A polypeptide, nucleic acid construct and pharmaceutical composition for treating cancer

Abstract

The present application relates to a kind of polypeptide, nucleic acid construct and pharmaceutical composition for treating cancer, the amino acid sequence of polypeptide is RRVCVHFHFSLSLR.Compared with prior art, the polypeptide of the present application can significantly inhibit cancer cell proliferation, can be used for further depth development to inhibit tumor cell growth and antitumor drug, provides the biological active lead molecule with extremely commercial value for the treatment of cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a polypeptide, nucleic acid construct and pharmaceutical composition for treating cancer. Background Technology

[0002] Gastric cancer is a common tumor of the digestive system and is one of the most deadly malignant tumors worldwide, especially in China. Its insidious course makes early diagnosis extremely difficult. More importantly, gastric cancer exhibits strong organ specificity and high heterogeneity during its progression due to genetic instability. Multiple tumor cell subpopulations with different biological characteristics often emerge, and key signaling molecules are frequently expressed heterogeneously, leading to differences in malignant biological characteristics such as tumor invasion and metastasis, and affecting prognosis to varying degrees. Furthermore, gastric cancer shows significant geographical variation, with over 70% of new gastric cancer cases worldwide occurring in developing countries, primarily in East Asia, including China, Japan, and South Korea. Compared to other tumors, international research on gastric cancer lags far behind, particularly in molecular subtyping, which remains very preliminary. Although researchers have made some progress in gastric cancer subtyping, little is known about its heterogeneity and underlying pathological mechanisms. Correspondingly, there is currently a lack of effective diagnostic and treatment methods for gastric cancer worldwide, making the situation extremely serious.

[0003] The highly conserved Hippo signaling pathway participates in regulating a series of life processes, including cell proliferation and apoptosis, and is closely related to the development and progression of various tumors. The core of the Hippo signaling pathway is a phosphorylation cascade: Hippo (MST1 / 2 in mammals) kinase regulates the phosphorylation of LATS1 / 2 kinase. Once activated, LATS1 / 2 further phosphorylates the transcriptional activator YAP / TAZ-TEAD, regulating its nuclear import and export, thereby controlling its pro-cancer activity. Therefore, key molecules in the Hippo signaling pathway are potential targets for anti-tumor drugs. Summary of the Invention

[0004] The purpose of this invention is to provide a polypeptide, nucleic acid construct, and pharmaceutical composition for treating cancer.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] A polypeptide for treating cancer, the structural formula of which is:

[0007]

[0008] The amino acid sequence of this polypeptide is RRVCVHFHFSLSLR, therefore, the structural formula of this polypeptide (PET polypeptide) can also be simplified to the following formula:

[0009]

[0010] A modified polypeptide, based on the polypeptide, wherein the modified polypeptide is a polypeptide obtained by modifying the N-terminus and / or C-terminus of the polypeptide of claim 1.

[0011] Furthermore, the N-terminus is acetylated, and the C-terminus is amidated.

[0012] Furthermore, the modified polypeptide also has disulfide bond modification.

[0013] A modified polypeptide, based on the aforementioned polypeptide, wherein the modified polypeptide comprises a cyclic structure with a benzene ring linking the first arginine and the fourth cysteine ​​of the polypeptide, and the structural formula of the modified polypeptide is as follows:

[0014]

[0015] The structural formula of this modified polypeptide (GPET polypeptide) can also be simplified to the following formula:

[0016]

[0017] A polynucleotide sequence, wherein the polynucleotide sequence is the coding sequence of the said polypeptide or modified polypeptide, or the complementary sequence of the coding sequence.

[0018] A nucleic acid construct containing the aforementioned polynucleotide sequence.

[0019] The use of a polypeptide or modified polypeptide in the preparation of a drug for treating or preventing diseases mediated by the Hippo signaling pathway.

[0020] A pharmaceutical composition for treating or preventing diseases mediated by the Hippo signaling pathway, the pharmaceutical composition comprising a polypeptide or modified polypeptide as described above.

[0021] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0022] Preferably, the disease is cancer; more preferably, the disease is gastric cancer, but does not exclude papillary renal cell carcinoma, medulloblastoma, lung cancer, pancreatic cancer, esophageal cancer, liver cancer, colon cancer, prostate cancer, and ovarian cancer.

