Polypeptide inhibitor of targeted MZF1 gene and application of polypeptide inhibitor in colon cancer treatment

Abstract

The invention relates to the technical field of biological medicine, in particular to a polypeptide inhibitor of a targeted MZF1 gene and application of the polypeptide inhibitor in colon cancer treatment, and a polypeptide compound named as 10PANX ACETATE is the first MZF1 gene efficient targeted inhibitor which is clearly recognized and has high uniqueness and innovativeness. In the field of biological medicines, the compound fills the blank of medicines for treating diseases caused by abnormal expression of MZF1. Through rigorous in-vitro and in-vivo animal experiments, the 10PANX ACETATE shows a remarkable inhibition effect on MZF1 overexpression cells, including inhibition of cell viability, proliferation and clone formation ability and down-regulation of MZF1 protein expression, and the 10PANX ACETATE shows a remarkable inhibition effect on MZF1 overexpression cells. In addition, the compound has a remarkable curative effect on the living body level, is high in safety and has no obvious toxic or side effect. Therefore, the 10PANX ACETATE provides a brand new drug candidate for prevention and treatment of MZF1 overexpression related diseases such as colon cancer, and has important clinical application value and wide market prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically, it relates to a polypeptide inhibitor targeting the MZF1 gene and its application in the treatment of colon cancer. Background Art

[0002] Colon cancer (CC) is one of the most common malignant tumors in the digestive system.

[0003] There are various treatment methods for colon cancer, including surgical resection, chemotherapy, radiotherapy, immunotherapy, and targeted drug therapy, etc. Early colon cancer patients can usually achieve a radical cure through surgical resection. However, due to the insidious onset of colon cancer, most patients are in the middle and late stages at the time of diagnosis and have missed the best opportunity for surgical radical cure. For middle and late stage colon cancer patients, chemotherapy and targeted drug therapy have become the main treatment methods. However, chemotherapy drugs have obvious toxic and side effects, and are prone to drug resistance after long-term application, which limits their clinical application effects. Targeted drug therapy has received extensive attention due to its strong targeting and small side effects.

[0004] Transcription factors, as a class of proteins that can regulate gene expression, play a crucial role in the occurrence, development, and prognosis of colon cancer. Among them, the MZF1 (Myeloid Zinc Finger 1) gene, as an important transcription factor, has been increasingly confirmed by more and more studies to be closely related to the malignant progression of colon cancer in recent years. The protein encoded by the MZF1 gene has a unique zinc finger structure, which can specifically bind to DNA and regulate the expression of downstream genes, thus playing an important role in processes such as cell proliferation, differentiation, apoptosis, migration, and invasion.

[0005] In colon cancer cells, the expression level of the MZF1 gene often abnormally increases. By regulating a series of genes related to cell proliferation, apoptosis, migration, and invasion, such as promoting the cell cycle process, inhibiting the expression of apoptosis-related genes, and upregulating genes related to tumor invasion and metastasis, etc., it further promotes the malignant proliferation, invasion, and metastasis of colon cancer cells. Therefore, targeted inhibition of the MZF1 gene has become a new strategy for colon cancer treatment and has broad application prospects.

[0006] However, at present, although the research on MZF1 in various malignant tumors is increasing, the research on small molecule inhibitors targeting MZF1 is still a key problem to be solved urgently, and no mature drugs have entered the clinical application stage. Especially in colorectal cancer, inhibitors directly targeting MZF1 have never been reported.

[0007] Therefore, the development of an efficient, specific, low-toxic and highly bioavailable MZF1 gene-targeted inhibitor is of great significance for improving the treatment effect of colorectal cancer and the quality of life of patients. Summary of the Invention

[0008] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a polypeptide inhibitor targeting the MZF1 gene and its application in the treatment of colon cancer. This polypeptide inhibitor can specifically bind to the MZF1 protein and inhibit its activity, thereby blocking the abnormal regulation of the downstream signaling pathway of MZF1, inhibiting the proliferation, invasion and metastasis of colon cancer cells, providing new drug candidates and treatment strategies for the treatment of colon cancer, and promising to bring better treatment effects and quality of life to colon cancer patients.

