, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, kidney cancer and bladder cancer.

[0016] The beneficial effects of the present invention are:

[0017] The present invention proposes the use of luteolin in the preparation of an inhibitor targeting IGF2BP3. Luteolin can hinder the binding of IGF2BP3 protein with target genes (c-Myc, c-Met, CDK6, etc.) to reduce its protein expression, thereby significantly inhibiting cell growth and inhibiting the growth of 4T1 mouse transplanted tumors. It is expected to be used as a good targeted inhibitor for the treatment of tumors with high IGF2BP3 expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of 5280637 in Example 1 of the present invention.

[0019] Figure 2 This is a computer fitting docking diagram of 5280637 and the KH1-2 domain of IGF2BP3 protein in Example 1 of the present invention.

[0020] Figure 3 This is a Coomassie Brilliant Blue staining image of the electrophoresis of the IGF2BP3 protein purified after eukaryotic cell expression in Example 2 of the present invention.

[0021] Figure 4 This is a graph showing the affinity test results of 10 small molecule compounds and IGF2BP3 protein in Example 3 of the present invention.

[0022] Figure 5 This is a graph showing the results of the inhibition of triple-negative breast cancer cell proliferation by 10 small molecule compounds in Example 4 of the present invention.

[0023] Figure 6 This is a result diagram of the effect of luteolin on the protein expression levels of IGF2BP3 target genes c-Myc, c-Met, and CDK6 in Example 5 of the present invention.

[0024] Figure 7 This is a result diagram of the effect of luteolin on the binding ability of IGF2BP3 protein with target genes c-Myc, c-Met, and CDK6 RNA in Example 6 of the present invention.

[0025] Figure 8 This is a graph showing the construction of a mouse tumor-bearing model in Example 7 of the present invention and the changes in tumor volume after luteolin treatment.

[0026] Fig. 9 This is a graph showing the immunohistochemistry results of c-Myc, c-Met, and CDK6 in tumor tissues after treatment with luteolin in Example 7 of the present invention. DETAILED DESCRIPTION

[0027] In order to better understand the technical content of the present invention, specific embodiments are described below in conjunction with the accompanying drawings.

[0028] Various aspects of the present invention are described in this disclosure with reference to the accompanying drawings, in which many illustrative embodiments are shown. The embodiments of the present disclosure are not necessarily intended to include all aspects of the present invention. It should be understood that the various concepts and embodiments introduced above, as well as those described in more detail below, can be implemented in any of many ways.

[0029] In existing studies, JQ1, 1-BET151, isoliquiritigenin, berberine, and d-ICD have been confirmed to be effective inhibitors of IGF2BP3. JQ1 and 1-BET15 can target all bromodomains of BET family members to inhibit IGF2BP3 gene expression and affect the growth of gliomas; isoliquiritigenin controls the progression of non-small cell lung cancer by inhibiting the methylation level at the N6 position and reducing the expression of IGF2BP3; berberine causes colorectal cancer cycle arrest by reducing the expression of IGF2BP3; d-ICD inhibits drug resistance by downregulating the expression of IGF2BP3 in hepatocellular carcinoma.

[0030] Although these inhibitors can inhibit tumor progression by reducing IGF2BP3 expression, they cannot directly target IGF2BP3 to inhibit its protein function. The drug effects are indirect and dependent on the expression of upstream molecules, which limits the effects of the drugs in a wider range of tumors.

[0031] The present invention uses computer virtual drug screening based on the IGF2BP3 protein structure, and selects small molecules with top scores from the ChemDiv drug target library through cascade screening with different precisions, and screens out 10 potential active compounds including luteolin (pubchem ID: 5280637) from the active small molecules. The binding ability of small molecules to proteins is verified by MST, and the tumor cell killing ability is detected by CCK-8 experiment. At the same time, it is verified on 4T1 cell transplanted tumor mice that mice given 5280637 can effectively inhibit tumor growth, thereby achieving an anti-tumor effect.

[0032] Furthermore, in an exemplary embodiment of the present invention, a use of luteolin in the preparation of an inhibitor drug targeting IGF2BP3 is provided.

[0033] As a preferred example, the structural formula of luteolin is shown in Formula I:

[0034]

[0035] As a preferred example, the use of luteolin is to inhibit the binding between IGF2BP3 protein and RNA, thereby reducing the protein expression of IGF2BP3 target gene.

[0036] As a preferred example, IGF2BP3 target genes include c-Myc, c-Met and CDK6

[0037] In another exemplary embodiment of the present invention, an inhibitor targeting IGF2BP3 is provided, comprising the aforementioned luteolin.

[0038] In another exemplary embodiment of the present invention, a pharmaceutical composition is provided, comprising the aforementioned inhibitor targeting IGF2BP3.