[0023] Compared with existing technologies, the peptides of this invention can significantly inhibit the proliferation of cancer cells and can be used for further in-depth development of drugs that inhibit tumor cell growth and fight tumors, providing a highly commercially valuable bioactive lead molecule for cancer treatment. Attached Figure Description

[0024] Figure 1To detect the effect of peptides on cell proliferation in a soft agarose cell colony formation assay, where A represents the statistical results of cell viability and B represents the staining results;

[0025] Figure 2 The half-inhibitory concentration (IC50) of GPET peptide against different gastric cancer cell lines. 50 );

[0026] Figure 3 This is the result of the pharmacokinetic dynamics of GPET peptides, i.e., the concentration half-life.

[0027] Figure 4 The image shows the effect of peptides on tumors in a mouse gastric cancer model. In the image, A is an image of a mouse gastric tumor, and B is the staining of mouse gastric tissue by immunohistochemistry.

[0028] Figure 5 The results of the kinetic experiment (binding constant) of GPET peptide and TEAD4;

[0029] Figure 6 PET peptides and PET mut Kinetic results of peptide-TEAD4 reaction;

[0030] Figure 7 PET peptides and PET mut Results of peptide inhibition of gastric cancer cell line HGC-27 proliferation. Detailed Implementation

[0031] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0032] Extensive research evidence has demonstrated the crucial role of the Hippo signaling pathway in organ size regulation, cancer development, tissue regeneration, and stem cell function regulation. Abnormal activation of the key effector molecules YAP / TEAD in the Hippo pathway is closely associated with the development and progression of various tumors. As upstream and core molecules in tumor suppressor gene regulation, dysregulation of the Hippo pathway can lead to uncontrolled proliferation and apoptosis inhibition. The peptides and their modified peptides in this invention can bind to the TEAD4 protein, thereby inhibiting downstream activation of the Hippo signaling pathway, demonstrating significant potential for application in tumor therapy.

[0033] Therefore, the present invention provides a polypeptide and its modified polypeptide, which can bind to TEAD4 protein and inhibit the activation of downstream Hippo signaling pathway, and has a significant inhibitory effect on cancer cell proliferation, and can be used for cancer treatment and prevention.

[0034] The amino acid sequence of the present invention can be a chemically synthesized product. For example, the amino acid sequence of the present invention can be synthesized using peptide chemical synthesis methods known in the art.

[0035] This invention also includes the coding sequence of the aforementioned polypeptide and its complementary sequence. Where appropriate, a recombinant expression vector can be used to prepare the polypeptide of this invention. The structural composition of the recombinant expression vector, its construction method, and expression method are well known in the art.

[0036] The modification methods of peptides are well known in the art.

[0037] The present invention also provides a pharmaceutical composition containing a therapeutically effective amount of the polypeptide of the present invention. As used herein, "effective amount" means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals. For example, for liquid formulations or compositions, the concentration of the polypeptide may be 20 ng / ml or more, such as 50 ng / ml or more, 80 ng / ml or more, 100 ng / ml or more, or higher.

[0038] Pharmaceutical compositions may contain pharmaceutically acceptable carriers. In this invention, "pharmaceutically acceptable carrier" refers to a carrier used for therapeutic administration, including various excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity after administration. Suitable carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in a composition may contain liquids such as water, saline, or buffer solutions. Additionally, these carriers may contain auxiliary substances such as fillers, lubricants, flow aids, wetting agents or emulsifiers, pH buffers, etc.

[0039] The pharmaceutical compositions of the present invention can be administered to the subject using various conventional and suitable methods in the art, including but not limited to: oral administration, subcutaneous injection, intramuscular injection, transdermal administration, local administration, implantation, sustained-release administration, etc.

[0040] This invention also provides the use of the peptides described herein in the preparation of medicaments for the treatment or prevention of Hippo signaling pathway-mediated diseases. Hippo signaling pathway-mediated diseases generally include various cancers known to be associated with abnormalities in the Hippo signaling pathway, including but not limited to papillary renal cell carcinoma, medulloblastoma, lung cancer, pancreatic cancer, esophageal cancer, liver cancer, gastric cancer, colon cancer, prostate cancer, and ovarian cancer.

[0041] In a preferred embodiment, the present invention provides the use of the polypeptide described herein in the treatment or prevention of gastric cancer or in the preparation of a medicament for the treatment or prevention of gastric cancer. In this document, gastric cancer can refer to any of the various classifications of gastric cancer known in the art.