[0009] To achieve the above object, the present invention adopts the following technical solutions: Application of a polypeptide compound in the preparation of an MZF1 gene-targeted inhibitor, wherein the polypeptide compound is 10PanxAcetate, and the structural formula of 10Panx Acetate is as Figure 2 shown.

[0010] A polypeptide inhibitor targeting the MZF1 gene, comprising an effective dose of 10Panx Acetate and a pharmaceutically acceptable carrier, and the polypeptide inhibitor is used for the treatment of colon cancer with overexpression of MZF1.

[0011] Furthermore, surface plasmon resonance (SPR) detection proves that 10PanxAcetate can bind to the MZF1 protein.

[0012] Furthermore, the colon cancer cells are HCT8 or SW480.

[0013] Furthermore, the IC50 (half inhibitory concentration) of 10Panx Acetate against colon cancer cells HCT8 and SW480 are 38.13 uM and 42.03 uM respectively.

[0014] Furthermore, the CCK8 proliferation assay proves that the inhibitory effect of 10Panx Acetate on the viability of HCT8 and SW480 colon cancer cells with overexpression of MZF1 is more significant than that on mock cells.

[0015] Furthermore, the inhibitory effect of 10Panx Acetate on the colony formation ability of HCT8 and SW480 colon cancer cells with overexpression of MZF1 is more significant than that on mock cells.

[0016] Furthermore, 10Panx Acetate can reduce the expression of the MZF1 gene in colon cancer cells.

[0017] Furthermore, animal experiments have shown that 10Panx Acetate has a significant therapeutic effect on MZF1 gene-related diseases at the in vivo level.

[0018] Therefore, 10Panx Acetate can play an anti-colon cancer role by inhibiting the expression of the MZF1 gene both in vitro and in vivo, and can be applied to the preparation of drugs for preventing and treating colon cancer.

[0019] The present invention has the following beneficial effects: The 10PANX ACETATE polypeptide compound provided by the present invention is the first clearly identified highly efficient targeted inhibitor of the MZF1 gene, with high uniqueness and innovation. In the field of biomedicine, this compound fills the gap in drugs for treating diseases caused by abnormal expression of MZF1. Through rigorous in vitro and in vivo animal experiments, 10PANX ACETATE shows a significant inhibitory effect on MZF1 overexpressing cells, including inhibiting cell viability, proliferation, colony formation ability, and downregulating the expression of MZF1 protein. In addition, this compound has a significant therapeutic effect at the in vivo level and high safety, without obvious toxic and side effects. Therefore, 10PANX ACETATE provides a new drug candidate for the prevention and treatment of MZF1 overexpression-related diseases such as colon cancer, with important clinical application value and broad market prospects. Its unique mechanism of action and significant therapeutic effect make this compound have outstanding advantages and potential in the field of biomedicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the three-dimensional structure and cavity of MZF1; wherein, part A is the three-dimensional structure of the MZF1 protein, and part B is a schematic diagram of the internal cavity of MZF1.

[0021] Figure 2 It is a chemical structural formula of the small molecule polypeptide 10 PanxAcetate with the highest docking score in the three binding pockets of the MZF1 protein and an interaction diagram with the MZF1 protein; wherein, part A is the chemical structural formula of the polypeptide 10 Panx Acetate; part B is an interaction diagram of the MZF1 protein and the polypeptide 10 Panx Acetate.

[0022] Figure 3 It is an interaction diagram of detecting the interaction between the polypeptide 10Panx Acetate and MZF1 by SPR experiment.

[0023] Figure 4Figure showing the effect of 10 Panx Acetate on the viability of colon cancer cells HCT8 and SW480 in toxicity tests.

[0024] Figure 5 Figure showing the effect of 10 Panx Acetate on the proliferation ability of HCT8 and SW480 cells with or without overexpression of MZF1 detected by CCK8 assay.

[0025] Figure 6 Figure showing the effect of 10 Panx Acetate on cell colony formation in plates (n = 3). Among them, part A is the figure showing the effect of 10 Panx Acetate on the colony formation ability of HCT8 cells; part B is the figure showing the effect of 10 Panx Acetate on the colony formation ability of SW480 cells: the left side is the representative picture, and the right side is the quantitative result; *P < 0.05 compared with the control group.