[0039] As a preferred example, the pharmaceutical composition further includes a pharmaceutically acceptable salt and an auxiliary material.

[0040] As a preferred example, the pharmaceutical composition is a drug for cancers with high IGF2BP3 expression.

[0041] As preferred examples, cancers with high expression of IGF2BP3 include: triple-negative breast cancer, gastric cancer, acute leukemia, lung cancer, colorectal cancer, oral cancer, esophageal cancer, hepatocellular carcinoma, Ewing sarcoma, meningioma, glioma, nasopharyngeal carcinoma, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, kidney cancer and bladder cancer.

[0042] For better understanding, the present invention is further described below in conjunction with several specific examples, which are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0043] The 5280637 in the following examples is luteolin.

[0044] Example 1

[0045] In this example, small molecule compounds targeting IGF2BP3 were preliminarily selected through supercomputer virtual screening.

[0046] Firstly, the crystal complex structure of IGF2BP3 KH 1-2 domain was downloaded from the PDB database (PDB ID: 6GQE). The hydrophobic pocket composed of Ala-209 Lys-213 Thr-217 Glu-262 Ile-263Lys-266Glu-267 connected to RNA was selected in the Schrödinger software, and the small molecule compounds in the ChemDiv database were virtually screened with the above hydrophobic pocket using the Glide docking algorithm. After the top 1000 compound molecules were screened through three stages (HTVS, SP and XP), the top 10 candidate compounds were finally obtained for subsequent experimental testing (Table 1).

[0047] Table 1 Number, name, affinity index and corresponding structural formula of the top 10 small molecule compounds based on computer virtual screening scores for IGF2BP3KH 1-2 domain and mRNA binding site

[0048]

[0049]

[0050]

[0051] The 10 compounds were analyzed and it was found from the nuclear magnetic resonance hydrogen spectrum that all the 10 compounds were small molecules with high purity. The corresponding structural formulas are shown in Table 1. Among them, 5280637 is luteolin ( Figure 1 ). For example, the computer fitting docking of 5280637 with the KH1-2 domain of IGF2BP3 protein Figure 2 shown.

[0052] Example 2

[0053] In this example, the IGF2BP3 purified protein was obtained by expressing the plasmid in eukaryotic cells.

[0054] 1. Query the mRNA sequence of IGF2BP3 gene on NCBI (Accession: NM_006547.3), retrieve its CDS region, design a plasmid with 6his tag according to the CDS sequence and send it to Kress Biotechnology Co., Ltd. for synthesis.

[0055] 2.IGF2BP3 protein expression and purification.

[0056] (1) HEK293t cells were grown in a 10 cm culture dish to a confluency of 60-70%, and transfected with 125 μL / dish of Lipofectamine 3000 (Invitrogen) and 12.5 μg of plasmid.

[0057] (2) 48 hours after transfection, cells were lysed using IP lysis buffer (Biyuntian: P0013), followed by nickel column purification using a His tag protein purification kit (Biyuntian: P2229S).

[0058] The results are as follows Figure 3 As shown, Figure 3 This is the staining image of the purified IGF2BP3 protein after electrophoresis.

[0059] from Figure 3 It can be seen that the electrophoresis band of the purified IGF2BP3 protein is single, indicating that a relatively pure IGF2BP3 protein is obtained.

[0060] Example 3

[0061] In this example, affinity detection was performed using microfluidics (MST) technology.

[0062] The purified IGF2BP3 protein was labeled with red fluorescence at a final concentration of 10 μM using the RED-NHS second-generation protein labeling kit (NANOTEMPER: MO-L011). The in vitro binding ability of 10 compounds to IGF2BP3 protein was tested using a microcalorimeter (Monolith NT.115Pico). The compound was dissolved in DMSO to 50 mM as a stock solution, and diluted with PBS to a final concentration of 15.26 nM, 30.52 nM, 61.04 nM, 122.07 nM, 244.14 nM, 488.28 nM, 976.56 nM, 1.95 μM, 3.91 μM, 7.81 μM, 15.63 μM, 31.25 μM, 62.5 μM, 125 μM, 250 μM, and 500 μM, 10 μL of each solution, and then 10 μL of purified IGF2BP3 protein solution labeled with red fluorescence was added, and the KD value was calculated by affinity fitting using the software.

[0063] Figure 4 This is the result of affinity test between IGF2BP3 protein and different small molecule compounds.

[0064] Depend on Figure 4 It can be seen that IGF2BP3 has micromolar affinity with 5280637, 5291488, and 13887800, and the Kd values ​​are 12.29μM, 101.21μM, and 149.59μM, respectively, indicating that IGF2BP3 has the strongest binding ability with 5280637.