[0042] Example 1:

[0043] Design and synthesis of PET peptides

[0044] Based on existing experimental results, this invention designs a polypeptide with the sequence RRVCVHFHFSLSLR. The polypeptide is synthesized by a polypeptide synthesis company using a publicly available solid-phase polypeptide synthesis method. This involves first covalently linking the hydroxyl-terminal amino acid of the desired peptide chain to an insoluble polymer resin. Then, this amino acid, bound to the solid support, is used as the amino group. After deprotection of the amino group, it reacts with an excess of activated carboxyl group components to lengthen the peptide chain. This process (condensation → washing → deprotection → neutralization and washing → next condensation) is repeated until the desired peptide chain length is achieved. Finally, the peptide chain is cleaved from the resin, purified, and the desired polypeptide is obtained with a purity greater than 98%. The structure of the polypeptide is as follows:

[0045]

[0046] Example 2:

[0047] Design and synthesis of GPET peptides

[0048] Starting with the polypeptide sequence RRVCVHFHFSLSLR from Example 1, the side chain thiol groups at the first R and fourth C positions are covalently linked by an S-benzene ring-S to form a cyclic peptide, as shown below.

[0049]

[0050] This cyclic peptide can be synthesized using a publicly available synthetic method, and the resulting polypeptide has a purity greater than 98%.

[0051] Example 3:

[0052] Effects of PET peptides on cell proliferation

[0053] 1. The experimental method is as follows:

[0054] Cell culture: HGC-27, MKN-45, AGS, MNK-28, SH-10-TC, SNU-216, BGC-823, BGC-803, AZ-521, and GES-1 cells (all obtained commercially available) were cultured in RPMI 1640 (Invitrogen) medium supplemented with 10% serum, 100 μg / ml penicillin, and 100 μg / ml streptomycin. Cells were cultured at 37°C with a carbon dioxide concentration of 5%.

[0055] Cell proliferation assay: Cell proliferation was detected using an ATP cell viability assay kit (Beyotime Biotechnology Co., Ltd.) (Luminescent Cell Viability Assay) Prepare cells with culture medium in a 96-well plate with opaque walls, 100 μl / well, 3000 cells / well. After adhesion the next day, add different concentrations of S-peptide (0 μg / ml, 10 μg / ml, 20 μg / ml, 30 μg / ml, etc.). Simultaneously prepare control wells containing only culture medium without cells to obtain background luminescence values. Cell viability is measured using CellTiter reagent after 48 hours. Equilibrate the plate and its contents to room temperature, which takes approximately 30 minutes. Add an equal volume of culture medium to each well. 100 μl of reagent. Mix the contents on a fixed-track shaker for 2 minutes to induce cell lysis, then incubate the plate at room temperature for 10 minutes to stabilize the fluorescence signal value and record the luminescence signal.

[0056] Soft agarose cell colony formation assay: Cells were formed when the cell density reached 10-1. 4 Then, the clones were inoculated onto soft agarose in 6-well plates, and clones with a diameter greater than 0.05 mm were counted 14 days later.

[0057] Cellular IC 50 Cell proliferation experiment results were obtained by processing data using Graphpad Prism software to obtain IC50 values. 50 Numerical value.

[0058] Data Analysis: The data were analyzed using SAS data analysis software package (9.1.3). The statistical data were expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) and Student's t-test were used to analyze continuous variables. Confidence intervals were defined as P < 0.05.

[0059] 2. The experimental reagents are as follows:

[0060] PET peptides and GPET peptides were dissolved in phosphate-buffered saline (PBS) for use in experiments.

[0061] Phosphate-buffered saline (PBS): Dissolve 0.2g KCl, 8g NaCl, 0.24g KH₂PO₄, and 1.44g Na₂HPO₄ in 800ml distilled water. Adjust the pH of the solution to 7.4 with HCl, and bring the volume to 1L. Autoclave or filter sterilize.

[0062] 3. Experimental Results and Analysis

[0063] Gastric cancer cell line was treated with phosphate-buffered saline (PBS) (control) and GPET peptide using a soft agarose cell colony formation assay. The results showed that GPET peptide significantly inhibited the proliferation of gastric cancer cells. Given the long duration of action in the soft agarose cell colony formation assay, this indicates that the inhibitory effect of GPET peptide on gastric cancer cell proliferation is sustained. Figure 1 ).

[0064] Normal gastric mucosal cells GES-1 and gastric cancer cell lines HGC-27, MKN-45, AGS, MNK-28, SH-10-TC, SNU-216, BGC-823, BGC-803, and AZ-521 were treated with GPET peptides at concentrations of 5 μg / ml, 10 μg / ml, 15 μg / ml, 20 μg / ml, and 25 μg / ml, respectively. After 48 hours, the cells were processed using Beyotime Biotechnology Co., Ltd. Reagents were used to determine cell viability. Cell IC50 was calculated. 50 ,like Figure 2 As shown, the IC50 of GPET peptide against the normal cell line GES-1 is... 50 The concentration was 13.1 μg / ml, while the IC50 against gastric cancer cell lines HGC-27, MKN-45, MNK-28, SNU-216, BGC-803, and AZ-521 was [not specified]. 50 The levels of GPET peptide were significantly lower than in normal cells, exhibiting a significant inhibitory effect on the proliferation of gastric cancer cells. Furthermore, in all three cell lines with even lower TEAD4 expression levels—MNK-28, BGC-803, and AZ-521—the IC50 for GPET peptide-mediated inhibition of proliferation was significantly lower. 50 All were significantly lower than GES-1.