[0026] Figure 7 Figure showing the effect of 10 Panx Acetate on the protein level of MZF1 (n = 3); the left side is the representative blot, and the right side is the quantitative result; *P < 0.05 compared with the control group.

[0027] Figure 8 Figure showing the effect of 10 Panx Acetate on the half-life level of MZF1 protein detected by treating HCT8 cells with or without overexpression of MZF1 with CHX for different times (n = 3); the left side is the representative blot, and the right side is the quantitative result; *P < 0.05 compared with the control group.

[0028] Figure 9 Schematic diagram showing the results of verifying the inhibition of the growth of xenograft tumors derived from colon cancer cells (HCT8 Mock and HCT8-LV-MZF1) by 10 Panx Acetate in an embodiment of the present invention: Among them, part A is the tumor image after treatment with normal saline in the control group or 10 Panx Acetate (10 mg / Kg) in the inhibitor group; part B is the quantitative analysis of the tumor weights of four groups of mice; part C is the quantitative analysis diagram of the tumor volumes of four groups of mice. Detailed implementation manners

[0029] The technical research solutions and effects of the present invention will be introduced in detail below in combination with specific embodiments and the accompanying drawings. For experimental methods without specific conditions, they are usually carried out according to conventional experimental protocol conditions, such as those described in textbooks, experimental guides, and product manuals, or according to the conditions recommended by the manufacturer, which are mastered or easily obtained by relevant technical researchers in this field. The following embodiments are only preferred embodiments of the present invention and do not limit the present invention. For relevant technical researchers in this field, various choices and optimizations can be made in terms of conditions and solutions for the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention. Example 1

[0030] The materials and methods involved in this example are as follows: (1) Cell culture and reagents: Human colon cancer cell lines HCT8 and SW480 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in 1640 (C11875500BT, GIBCO, USA) or L15 (C11415064 BT, GIBCO, USA) medium containing 10% fetal bovine serum (FSP500, Exce11 Bio, China). The culture temperature in the incubator was 37°C and 5% CO2. Cells in the logarithmic growth phase were used for the experiment. 10Panx Acetate (T21839L) was purchased from Shanghai TargetMol Biotechnology Co., Ltd. (TargetMol, China). The primary antibodies were as follows: anti-MZF1 (A10356, Abclonal, China), anti-β-tubulin (66240-1-Ig, Proteintech, USA). The secondary antibodies used in Western blotting were all purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.

[0031] (2) Molecular docking: The amino acid sequence of MZF1 was downloaded from the National Center for Biotechnology Information (NCBI) of the United States, and its three-dimensional structure was predicted using the artificial intelligence model AlphaFold 3 (AF3) ( Figure 1 A). After consulting relevant literature, it was found that there is currently no specific report on the active site of the MZF1 protein. Therefore, we selected two cavities of MZF1 that may be related to protein activity for virtual screening through predictive analysis ( Figure 1B). Using the Schrodinger molecular docking simulation software, the docking grid box center and the docking grid box were set, and a ligand database (in SMILES format) was obtained from Shanghai TargetMol Co., Ltd. Compounds with strong binding force and many interacting residues were screened out according to the ascending order of the docking score, and the interactions between the protein and the ligand were further analyzed.

[0032] (3) Detection of the half-maximal inhibitory concentration (IC50): HCT8 and SW480 cells in the logarithmic growth phase were seeded into 96-well plates at a density of 6,000 cells / well. After adherence, 10 Panx Acetate was added respectively to make the final concentration reach 6.25, 12.5, 25, 50, 100 μM. Each group was set with 3 replicate wells, and the final volume per well was 100 μl. The control group was added with an equal volume of solvent-containing medium. After culturing for 48 h, a detection solution mixed by blank culture and CCK8 in a ratio of 9:1 was added, 100 μl per well. After culturing in a cell incubator for 1.5 h, the absorbance of each well was detected at a wavelength of 450 nm using a WD-2102A type automatic microplate reader. The cell survival rate and the IC50 value were calculated according to the absorbance. Cell survival rate = [(experimental group - blank group) / (control group - blank group)] × 100%.

