Use of natural compounds targeting colon cancer drug resistance associated proteins

Abstract

The application belongs to the technical field of biological medicine, and provides application of a natural compound targeting colon cancer drug resistance related protein, in particular, application in preparation of a drug for preventing or treating colon cancer; the application screens out a natural small-molecule compound hesperetin dihydrochalcone targeting colon cancer drug resistance related protein TAGLN, and provides a new scheme for reversing colon cancer drug resistance, so as to solve the situation that no drug is available for the target point. In addition, the application further provides a scheme in which hesperetin dihydrochalcone is combined with oxaliplatin, and the scheme can significantly enhance the killing effect of oxaliplatin on drug-resistant colon cancer, thereby providing a new choice for clinical treatment of colon cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of a natural compound that targets a protein associated with drug resistance in colon cancer. Background Technology

[0002] Colorectal cancer is one of the leading causes of cancer-related deaths worldwide. Chemotherapy is one of the main treatments for colorectal cancer, with platinum-based drugs (such as oxaliplatin) being widely used. However, during treatment, tumor cells easily develop drug resistance, leading to chemotherapy failure, disease recurrence, and patient death. Overcoming chemotherapy resistance is one of the greatest challenges currently facing clinical treatment of colorectal cancer.

[0003] Existing reports have confirmed that the expression of the cytoskeleton-associated protein TAGLN gradually increases during the progression of colorectal cancer and is closely associated with drug resistance and metastasis. TAGLN expression activates the p38 MAPK signaling pathway, leading to resistance of colorectal cancer cells to chemotherapeutic drugs such as oxaliplatin. Therefore, TAGLN is a key pivotal protein in colorectal cancer chemotherapy resistance and a highly promising new target for anticancer drugs. The advantage of TAGLN as a potential target lies in the fact that it is largely absent from normal colonic epithelial cells, but its expression level rises dramatically in advanced colorectal cancer. Furthermore, TAGLN knockout mice did not show abnormalities in survival or reproduction, indicating that targeting TAGLN is extremely safe compared to upstream pivotal targets.

[0004] Although the role of TAGLN in mediating chemotherapy resistance has been preliminarily revealed, there is currently a lack of small molecule inhibitors that can specifically target the TAGLN protein and effectively regulate its function, and no effective strategy for reversing chemotherapy resistance in colorectal cancer by inducing TAGLN protein degradation has been disclosed. Summary of the Invention

[0005] The purpose of this invention is to provide the application of natural compounds that target colorectal cancer drug resistance-related proteins, thereby addressing the problems raised in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: the application of a natural compound targeting colorectal cancer drug resistance-related proteins in the preparation of drugs for the prevention or treatment of colorectal cancer, wherein the natural compound is neohesperidin dihydrochalcone.

[0007] Furthermore, the natural compound is used to significantly degrade colorectal cancer drug resistance-related proteins.

[0008] Furthermore, the colorectal cancer drug resistance-related protein is the TAGLN protein.

[0009] Furthermore, the drug is a drug that reverses chemotherapy resistance in colon cancer.

[0010] Furthermore, the drug resistance refers to resistance to oxaliplatin.

[0011] Furthermore, the screening method for the natural compounds includes the following steps:

[0012] Using molecular docking virtual screening technology, a library of natural small molecule compounds related to colorectal cancer drug resistance was virtually screened to identify a set of potential binding compounds.

[0013] Based on a set of potential binding compounds, in vitro cell experiments were conducted to detect the effect of each compound in the set on the expression of colorectal cancer drug resistance-related proteins, and to obtain an effective subset of compounds that can inhibit the expression of colorectal cancer drug resistance-related proteins in a dose-dependent manner.

[0014] Based on a subset of effective compounds, binding validation experiments were conducted to screen for natural compounds that can target and bind to colorectal cancer drug resistance-related proteins.

[0015] Another object of the present invention is to provide a medicament for the prevention or treatment of colon cancer, comprising a pharmaceutically acceptable carrier, and further comprising a natural compound and oxaliplatin; said natural compound being neohesperidin dihydrochalcone.

[0016] Furthermore, the concentration of the natural compound is 5-20 μM.

[0017] This invention provides a novel approach to reversing colorectal cancer drug resistance by screening for neohesperidin dihydrochalcone, a natural small molecule compound that targets the colorectal cancer drug resistance-associated protein TAGLN, thus addressing the lack of available drugs for this target. Furthermore, this invention also provides a combination of neohesperidin dihydrochalcone and oxaliplatin, which significantly enhances the killing effect of oxaliplatin on drug-resistant colorectal cancer, offering a new option for the clinical treatment of colorectal cancer. Attached Figure Description

[0018] Figure 1 A schematic flowchart illustrating the screening method for natural compounds that target and bind to colorectal cancer drug resistance-related proteins provided in this embodiment of the invention;

[0019] Figure 2 The figure shows the identification results of potential compounds that may bind to TAGLN; in the figure, a is a schematic diagram of multiple docking screening of 3057 natural compound libraries; b is the identification results of 13 potential compounds that may bind to TAGLN.