[0065] These results indicate that GPET peptides can significantly inhibit the proliferation of gastric cancer cells, while having limited inhibitory effect on the proliferation of normal cells. This inhibitory effect is more pronounced in cell lines with low TEAD4 expression levels.

[0066] Example 4:

[0067] Pharmacokinetics of GPET peptides

[0068] Pharmacokinetics: Blood samples were collected from mice after drug administration, and the obtained plasma was analyzed using liquid chromatography-mass spectrometry (LC-MS). Based on the drug concentration in the mouse blood at different time points, Graphpad software was used to calculate and analyze the absorption, distribution, metabolism, and excretion of the drug in vivo, obtaining relevant parameters of pharmacokinetics. The concentration half-life was 4.58 hours. Figure 3 As shown.

[0069] Example 5:

[0070] Effects of GPET peptides on tumors in a mouse gastric cancer model

[0071] 1. The experimental method is as follows:

[0072] MNNG-induced establishment of a mouse gastric cancer model

[0073] Mice were housed in a specialized culture chamber for infectious disease animal models, subjected to a circadian rhythm of 12 hours of light and 12 hours of darkness. MNNG (100 mg / ml) was added to drinking water and administered continuously for 14 days, followed by a 14-day pause in the circadian rhythm, and fed for 6 weeks to induce gastric cancer. GPET peptides were injected into mice using PBS as the drug solvent, while control mice were injected with PBS. After another 6 weeks of feeding, the mice were sacrificed for analysis.

[0074] Animal breeding and animal experiments are conducted in accordance with the relevant charter and animal welfare policies of the Animal Management Committee of the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences.

[0075] Immunohistochemistry: Tissue samples were processed according to BD Pharmingen TM The IHC Zinc Fixative Handbook (Handbook No.: 550523) describes zinc fixation (BD Biosciences) followed by paraffin embedding. Tissue sections (5 μm thick) are fixed by heat, dewaxed in xylene for 5 minutes, then dewaxed again with fresh xylene, for a total of 3 dewaxing cycles. Anhydrous ethanol is applied twice for 5 minutes each. 90% ethanol is applied twice for 5 minutes each, followed by 70% ethanol once for 5 minutes. Distilled water is applied twice for 5 minutes each. Depending on the antigen and antibody, sections can be placed in the following antigen retrieval solutions: 10 mM sodium citrate, pH 6.0; or 1 mM EDTA, pH 8.0; or 10 mM Tris, pH 10.0. The sections are heated at 95°C for 12 minutes and then slowly cooled to room temperature over approximately 30 minutes. 5% skim milk is added for blocking for 60 minutes.

[0076] From the initial sealing step onwards, it is crucial to maintain sample moisture and prevent drying, as this can easily lead to high background. Dilute the primary antibody appropriately, incubate overnight at 4°C with gentle shaking, recover the primary antibody, add PBST, and wash for 5 minutes. After aspirating the washing buffer, add more washing buffer and wash for 5 minutes. Repeat this washing process three times. Dilute the horseradish peroxidase (HRP), biotin, or alkaline phosphatase (AP) labeled secondary antibody at an appropriate ratio. Incubate at room temperature or 4°C on a side-shaking rack with gentle shaking for one hour. Recover the secondary antibody, add PBST washing buffer, and wash for 5 minutes on a side-shaking rack with gentle shaking. After aspirating the washing buffer, add more washing buffer and wash for 5 minutes. Repeat this washing process three times. Use DAB for subsequent detection; after DAB staining, perform HE staining. Finally, dehydrate, clear, and mount with neutral resin.

[0077] 2. The experimental reagents are as follows:

[0078] GPET peptides are prepared and diluted with phosphate-buffered saline (PBS) for use in experiments.