[0033] (4) Detection of cell colony formation by crystal violet staining: HCT8 and SW480 cells in the logarithmic growth phase were seeded into 6-well plates at a density of 300 cells / well. After cell adherence, a drug-containing medium was added and the cells were continuously cultured until the 14th day. The medium was changed every 3 days in the middle and the cell state was observed. After colony formation, 1 ml of 4% paraformaldehyde was added to each well to fix for 30 min, then washed 3 times with PBS. Subsequently, 1 ml of crystal violet staining solution was added and stained for 20 min, washed 3 times with PBS, and photographed with a camera after air drying.

[0034] (5)Western Blot detection of the expression level of MZF1 protein: After the cells were treated under different conditions, the cells were lysed with RIPA lysis buffer (KeyGEN Bio TECH, China, #KGB5204-100) containing protease and phosphatase inhibitors (NCM Biotech, China, #P002). After vigorous shaking on ice for 30 min, the mixture was centrifuged at 12,000 rpm for 15 min at 4 °C, and the supernatant was collected. Subsequently, after determining the protein concentration with a BCA kit, 1 / 3 volume of 4X loading buffer was added to the remaining protein and mixed. Then the cells were placed in a 100 °C metal bath and boiled for 10 min. The proteins were electrophoretically separated on a 10% SDS polyacrylamide gel and transferred to a 0.2 μm polyvinylidene difluoride (PVDF) membrane (Millipore, #ISEQ00010, Ireland). After blocking with 5% skim milk for 2 h, the PVDF membrane was incubated with the primary antibody overnight at 4 °C. The next day, the primary antibody was recovered, and the membrane was washed three times with TBST, and then incubated with the secondary antibody at room temperature for 2 h. The PVDF membrane was photographed using a gel imaging system to observe the expression of the target protein.

[0035] (6)Construction of a nude mouse tumor xenograft model: A colon cancer xenograft model was established using 5-week-old female nude mice. HCT8 cells overexpressing or not overexpressing MZF1 were digested with trypsin (25200072, Gibco, USA), resuspended with PBS, and made into a uniform single-cell suspension. 100 μL (5 x 106 HCT8 cells) of the cell suspension was subcutaneously injected into the back of each nude mouse. Seven days later, tumor xenografts formed subcutaneously in the nude mice. The control group and the drug administration group were intraperitoneally injected with PBS or 10 mg / Kg of 10PanxAcetate, respectively, once every 3 days, and the tumor growth was measured and recorded. The volume calculation formula for each tumor was: V = length * width2 / 2. Fourteen days after treatment (at the end of treatment), the mice were sacrificed, and the tumors, liver, spleen, and kidneys were taken for corresponding staining, and the tumor growth was evaluated during this period.

[0036] (7) Surface plasmon resonance (SPR): SPR was used to detect the interaction between small molecule inhibitors and MZF1. First, the sensor chip was pretreated. Immediately before use, 400 mM EDC and 100 mM NHS were mixed to prepare the activator, and then the CM5 sensor chip was activated at a flow rate of 10 μL / min for 420 seconds. Then, the ligand immobilization operation was performed. The MZF1 protein was diluted to 20 μg / mL and injected into the sample channel (Fc2). The flow rate was maintained at 10 μL / min to achieve an immobilization level of approximately 3900 RU. This step was not required for the reference channel (Fc1); 1 M ethanolamine hydrochloride was then used to block the chip surface at the same flow rate for 420 seconds to quench the residual active sites. In the multi-cycle analyte detection stage, 10panx Acetate needs to be diluted with running buffer to six concentration gradients of 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM and 0 μM, and injected into the Fc1-Fc2 channel in order from low to high concentration, with a flow rate of 30 μL / min. Each cycle contains a 120-second binding period and a 300-second dissociation period. No additional regeneration is required between cycles, and the sensor surface is reset by natural dissociation. All operations need to be performed in a low-temperature environment, and the sensor map data is recorded in real time by the instrument software to analyze the molecular interaction dynamics.