[0065] Example 4

[0066] In this example, the CCK8 method was used to detect and calculate the half-inhibitory concentrations of 10 small molecule compounds on triple-negative breast cancer MDA-MB-231 cells with high expression of IGF2BP3. The specific steps are as follows:

[0067] (1) Cell plating: When the cells grow to a confluency of 70-80%, digest them with trypsin for 5 min, add DMEM complete medium containing 10% serum to terminate digestion, and centrifuge at 1000 rpm for 5 min. Discard the supernatant, resuspend the cells in complete medium, count them, and adjust the cell density to 1×10 5 Cells were seeded into 96-well plates, 100uL of cell suspension was added to each well, and the plates were cultured at 37°C until they adhered to the wall.

[0068] (2) Cell administration: Small molecule compounds of different concentrations and types (0 μM, 0.1 μM, 1 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM, 200 μM) were added to the wells containing cells.

[0069] (3) CCK-8 experiment: 48 hours after drug addition, the original culture medium was discarded and CCK-8 solution (DMEM culture medium: CCK-8 = 9:1) was added. The 96-well plate was placed in an incubator and incubated for 2 hours. The absorbance at 450 nm was measured by an ELISA reader to calculate the relative cell viability.

[0070] Relative cell viability = [(As-Ab) / (Ac-Ab)] × 100%

[0071] Wherein, As: absorbance of the experimental group (including cells, culture medium, CCK-8 and drugs); Ac: absorbance of the control group (including cells, culture medium, CCK-8, but not drugs); Ab: absorbance of the blank group (including culture medium, CCK-8, but not cells and drugs).

[0072] Figure 5 This is the result of CCK-8 detection of the growth inhibition of MDA-MB-231 cells, a triple-negative breast cancer cell line with high expression of IGF2BP3, by small molecule compounds of different concentrations.

[0073] Results from Figure 5 As shown, 5280637, 5291488, and 3803553 all had growth inhibitory effects on MDA-MB-231 cells, with IC50 values ​​of 9.397 μM, 24.013 μM, and 28.486 μM, respectively.

[0074] Based on the results of Examples 1, 2, 3, and 4, it was preliminarily determined that 5280637 is the most effective small molecule inhibitor highly targeting IGF2BP3 for the treatment of triple-negative breast cancer.

[0075] Example 5

[0076] In this example, protein immunoblotting experiments were performed to verify the effect of 5280637 on the protein expression levels of the downstream target genes c-Myc, c-Met, and CDK6 directly regulated by IGF2BP3.

[0077] Small molecule compound 5280637 was added to DMEM medium containing 10% serum at final concentrations of 0 μM, 1 μM, 5 μM, and 10 μM, respectively, and MDA-MB-231 and BT-549 cells were cultured for 24 hours, the medium containing small molecule compounds was discarded, and the cells were washed 3 times with pre-cooled PBS buffer, and the total cell protein was extracted. The total cell quantification was performed using the BCA kit (Biyuntian: P0011). The above proteins were subjected to electrophoresis and membrane transfer. Antibodies were incubated with c-Myc, c-Met, and CDK6 antibodies and corresponding secondary antibodies, and then exposed for color development.

[0078] The results are as follows Figure 6 As shown, 5280637 inhibited the protein expression of c-Myc, c-Met, and CDK6 in a dose-dependent manner, indicating that 5280637 can successfully interfere with the protein expression of IGF2BP3 target genes.

[0079] Example 6

[0080] In this example, RNA binding protein immunoprecipitation experiments were performed to verify the effect of 5280637 on the RNA binding ability of downstream target genes c-Myc, c-Met, and CDK6 directly regulated by IGF2BP3.

[0081] The specific steps are as follows: 4 dishes of MDA-MB-231 cells were grown to 70-80% confluence in 10 cm culture dishes, and 5280637 10 μM and an equal volume of DMSO were added to 2 dishes of cells for 48 hours. The cells were collected and lysed using the Magna RIP kit (Merck: 17-700) and incubated with 5 μg of anti-IGF2BP3 or anti-IgG at 4°C overnight. The next day, the RNA-protein complex was captured using protein A / G magnetic beads and incubated for 2 hours. After washing and elution with different concentrations of salt solution, RNA was purified by column (CW0599T), and the collected RNA was reverse transcribed and analyzed by qRT-PCR.

[0082] The results are as follows Figure 7 As shown, compared with the control group, after the addition of 5280637 treatment, the abundance of c-Myc, c-Met, and CDK6 RNA enrichment by IGF2BP3 was significantly reduced, which directly reflects the inhibition of 5280637 on the RNA binding function of IGF2BP3.

[0083] Example 7

[0084] This example verifies the therapeutic effect of 5280637 on triple-negative breast cancer xenografts through an in vivo administration experiment in tumor-bearing mice.