[0079] 3. Experimental Results and Analysis:

[0080] The results are as follows Figure 4 As shown. Figure 4 The results showed that mice treated with GPET peptides had significantly smaller gastric tumors than the control group. Figure 4 Similarly, immunohistochemical analysis of mouse stomach tissue using Ki67, an indicator of cell proliferation, showed that the control group had the highest Ki67 positivity rate and the most active cell proliferation, while the experimental group using GPET peptides showed a significantly lower Ki67 positivity rate and a marked decrease in cell proliferation. Figure 4 (B) Similarly, in terms of tissue morphology, the gastric tissue of the control group mice showed significant abnormal proliferation, while the gastric tissue morphology of the experimental group mice treated with GPET peptides was basically normal. This indicates that GPET peptides have a significant inhibitory effect on the proliferation of gastric tumor cells in a gastric cancer model mouse.

[0081] Example 6:

[0082] Target of GPET peptide

[0083] 1. The experimental method is as follows:

[0084] Protein purification: The in vitro protein purification method for TEAD4 was the same as that in the published literature S.Jiao., et al. (2014). Cancercell 25(2):66-180.

[0085] Kinetic detection: The kinetic parameters between TEAD4 protein and GPET peptide were detected using a micro-thermophoresis apparatus (MST). TEAD4 protein labeled with a fluorescent dye was added to the capillary of the MST, and different concentrations of GPET peptide were added to the sample cell. The affinity parameters of the interaction between these two molecules were obtained by detecting the effect of different concentrations of the conjugate molecules on the distribution of the thermophoretic equilibrium state of the fluorescent molecules.

[0086] 2. Experimental Results and Analysis:

[0087] Experimental results are as follows Figure 5 As shown in the figure. Kinetic analysis showed that the GPET peptide binds directly to the TEAD4 protein with an affinity of 3.35 μM.

[0088] Example 7:

[0089] Key sites for PET peptide function

[0090] 1. The experimental method is as follows:

[0091] A mutant peptide with the sequence RRVCVAAAASLSLR was designed and synthesized by a peptide synthesis company using a publicly available solid-phase peptide synthesis method. The peptide purity was greater than 98%. It was dissolved in phosphate-buffered saline (PBS) before use in experiments.

[0092] The protein purification and kinetic detection experiments were the same as in Example 6.

[0093] The cell proliferation experiment was the same as in Example 3.

[0094] 2. Experimental Results and Analysis:

[0095] Based on the structural simulation of the PET / GPET peptide complex with TEAD4, the HFHF site in the PET / GPET peptide may be directly related to its interaction with TEAD4. Therefore, a mutant peptide was designed to mutate HFHF to AAAA, named PET. mut .

[0096] Experimental results are as follows Figure 6 As shown. Kinetic analysis indicated that the PET peptide binds directly to the TEAD4 protein, with an affinity of 11.50 μM. PET mut The peptide does not bind to TEAD4.

[0097] Using PET peptides and PET mut The gastric cancer cell line HGC-27 was treated with peptides at concentrations of 5 μg / ml, 10 μg / ml, 20 μg / ml, and 40 μg / ml, respectively. After 48 hours, samples were collected from Beyotime Biotechnology Co., Ltd. Reagents are used to determine cell viability. For example... Figure 7 As shown, PET peptides have a significant inhibitory effect on the proliferation of gastric cancer cell line HGC-27, but PET... mut The peptide did not inhibit the proliferation of HGC-27 cells.

[0098] The above results indicate that the HFHF sequence in the GPET / PET peptide plays a crucial role in the peptide binding to TEAD4 and inhibiting the proliferation of gastric cancer cells.

[0099] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A polypeptide for treating cancer, characterized in that, The structural formula of the polypeptide is: 。 2. A modified polypeptide, based on the polypeptide of claim 1, characterized in that, The modified polypeptide is a polypeptide obtained by modifying the N-terminus and / or C-terminus of the polypeptide described in claim 1, wherein the N-terminus is acetylated and the C-terminus is amidated.

3. A modified polypeptide, based on the polypeptide of claim 1, characterized in that, The modified polypeptide is: The modified polypeptide of claim 1 has a cyclic structure formed by a benzene ring linking the first arginine and the fourth cysteine. The structural formula of the modified polypeptide is as follows: 。 4. A polynucleotide, characterized in that, The polynucleotide encodes the polypeptide or modified polypeptide as described in any one of claims 1 to 3.

5. A nucleic acid construct, characterized in that, The nucleic acid construct contains the polynucleotide as described in claim 4.

6. The use of a polypeptide or modified polypeptide as described in any one of claims 1 to 3 in the preparation of a medicament for treating or preventing gastric cancer.

7. A pharmaceutical composition for treating or preventing gastric cancer, characterized in that, The pharmaceutical composition contains the polypeptide or modified polypeptide as described in any one of claims 1 to 3.

8. A pharmaceutical composition for treating or preventing gastric cancer according to claim 7, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

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