[0037] The test results involved in this embodiment are as follows: (1) Detection of peptide 10Panx Acetate binding to MZF1 protein: Molecular docking screening of peptides, compounds or natural compounds targeting MZF1 protein. The ligand was allowed to simulate binding with the protein target, and the final docking results were scored based on the skeleton structure, interaction force, etc. The peptide 10Panx Acetate scored the highest. The chemical structure of 10Panx Acetate and its interaction with MZF1 protein are shown in Figure 2. Figure 2 As shown, the chemical structure of 10Panx Acetate is as follows Figure 2 As shown in A, its interaction diagram with MZF1 protein is shown in Figure 2 As shown in B, it is mainly bound by hydrogen bonds and hydrophobic bonds, with a binding energy of 58.393 kcal / mol. The SPR technology was used to detect the binding activity of the screened small molecule inhibitors with MZF1. The results showed that after the MZF1 protein was fixed, its binding affinity constant with 10Panx Acetate reached 65.8μM ( Figure 3 ). This data conclusively proves that there is a direct and significant binding interaction between 10Panx Acetate and MZF1 protein.

[0038] (2)Detection of the IC50 of 10Panx Acetate against colon cancer cells (n = 3): The Cell Counting Kit-8 (BS350A, Biosharp, China) was used to detect cell viability. SW480 and HCT8 cells were respectively seeded into 96-well plates at a cell density of 6000 cells / well. After the cells adhered, they were treated with different concentrations of 10Panx Acetate for an appropriate time. After adding the CCK8 reagent, the absorbance at 450 nm was measured. The cell viability and IC50 values were calculated based on the absorbance values. The results of the toxicity test showed that 10Panx Acetate could significantly inhibit the viability of colon cancer cells at the μM level, and its IC50 value for HCT8 was 38.13 μM( Figure 4 A), and the IC50 value for SW480 was 42.03 μM( Figure 4 B).

[0039] (3)Detection of the inhibitory effect of 10Panx Acetate on the proliferation ability of colon cancer cells overexpressing MZF1: To further explore the biological role of 10Panx Acetate targeting MZF1 in colon cancer, the inventors adopted a series of in vitro cell function experiments, including the CCK8 experiment and the plate colony formation experiment, to evaluate the effect of 10Panx Acetate on the proliferation ability of colon cancer cells. First, the CCK8 experiment was carried out. Cells were seeded into 96-well plates at a density of 3000 cells / well. After adhesion, different drug treatments were performed, and their cell proliferation ability was continuously detected for 96 hours. The growth curve results showed that whether it was HCT8( Figure 5 A) or SW480( Figure 5 B), 10Panx Acetate had a greater growth inhibitory effect on cells overexpressing MZF1, and the difference was statistically significant.

[0040] To further verify the effect of the polypeptide on proliferation, the inventors used HCT8 or SW480 cells stably transfected with the MZF1 expression vector or the empty vector to perform the plate colony formation experiment, and then treated them with or without 10Panx Acetate. The experiment showed that compared with the empty vector group, 10Panx Acetate more effectively inhibited the proliferation of tumor cells in the MZF1 overexpression group. Figure 6 A is the HCT8 cell line, Figure 6 B is the SW480 cell line. The left is the representative image, and the right is the column statistical chart.

[0041] (4)Test on the effect of 10Panx Acetate on the protein level and half-life of MZF1 in colon cancer cells: Immunoblotting was used to evaluate the effect of the polypeptide 10Panx Acetate on the MZF1 protein. The results are as Figure 7As shown, the immunoblot results and statistical data indicate that 10Panx Acetate inhibits the expression of MZF1 protein in both HCT8 and SW480 cells. To further investigate the mechanism by which 10Panx Acetate regulates MZF1 protein expression, the inventors conducted a CHX pulse-chase experiment to determine the half-life and degradation rate of the protein ( Figure 8 ). The results showed that 10Panx Acetate promoted the degradation of MZF1.