[0020] Figure 3 Figure showing the results of verifying the effect of candidate compounds on TAGLN protein expression levels;

[0021] Figure 4 A molecular docking model for TAGLN and NhDC;

[0022] Figure 5 The image shows the results of surface plasmon resonance detection of the binding between TAGLN and NhDC.

[0023] Figure 6 The image shows the results of the cell thermal displacement assay for detecting the binding of TAGLN and NhDC.

[0024] Figure 7 A comparative diagram showing the cytotoxic effects of oxaliplatin on drug-resistant colon cancer cells at the cellular level in each group;

[0025] Figure 8 A comparative graph showing the antitumor effects of NhDC sensitizing oxaliplatin in different groups at the animal level. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0027] Virtual screening based on molecular docking can perform calculations on compound databases containing thousands or even tens of thousands of small molecules to identify small molecule compounds that may bind to the target protein. Subsequent low-throughput experiments can verify the impact of potential binding small molecules on the stability of the target protein, and activating / inhibiting compounds can be screened for further research based on the research objectives. Considering the superior tolerance and safety of natural small molecules in humans, this invention performed docking calculations between 3057 natural small molecules and the TAGLN protein, virtually screening out 13 potential binding compounds. The effects of the obtained compounds on TAGLN protein expression were further evaluated. Among them, neohesperidin dihydrochalcone (NhDC) showed significant dose-dependent inhibition of TAGLN, and is expected to serve as an oxaliplatin sensitizer in the treatment of colorectal cancer. NhDC is extracted from natural citrus and has excellent biocompatibility, clear anti-inflammatory and hepatoprotective functions, and other clinical advantages. Its potential to reverse chemotherapy resistance will further enhance its development value.

[0028] Specifically, in one embodiment of the present invention, a method for screening natural compounds that target and bind to colorectal cancer drug resistance-related proteins is provided, comprising the following steps:

[0029] S1. Using molecular docking virtual screening technology, a library of natural small molecule compounds related to colorectal cancer drug resistance is virtually screened to identify a set of potential binding compounds.

[0030] S2. Based on the set of potential binding compounds, conduct in vitro cell experiments to detect the effect of each compound in the set of potential binding compounds on the expression of colorectal cancer drug resistance-related proteins, and obtain an effective subset of compounds that can dose-dependently inhibit the expression of colorectal cancer drug resistance-related proteins, such as neohesperidin dihydrochalcone, 1F-fructofuranosylnystose, and forsythoside B, etc.

[0031] S3. Based on a subset of effective compounds, conduct binding verification experiments to screen for natural compounds that can target and bind to colorectal cancer drug resistance-related proteins.

[0032] Screening revealed that neohesperidin dihydrochalcone can significantly degrade colorectal cancer drug resistance-related proteins (such as TAGLN protein), and is a small molecule that targets and degrades TAGLN. It can synergize with oxaliplatin for the treatment of colorectal cancer, thereby significantly enhancing the killing effect of oxaliplatin on colorectal cancer cells.

[0033] In another embodiment of the present invention, the use of a natural compound targeting a colorectal cancer drug resistance-related protein in the preparation of a drug for the prevention or treatment of colorectal cancer is also provided, wherein the natural compound is any one of neohesperidin dihydrochalcone, 1F-fructofuranosylnesose, and forsythoside B. Preferably, the natural compound is neohesperidin dihydrochalcone.

[0034] In another embodiment of the invention, a drug for the prevention or treatment of colon cancer is also provided, comprising a pharmaceutically acceptable carrier, and further comprising a natural compound and oxaliplatin; said natural compound is at least one selected from neohesperidin dihydrochalcone, 1F-fructofuranosylnesose, and forsythoside B; wherein the drug is a drug for reversing chemotherapy resistance in colon cancer. Preferably, the resistance is resistance to oxaliplatin; the natural compound is neohesperidin dihydrochalcone; and the concentration of the natural compound is 5-20 μM.

[0035] Example 1: As Figure 1 As shown, this embodiment provides a method for screening natural compounds that target and bind to the TAGLN protein in colorectal cancer, specifically including the following steps:

[0036] S1. Virtual screening of potential small molecule compounds that bind to TAGLN:

[0037] Protein preparation: TAGLN was prepared using the Schrödinger Protein Preparation Wizard module, including bond allocation and hydrogenation. Water molecules and cofactors were removed from the protein. The PROPKA method was used to optimize the hydrogen bond network of protein amino acid residues at pH 7.0. Finally, OPLS_4 force field optimization was performed to minimize the protein energy, optimizing only hydrogen atoms, with RMSD converging at 0.3 Å. Sitemap prediction of TAGLN binding pockets was used, and the top-ranked site was selected for docking box generation.

