ST3GAL1-targeting anti-tumor small molecule compound and application thereof

Abstract

The invention discloses an ST3GAL1-targeting anti-tumor small molecule compound and application thereof, the small molecule compound is N '-[1-(4-fluorophenyl)-1H-pyrazolo [3, 4-d] pyrimidine-4-yl]-4-methoxybenzoyl hydrazine, and the structure of the small molecule compound is as shown in the following formula I: # imgabs0 #. The ST3GAL1-targeting anti-tumor small molecule compound is obtained through virtual screening, and the ST3GAL1-targeting anti-tumor small molecule compound has the advantages that the ST3GAL1-targeting anti-tumor small molecule compound can be used for preparing anti-tumor drugs; the polypeptide can inhibit the expression of ST3GAL1, and has the potential of being applied to tumor treatment and preparation of anti-tumor drugs. Experiments show that the small molecule compound provided by the invention has a remarkable inhibition effect on a human pancreatic cancer PANC-1 cell line and a human in-situ pancreatic cancer BxPC-3 cell line after acting for 48 hours, and is dose-dependent, the IC50 value of the small molecule compound to PANC-1 cells is 42.17 mu M, and the IC50 value of the small molecule compound to BxPC-3 cells is 46.83 mu M.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and medicine, and in particular to an anti-tumor small molecule compound targeting ST3GAL1 and application thereof. Background Art

[0002] Malignant tumors, as one of the major threats to global health, have complex pathogenesis and high heterogeneity, which poses great challenges to clinical treatment. With the in-depth study of tumor biological characteristics, scientists have gradually realized that a variety of molecules and signaling pathways in the tumor microenvironment play a key role in the occurrence, development and metastasis of tumors. Therefore, targeted therapy for these key molecules or pathways has become an important direction of current anti-tumor research. Computer-aided drug design has greatly reduced the R&D cost of traditional drugs, shortened the cycle, and has strong targeting, making it a powerful tool for the development of tumor targeted therapeutic drugs.

[0003] ST3GAL1 (ST3β-galactoside α-2,3-sialyltransferase 1) is an important glycosyltransferase that plays a central role in the sialylation of cell surface glycoproteins and glycolipids. In recent years, more and more studies have shown that abnormal expression of ST3GAL1 is associated with the progression and chemoresistance of various malignant tumors. ST3GAL1 promotes the growth, invasion and metastasis of tumor cells by catalyzing the sialylation modification of specific proteins or lipids, affecting biological processes such as cell-to-cell adhesion, signal transduction and immune escape. [1,2] .

[0004] Currently, anti-tumor targeted inhibitors are widely used in clinical tumor treatment due to their good effects and relatively small side effects, but there are still some limitations, such as the easy development of drug resistance, the complexity and diversity of targets. To date, there are few studies on glycosylated proteins, and no studies have reported the emergence of inhibitors targeting ST3GAL1. Therefore, it is of great significance to find ST3GAL1 inhibitors and develop an anti-tumor drug targeting ST3GAL1 to provide more reliable solutions for cancer treatment.

[0005] References:

[0006] [1]Yeo HL,Fan TC,Lin RJ,et al.Sialylation of vasorin by ST3Gal1facilitates TGF-β1-mediated tumor angiogenesis and progression[J].Int JCancer,2019,144(8):1996-2007.

[0007] [2]Wu Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide an anti-tumor small molecule compound targeting ST3GAL1 and its application in view of the deficiencies in the above-mentioned prior art.

[0009] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: In the first aspect of the present invention, an anti-tumor small molecule compound targeting ST3GAL1 is provided, and the small molecule compound is: N'-[1-(4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]-4-methoxybenzohydrazide, and its structure is shown in the following formula I:

[0010]

[0011] Preferably, the small molecule compound can inhibit the expression of ST3GAL1.

[0012] Preferably, the small molecule compound is obtained by virtual screening, and the virtual screening method is:

[0013] The human ST3GAL1 protein sequence was obtained, homology modeling was performed using Swiss-Model, the modeled protein structure was optimized using Gromacs, virtual screening was performed using AutoDock Vina software, and molecular dynamics simulation was performed using Desmond to obtain the small molecule compound.