[0042] (5) Test for the inhibitory effect of 10Panx Acetate on the growth of mouse colon cancer cells: To verify the therapeutic effect of 10Panx Acetate on the growth of colon cancer tumors in vivo, the inventors constructed xenograft tumor models derived from HCT8 Mock and HCT8-LV-MZF1 cells, and treated them with normal saline and 10Panx Acetate (10 mg / Kg), respectively. After 2 weeks of treatment (at the end of the treatment), the mice were sacrificed, and the tumors were taken to evaluate the tumor growth. The results are as Figure 9 shown. Compared with the control group, the tumor growth in the HCT8-LV-MZF1 group treated with 10PanxAcetate was significantly inhibited, while the tumor growth in the HCT8 Mock group was also inhibited to a certain extent, but the effect was not as significant as that in the HCT8-LV-MZF1 group. Through quantitative analysis of the tumor weight and volume ( Figure 9 B, C), it was found that the tumor volume inhibition rate ≥ 50% and the tumor weight inhibition rate ≥ 40%. The inventors further confirmed that the inhibitory effect of 10PanxAcetate on the tumors in the LV-MZF1 group was more obvious.

[0043] As can be seen from the above examples, the 10PANX ACETATE polypeptide compound disclosed in the present invention, as an innovative MZF1 gene-targeted inhibitor, exhibits excellent inhibitory effects. Through computer simulation technology and virtual screening by molecular docking, the inventors successfully screened out this polypeptide with high affinity from a large compound library. In subsequent cell experiments and animal experiments, 10PANX ACETATE not only specifically bound to the MZF1 protein, but also significantly down-regulated the expression of the MZF1 protein by promoting the protein degradation pathway, effectively inhibiting the proliferation, colony formation ability of colon cancer cells and the growth of tumors. These series of experimental results fully verify the high efficiency and specificity of 10PANX ACETATE as an MZF1 gene-targeted inhibitor, providing a new drug candidate for the treatment of colon cancer and promising more significant therapeutic effects for patients.

[0044] This patent application first discovered that 10PANX ACETATE has shown great potential in the treatment of colon cancer with overexpression of MZF1. This polypeptide compound not only showed significant inhibitory effects on colon cancer cells in in vitro experiments, but also successfully slowed down the tumor growth rate at the in vivo level, and had no obvious effect on the body weight of mice, showing good safety and tolerance. This discovery not only fills the gap in therapeutic drugs for diseases caused by abnormal expression of MZF1, but also provides new ideas and methods for the prevention and treatment of MZF1 gene-related diseases. The unique mechanism of action and significant efficacy of 10PANX ACETATE make it have broad application prospects and important clinical value in the biomedical field, and are expected to bring a revolutionary breakthrough in the treatment of MZF1 overexpression-related diseases such as colon cancer.

[0045] The above are only the preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions falling within the idea of the present invention belong to the protection scope of the present invention. It should be pointed out that for those of ordinary skill in the art, several improvements and refinements made without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. Use of a polypeptide compound in the preparation of a MZF1 gene targeting inhibitor, characterized in that: The polypeptide compound is 10Panx Acetate.

2. The use according to claim 1, characterized in that: The MZF1 gene targeted inhibitor is used to treat colorectal cancer with overexpression of MZF1.

3. The use according to claim 1 or 2, characterized in that: The binding affinity constant of 10Panx Acetate to MZF1 protein is 65.8μM.

4. The use according to any one of claims 1 to 3, characterized in that: The half-inhibitory concentrations of 10Panx Acetate on colorectal cancer cells HCT8 and SW480 were 38.13μM and 42.03μM, respectively.

5. The use according to any one of claims 1 to 3, characterized in that: 10Panx Acetate inhibits the expression of MZF1 by promoting its degradation.

6. The use according to any one of claims 1 to 3, characterized in that: 10Panx Acetate was able to inhibit the growth of colon cancer xenografts with high MZF1 expression in in vivo experiments, with tumor volume inhibition rates ≥50% and tumor weight inhibition rates ≥40%.

7. A polypeptide inhibitor targeting the MZF1 gene, characterized in that: The invention comprises an effective dose of 10 Panx Acetate and a pharmaceutically acceptable carrier, and the polypeptide inhibitor is used for treating colon cancer with overexpression of MZF1.

8. The polypeptide inhibitor according to claim 7, characterized in that The polypeptide inhibitor is administered by intraperitoneal injection.

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