[0038] Virtual screening: Molecular docking was performed using the Virtual Screening Workflow module in Schrödinger. The virtual screening process employed four modes sequentially: HTVS (High-throughput Screening), SP (Standard Precision Screening), XP (Ultra-Precision Screening), and MM-GBSA binding free energy rescore. After deduplication and removal of compounds with MM-GBSA scores higher than -50 kcal / mol, 13 small molecule compounds that might bind to the TAGLN protein were ultimately obtained.

[0039] S2. NhDC was identified as the target compound for TAGLN:

[0040] NhDC was found to inhibit TAGLN protein expression: Thirteen small molecule compounds were screened and treated with HCT116 cells at concentrations of 0, 5, 10, and 20 μM for 24 h. The effect on TAGLN protein expression was detected by Western Blot.

[0041] Surface plasmon resonance (SPR) detection of NhDC-TAGLN binding rate: Human TAGLN recombinant protein was immobilized on the chip surface, while NhDC solution flowed across the chip surface. When molecules bind and dissociate, changes in the chip surface mass cause changes in the resonance angle. The sensor image records the signal values ​​under these interactions in real time, and the intermolecular interaction information can be obtained from this change curve. Specifically, PBS buffer, water bottles, and waste bottles were placed in the left and right trays respectively, and the corresponding inlet tubes were inserted. Holding the CM5 chip with the labeled side facing up, the chip was gently pushed into the slot according to the arrow direction on the chip, and finally the chip was closed. Channel 4 of the chip was activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide at a flow rate of 10 μL / min. The TAGLN protein was diluted with sodium acetate to 50 μg / mL and immobilized in channel 4 of the chip at a flow rate of 10 μL / min. The channel was then closed with ethanolamine at a flow rate of 10 μL / min. NhDC was diluted to several concentrations in a 96-well plate and coupled to TAGLN protein from low to high concentrations via a microarray. The flow rate was 30 μL / min, and the duration was 150 s. After each concentration point, the microarray was regenerated for 5 min with 10 mM glycine hydrochloride (pH=2.0) solution, and this process was repeated until all corresponding concentrations of the analytes were obtained. The data were globally fitted to a 1:1 Langmuir binding model using Biacore Insight evaluation software to obtain the binding and dissociation constants.

[0042] Cellular thermal displacement assays further validated the binding of native intracellular TAGLN to NhDC: HCT116 cells were exposed to DMSO or NhDC for 24 hours, cells were collected, and washed with PBS containing protease inhibitors. Cells were resuspended in PBS to a final density of 2 × 10⁻⁶. 7 / mL. Each group of cells was aliquoted into 10 PCR tubes and heated for 3 minutes at a specified temperature gradient (37℃-70℃) to denature the proteins. Cells were lysed, centrifuged at 20000×g for 20 min at 4℃, and the supernatant was collected. An equal volume of 2×SDS loading buffer was added to the supernatant, and TAGLN protein expression was detected by Western blotting. Thermal displacement curves were plotted using GraphPad Prism software, and thermostability analysis was performed at 50% temperature (Tm50).

[0043] S3 and NhDC-sensitized oxaliplatin's killing effect on drug-resistant colon cancer cells:

[0044] Oxaliplatin-resistant HCT116 / DDP cells were divided into four groups: control group, NhDC alone group, oxaliplatin alone group, and NhDC combined with oxaliplatin group. Cell viability was assessed by CCK-8 assay after 24 h of treatment.

[0045] Twenty Balb / c nude mice were randomly divided into four groups and subcutaneously injected with 1*10 6 One week later, HCT116 / DDP cells were administered with saline, NhDC alone, oxaliplatin alone, and a combination of NhDC and oxaliplatin. Subcutaneous tumors were harvested 28 days later and their size was measured.

[0046] Example 2: NhDC was identified as one of the potential binding compounds for TAGLN.

[0047] This embodiment aims to screen for target compounds that may affect the stability of the TAGLN protein. The results are as follows: Figure 2 As shown in a and b, through multiple docking screening from a library of 3057 natural compounds, 13 potential compounds that may bind to TAGLN were finally obtained. Among them, the top three were 1F-Fructofuranosylnystose, Neohesperidin Dihydrochalcone, and Forsythoside B.