[0014] Preferably, the virtual screening method is:

[0015] S1. Protein sequence acquisition and modeling: The human ST3GAL1 protein sequence was obtained from the Uniprot database, and Swiss-Model was used for three-dimensional structural homology modeling;

[0016] S2. Structural optimization: The modeled protein structure was optimized using Gromacs software to ensure the accuracy and reliability of the model;

[0017] S3. Virtual screening: AutoDock Vina software was used for three rounds of virtual screening, using different exhaustive indices in each round, and the top 10% of the compounds with docking scores in each round were retained to obtain candidate compounds;

[0018] S4. Molecular dynamics simulation: The selected candidate compounds were subjected to molecular dynamics simulation using Desmond software to verify the binding stability of the candidate compounds with ST3GAL1, and the candidate compounds with qualified stability were screened to obtain the small molecule compounds.

[0019] Preferably, the Gromacs software version is 2023.1, the AutoDock Vina software version is 1.2.3, and the Desmond software version is 2023.4.

[0020] The second aspect of the present invention provides a use of the above-mentioned small molecule compound N'-[1-(4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]-4-methoxybenzohydrazide and a pharmaceutically acceptable salt thereof in the preparation of anti-tumor drugs.

[0021] Preferably, the anti-tumor drug is obtained by combining the small molecule compound as an active ingredient with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants.

[0022] Preferably, the tumor is a tumor disease associated with ST3GAL1.

[0023] Preferably, the tumors include lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, colorectal cancer, pancreatic cancer, and bile duct cancer.

[0024] The third aspect of the present invention provides a use of the small molecule compound and a pharmaceutically acceptable salt thereof as described above in the preparation of an ST3GAL1 inhibitor.

[0025] The beneficial effects of the present invention are:

[0026] The present invention obtains an antitumor small molecule compound targeting ST3GAL1 through virtual screening, which can inhibit the expression of ST3GAL1 and has the potential to be used in tumor treatment and the preparation of antitumor drugs; the experiments of the present invention show that the small molecule compound provided by the present invention has a significant inhibitory effect on human pancreatic cancer PANC-1 cell line and human in situ pancreatic cancer BxPC-3 cell line after 48 hours of action, and is dose-dependent, wherein the IC50 value for PANC-1 cells is 42.17 μM, and the IC50 value for BxPC-3 cells is 46.83 μM. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1The results of CCK-8 proliferation assay of PANC-1 cells;

[0028] Figure 2 The results of CCK-8 proliferation assay of BxPC-3 cells;

[0029] Figure 3 The CCK-8 results of PANC-1 cells treated with compound C312-ST01 for 48 hours;

[0030] Figure 4 The CCK-8 results of BxPC-3 cells treated with compound C312-ST01 for 48 hours. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0032] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0033] The test methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. In the following examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0034] The present invention provides an anti-tumor small molecule compound targeting ST3GAL1, which is obtained by virtual screening. The virtual screening method comprises the following steps: obtaining a human ST3GAL1 protein sequence, performing homology modeling using Swiss-Model, optimizing the structure of the modeled protein structure using Gromacs, performing three rounds of virtual screening using AutoDock Vina software, retaining the top 10% of the docking scores in each round of screening, and finally performing molecular dynamics simulation using Desmond to evaluate the binding stability with the ST3GAL1 protein, thereby finally screening the obtained small molecule compound.

[0035] The small molecule compound is: N'-[1-(4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]-4-methoxybenzoic acid hydrazide, with a molecular formula of C 19 H 15 FN6O2, codenamed C312-ST01, has a structure as shown in the following formula I:

[0036]

[0037] The public information of this compound can be found at the following website:

[0038] https: / / pubchem.ncbi.nlm.nih.gov / #query=K405-4012.

[0039] Among them, small molecule compounds can inhibit the expression of ST3GAL1.

[0040] In a preferred embodiment, the virtual screening method is:

[0041] S1. Protein sequence acquisition and modeling: The human ST3GAL1 protein sequence was obtained from the Uniprot database, and Swiss-Model was used for three-dimensional structural homology modeling;

[0042] S2. Structural optimization: The modeled protein structure was optimized using Gromacs software to ensure the accuracy and reliability of the model;

[0043] S3. Virtual screening: AutoDock Vina software was used for three rounds of virtual screening, using different exhaustive indices in each round, and the top 10% of the compounds with docking scores in each round were retained to obtain candidate compounds;

[0044] S4. Molecular dynamics simulation: The selected candidate compounds were subjected to molecular dynamics simulation using Desmond software to verify the binding stability of the candidate compounds with ST3GAL1, and the candidate compounds with qualified stability were screened to obtain small molecule compounds.