[0085] 1. Model establishment: Take mouse triple-negative breast cancer 4T1 cells in the logarithmic growth phase, digest them, and resuspend them in an appropriate amount of PBS; after counting with a cell counter, adjust the cell density to 5×10 6 / mL, use a 1mL sterile syringe to draw the cell suspension and inoculate it subcutaneously on the back of the right hind limb of nude mice, injecting 100μL of cell suspension into each mouse. When the tumor grows to about 50mm 3 The mice were evenly divided into two groups, a 5280637 administration group (50 mg / kg) and a DMSO+PBS equal dose control group, with 5 mice in each group. The drug was administered by intraperitoneal injection once every 2 days for a total of 2 weeks. The body weight and tumor volume of the mice were recorded every 3 days, and the status of the mice was observed. After the modeling was completed, the mice were killed by cervical dislocation, and the tumors, heart, liver, spleen, lungs, and kidneys were dissected and photographed.

[0086] Figure 8 Figure 2 shows tumor photos and tumor volume change curves of different groups of tumor-bearing mice. Figure 8 It can be seen that 5280637 can effectively inhibit the growth of tumors in mice bearing triple-negative breast cancer xenografts.

[0087] 2. Target verification: Mouse tumor specimens were sliced ​​and immunohistochemically stained to detect the expression of IGF2BP3, NF1, MET, and Ki67. After dewaxing and antigen repair, the sections were incubated with the corresponding antibodies for IGF2BP3, NF1, and MET. After the samples were stored at 4°C overnight, they were treated with the corresponding secondary antibodies at 37°C for 1 hour. After washing with PBS twice, DAB was used for color development for 1 minute, followed by hematoxylin staining of the nucleus for 5 minutes. After drying and sealing, the sections were observed and images were captured under a microscope.

[0088] The results are as follows Fig. 9 As shown, 5280637 reduced the protein expression of IGF2BP3 target genes c-Myc, c-Met, and CDK6 in mouse tumors without affecting the expression of IGF2BP3 protein, thereby inhibiting the proliferation ability of the tumor.

[0089] Through supercomputer virtual drug screening, the top ten compounds in binding ability were selected after cascade screening, and the calculated binding energies were all <-7.5kcal / mol, indicating tight binding ability; at the same time, through MST experiments, it was found that three compounds including 5280637 had real physical binding with the purified IGF2BP3 protein; then small molecule compounds were added in a gradient manner to human triple-negative breast cancer cells to verify the tumor inhibitory effect, and the results showed that three compounds including 5280637 had tumor killing ability, among which 5280637 had the smallest IC50 value and the best tumor killing ability; at the same time, 4T1 cell transplanted tumor mice verified that 5280637 still had tumor suppressor ability in mice, and no serious organ toxicity and side effects were found at the same dose.

[0090] In summary, there is a strong affinity between luteolin and IGF2BP3 at the micromolar level. As an IGF2BP3 inhibitor, luteolin can not only significantly inhibit the proliferation ability of MDA-MB-231 cells, but also has a good inhibitory effect on tumor growth in animal models. Moreover, this effect is highly targeted at inhibiting the protein function of IGF2BP3, thereby reducing damage to normal organs and tissues.

[0091] Therefore, luteolin can be used to develop drugs for treating tumors with high IGF2BP3 expression, and has good prospects for therapeutic applications.

[0092] Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. A person with ordinary knowledge in the technical field to which the present invention belongs may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be determined by the definition of the claims.

Claims

1. Use of luteolin in the preparation of inhibitor drugs targeting IGF2BP3.

2. The use according to claim 1, characterized in that The structural formula of the luteolin is shown in Formula I:

3. The use according to claim 1, characterized in that The purpose of the luteolin is to inhibit the binding between the IGF2BP3 protein and RNA, thereby reducing the protein expression of the IGF2BP3 target gene.

4. The use according to claim 3, characterized in that The IGF2BP3 target genes include c-Myc, c-Met and CDK6.

5. An inhibitor targeting IGF2BP3, characterized in that: Contains the luteolin according to any one of claims 1 to 3.

6. A pharmaceutical composition, characterized in that The invention comprises the inhibitor targeting IGF2BP3 as claimed in claim 5.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition is a drug for treating cancers with high IGF2BP3 expression.

8. The pharmaceutical composition according to claim 7, characterized in that The cancers with high expression of IGF2BP3 include: triple-negative breast cancer, gastric cancer, acute leukemia, lung cancer, colorectal cancer, oral cancer, esophageal cancer, hepatocellular carcinoma, Ewing sarcoma, meningioma, glioma, nasopharyngeal carcinoma, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, kidney cancer and bladder cancer.

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