[0048] Example 3: NhDC reduces TAGLN protein expression in a dose-dependent manner

[0049] This embodiment aims to verify the effect of the candidate compound on the expression level of TAGLN protein. Human HCT-116 colon cancer cell line was cultured and divided into different groups, each treated with different concentrations of the target compound (0 μM, 5 μM, 10 μM, 20 μM) for 24 hours. Cells were collected, and the expression level of TAGLN protein was detected by Western blotting. The results are as follows: Figure 3 As shown, compared with the control group (0 μM), the TAGLN protein expression level in the NhDC-treated group was significantly reduced with increasing drug concentration, exhibiting a clear dose-dependent relationship. This indicates that NhDC can effectively downregulate TAGLN protein.

[0050] Example 4: Verification of direct binding of NhDC to TAGLN protein

[0051] This embodiment aims to demonstrate the direct physical binding between NhDC and the TAGLN protein. For example... Figure 4As shown, the molecular docking model illustrates the simulated morphology of TAGLN binding to NhDC. Subsequently, surface plasmon resonance (SPR) was used to detect the binding of TAGLN to NhDC in vitro. Purified TAGLN protein was immobilized on a chip, and different concentrations of NhDC were allowed to flow through the chip surface; binding and dissociation signals were then detected. Experimental results are shown below. Figure 5 As shown, NhDC can effectively bind to TAGLN protein, and its equilibrium dissociation constant KD value was measured to be 3.92e-06 M, indicating that the two have high affinity.

[0052] In addition, a cell thermal displacement assay was used to detect the binding of native TAGLN to NhDC within cells: colon cancer cells were treated with NhDC or a solvent (DMSO) control, the cells were heated at different temperatures, and then the remaining amount of TAGLN protein in the cell lysate was measured. Results are as follows: Figure 6 As shown, the thermal stability of TAGLN protein was significantly improved in cells treated with NhDC, and the melting temperature Tm value shifted significantly, which provides strong evidence for the direct binding of the two in cells.

[0053] Example 5: NhDC sensitizes oxaliplatin at the cellular level to kill drug-resistant colon cancer cells.

[0054] This embodiment aims to verify whether NhDC can reverse the resistance of colon cancer cells to oxaliplatin in vitro. The results are as follows... Figure 7 As shown, the combination therapy of NhDC and oxaliplatin significantly inhibited the activity of drug-resistant colon cancer cells compared to any single-drug therapy, and this was statistically significant. This demonstrates that NhDC can effectively reverse chemotherapy resistance in colon cancer at the cellular level.

[0055] Example 6: The antitumor effect of NhDC sensitizing oxaliplatin in animals

[0056] This embodiment aims to verify whether NhDC can enhance the efficacy of oxaliplatin in living animals. A nude mouse colon cancer xenograft model was constructed. After the tumors reached a certain size, the mice were randomly divided into four groups: a solvent control group, an NhDC-only group, an oxaliplatin-only group, and a combination of NhDC and oxaliplatin. Drug administration was performed periodically, and tumor volume was measured. The results are as follows: Figure 8 As shown, the tumor volume in the NhDC and oxaliplatin combination therapy group was significantly smaller than that in the other groups; this fully demonstrates that the technical solution provided in this embodiment of the invention has excellent sensitization effect in vivo.

[0057] In summary, the embodiments of the present invention have screened out a natural small molecule compound, neohesperidin dihydrochalcone, which can target the colorectal cancer drug resistance-related protein TAGLN. It can effectively target and degrade the TAGLN protein and can synergize with oxaliplatin for the treatment of colorectal cancer, so as to significantly enhance the killing effect of oxaliplatin on colorectal cancer cells, providing a new option for the clinical treatment of colorectal cancer.

[0058] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the appended claims.

Claims

1. The use of a natural compound targeting a colorectal cancer drug resistance-related protein in the preparation of a drug to reverse oxaliplatin chemotherapy resistance in colorectal cancer, characterized in that, The natural compound is neohesperidin dihydrochalcone.

2. The application according to claim 1, characterized in that, The natural compound is used to significantly degrade colorectal cancer drug resistance-related proteins.

3. The application according to claim 1 or 2, characterized in that, The colorectal cancer drug resistance-related protein is the TAGLN protein.

4. The application according to claim 1, characterized in that, The screening method for the natural compounds includes the following steps: Using molecular docking virtual screening technology, a library of natural small molecule compounds related to colorectal cancer drug resistance was virtually screened to identify a set of potential binding compounds. Based on a set of potential binding compounds, in vitro cell experiments were conducted to detect the effect of each compound in the set on the expression of colorectal cancer drug resistance-related proteins, and to obtain an effective subset of compounds that can inhibit the expression of colorectal cancer drug resistance-related proteins in a dose-dependent manner. Based on a subset of effective compounds, binding validation experiments were conducted to screen for natural compounds that can target and bind to colorectal cancer drug resistance-related proteins.

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