[0045] In a preferred embodiment, the Gromacs software version is 2023.1, the AutoDock Vina software version is 1.2.3, and the Desmond software version is 2023.4.

[0046] The present invention also provides a use of the above small molecule compound N'-[1-(4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]-4-methoxybenzohydrazide and a pharmaceutically acceptable salt thereof in the preparation of anti-tumor drugs.

[0047] In a preferred embodiment, the anti-tumor drug is obtained by combining a small molecule compound as an active ingredient with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants.

[0048] In a preferred embodiment, the tumor is a tumor disease associated with ST3GAL1.

[0049] In a preferred embodiment, the tumors include lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, colorectal cancer, pancreatic cancer, and bile duct cancer.

[0050] The present invention also provides use of the above small molecule compound and a pharmaceutically acceptable salt thereof in the preparation of an ST3GAL1 inhibitor.

[0051] The above is the overall concept of the present invention, and detailed embodiments and comparative examples are provided below on the basis of the overall concept of the present invention to further illustrate the present invention.

[0052] Example 1 Virtual screening of small molecule compound C312-ST01

[0053] S1. Protein sequence acquisition and modeling: The human ST3GAL1 protein sequence was obtained from the Uniprot database (URL: https: / / www.uniprot.org), and three-dimensional structure homology modeling was performed using Swiss-Model (URL: https: / / swissmodel.expasy.org);

[0054] S2. Structural optimization: The modeled protein structure was optimized using Gromacs software (version 2023.1) to ensure the accuracy and reliability of the model;

[0055] S3. Virtual screening: Three rounds of virtual screening were performed using AutoDock Vina software (version 1.2.3), using different exhaustive indices in each round, and retaining the top 10% of the compounds with docking scores in each round;

[0056] S4. Molecular dynamics simulation: The selected candidate compounds were subjected to molecular dynamics simulation using Desmond software (version 2023.4) to verify the binding stability of the compounds with ST3GAL1;

[0057] S5. Final screening compounds: Potential inhibitors were screened through the above process: small molecule compound C312-ST01, i.e., N'-[1-(4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]-4-methoxybenzohydrazide, whose structural formula is shown in Formula I:

[0058]

[0059] Example 2 Antitumor activity experiment of small molecule compound C312-ST01

[0060] In this example, the common CCK-8 method was used to perform a tumor cell proliferation inhibition experiment on the small molecule compound C312-ST01 obtained by virtual screening in Example 1.

[0061] The molecular formula of this small molecule compound is C 19 H 15 FN6O2, codenamed C312-ST01, was purchased from Taoshu Biological Co., Ltd.;

[0062] The sources of the main materials used below are as follows:

[0063] Human pancreatic cancer PANC-1 cells and human orthotopic pancreatic cancer BxPC-3 cells were purchased from Beina Biotechnology with catalog numbers BNCC339759 and BNCC338477, respectively; DMEM basal medium and RPMI-1640 basal medium were purchased from Meilun Biotechnology with catalog numbers MA0212 and MA0215, respectively; penicillin-streptomycin solution (100×) was purchased from Biosharp with catalog number BL505A; serum (FBS) was purchased from CellMax with catalog number SA211.02; trypsin-EDTA digestion solution was purchased from Solebao Biotechnology Co., Ltd. with catalog number T1300; CCK-8 reagent was purchased from Yacoin Biotechnology Co., Ltd. with catalog number ATWF06121.

[0064] The experimental method includes the following steps:

[0065] 1. Sample preparation: Dissolve compound C312-ST01 in DMSO to a 100 mM solution as a stock solution. In subsequent experiments, dilute it to different concentrations with the corresponding complete culture medium to treat cells according to experimental requirements;

[0066] 2. Cell lines: human pancreatic cancer PANC-1 cells, human orthotopic pancreatic cancer BxPC-3 cells;

[0067] 3. Culture medium: The culture medium for human pancreatic cancer PANC-1 cells is DMEM basal culture medium + FBS + double antibodies, and the culture medium for human orthotopic pancreatic cancer BxPC-3 cells is RPMI-1640 basal culture medium + FBS + double antibodies.

[0068] 4. Experimental method: CCK-8 method.

[0069] PANC-1 and BxPC-3 cells were seeded in 96-well plates, with 3000 cells in each well. After 24 hours, they were treated with the small molecule compound C312-ST01 at concentrations of 25, 50, and 100 μM, respectively. After 0, 24, and 48 hours, culture medium containing 10 μL CCK-8 reagent was added to each well, respectively. After incubation in the incubator for 1 hour, the OD value was measured at 450 nm on a microplate reader.

[0070] 5. Experimental results:

[0071] Reference Figure 1, are the results of the CCK-8 proliferation assay of PANC-1 cells. CCK-8 assay was performed after the compound C312-ST01 was treated for 0, 24, and 48 hours. The experimental results showed that C312-ST01 had no obvious effect on PANC-1 cells after 24 hours of treatment, but had a significant inhibitory effect on PANC-1 cells after 48 hours of treatment, and it was dose-dependent.

[0072] Reference Figure 2 , are the results of CCK-8 proliferation assay of BxPC-3 cells. CCK-8 assay was performed after the BxPC-3 cells were treated with compound C312-ST01 for 0, 24, and 48 hours. The experimental results showed that C312-ST01 had a significant inhibitory effect on BxPC-3 cells after 24 and 48 hours of treatment, and it was dose-dependent.

[0073] Reference Figure 3 , is the CCK-8 result of PANC-1 cells treated with compound C312-ST01 for 48 hours. It can be seen that when the concentration of C312-ST01 reaches 42.17 μM, half of the PANC-1 cells can be killed.

[0074] Reference Figure 4 , is the CCK-8 result of BxPC-3 cells treated with compound C312-ST01 for 48 hours. It can be seen that when the concentration of C312-ST01 reaches 46.83 μM, half of the BxPC-3 cells can be killed.

[0075] Based on the above results, it can be seen that compound C312-ST01 has a significant inhibitory effect on PANC-1 and BxPC-3 cells in a dose-dependent manner. The IC50 value of C312-ST01 for PANC-1 cells is 42.17 μM, and the IC50 value for BxPC-3 cells is 46.83 μM.

[0076] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. An anti-tumor small molecule compound targeting ST3GAL1, characterized in that: The small molecule compound is: N'-[1-(4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]-4-methoxybenzohydrazide, and its structure is shown in the following formula I:

2. The anti-tumor small molecule compound targeting ST3GAL1 according to claim 1, characterized in that: The small molecule compound can inhibit the expression of ST3GAL1.

3. The anti-tumor small molecule compound targeting ST3GAL1 according to claim 1, characterized in that: The small molecule compound is obtained by virtual screening, and the virtual screening method is: The human ST3GAL1 protein sequence was obtained, homology modeling was performed using Swiss-Model, the modeled protein structure was optimized using Gromacs, virtual screening was performed using AutoDock Vina software, and molecular dynamics simulation was performed using Desmond to obtain the small molecule compound.

4. The anti-tumor small molecule compound targeting ST3GAL1 according to claim 3, characterized in that: The virtual screening method is: S1. Protein sequence acquisition and modeling: The human ST3GAL1 protein sequence was obtained from the Uniprot database, and Swiss-Model was used for three-dimensional structural homology modeling; S2. Structural optimization: The modeled protein structure was optimized using Gromacs software to ensure the accuracy and reliability of the model; S3. Virtual screening: AutoDock Vina software was used to perform three rounds of virtual screening, using different exhaustive indices in each round, and the top 10% of the compounds in each round of docking scores were retained to obtain candidate compounds; S4. Molecular dynamics simulation: The selected candidate compounds were subjected to molecular dynamics simulation using Desmond software to verify the binding stability of the candidate compounds with ST3GAL1, and the candidate compounds with qualified stability were screened to obtain the small molecule compounds.

5. The anti-tumor small molecule compound targeting ST3GAL1 according to claim 4, characterized in that: The Gromacs software version is 2023.1, the AutoDock Vina software version is 1.2.3, and the Desmond software version is 2023.

4.

6. Use of the small molecule compound N'-[1-(4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]-4-methoxybenzohydrazide and its pharmaceutically acceptable salt as claimed in any one of claims 1 to 5 in the preparation of anti-tumor drugs.

7. The use according to claim 6, characterized in that: The anti-tumor drug is obtained by combining the small molecule compound as an active ingredient with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants.

8. The use according to claim 6, characterized in that: The tumor is a tumor disease associated with ST3GAL1.

9. The use according to claim 6, characterized in that: These tumors include lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, colorectal cancer, pancreatic cancer, and bile duct cancer.

10. Use of the small molecule compound according to any one of claims 1 to 5 and a pharmaceutically acceptable salt thereof in the preparation of an ST3GAL1 inhibitor.

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