Polypeptide and application thereof in biological pharmacy

Abstract

The invention discloses a polypeptide and application thereof in biological pharmacy, and requests to protect a polypeptide with an amino acid sequence of HGAFTTES (SEQ ID NO.1), and the polypeptide with the sequence is reported for the first time. An activity research shows that the polypeptide can effectively inhibit caenorhabditis elegans paralysis of an Alzheimer disease model, and has remarkable Alzheimer disease resisting activity. In addition, the polypeptide has obvious inhibitory activity on gastric cancer, breast cancer, lung cancer, esophageal cancer and hepatoma carcinoma cells, and especially has extremely strong inhibitory activity on gastric cancer and hepatoma carcinoma cells. Therefore, the polypeptide with the amino acid sequence of HGAFTTES has the prospect of being developed into anti-Alzheimer disease or anti-tumor drugs.

Description

Technical Field

[0001] The present invention belongs to the field of polypeptides, and relates to novel polypeptides and their applications, specifically to a polypeptide and its application in biopharmaceuticals. Background Art

[0002] In recent years, the research and development of small molecule drugs have become increasingly difficult, and macromolecule drugs such as antibodies tend to be homogenized. With the iterative update of peptide synthesis technology and the in-depth study of disease physiology, polypeptide drugs, as "unique drugs" between small and large molecules, have developed rapidly. Due to their advantages such as significant efficacy and strong specificity, polypeptide drugs are widely used in the prevention, diagnosis and treatment of tumors, cardiovascular and cerebrovascular diseases, diabetes, etc. They have broad development prospects and have become one of the hotspots in the field of new drug research and development.

[0003] Dementia is a syndrome centered on cognitive decline, mainly manifested by the gradual decline of memory, thinking, language and social abilities, affecting the daily living ability of patients. Common types include Alzheimer's disease, vascular dementia and Lewy body dementia, and the number of dementia cases globally has exceeded 50 million. Alzheimer's disease (AD) is related to the deposition of amyloid β-protein (Aβ) plaques and neurofibrillary tangles, the loss of neurons and neuroinflammation. Vascular dementia is mostly caused by cerebrovascular diseases, resulting in insufficient blood supply to the brain and affecting cognitive function. Although current treatments mainly focus on symptom management, commonly used drugs such as cholinesterase inhibitors and NMDA receptor antagonists can temporarily improve the cognitive ability of patients, but these drugs cannot prevent the progression of the disease, and there are individual effect differences and side effects, bringing great challenges to the treatment of dementia. There is an urgent need to develop new effective treatment strategies.

[0004] Tumors are formed due to abnormal cell proliferation and can be classified into benign tumors and malignant tumors (cancer). The latter is invasive and metastatic, posing a serious threat to human health. The occurrence of cancer is usually related to gene mutations, environmental factors, and lifestyle choices. Clinically, the treatment methods for cancer mainly include surgical resection, radiotherapy, chemotherapy, targeted therapy, and immunotherapy, etc. Surgical resection is the most direct treatment method, but for advanced cancer or metastatic cancer, the effect of resection surgery is often limited. In addition, the damage of chemotherapy and radiotherapy to normal cells, as well as the heterogeneity and drug resistance of tumors, make single treatment regimens often ineffective. Moreover, radiotherapy kills cancer cells through high-energy rays and often needs to be used in combination with other treatment methods. Targeted therapy designs drugs based on specific molecular characteristics of cancer cells. Although it improves the precision of treatment, some patients may still face drug resistance due to changes in tumor characteristics. In recent years, immunotherapy has made some remarkable progress by activating the patient's own immune system to identify and attack cancer cells. Successful cases of this method have significantly improved the survival rate of patients with certain types of cancer, but not all patients can benefit from it, and there are still problems of side effects and uneven efficacy.

[0005] The applicant has discovered a novel polypeptide with excellent activity and hereby proposes this invention. Summary of the Invention

[0006] The first object of the present invention is to provide a polypeptide with an amino acid sequence as shown in SEQ ID NO.1, the second object is to provide the application of this polypeptide in the preparation of drugs for treating Alzheimer's disease, and the third object is to provide the application of this polypeptide in the preparation of drugs for treating cancer.

[0007] The above objects of the present invention are achieved by the following technical solutions:

[0008] A polypeptide, whose amino acid sequence is as shown in SEQ ID NO.1.

[0009] The application of the polypeptide shown in SEQ ID NO.1 in the preparation of drugs for treating Alzheimer's disease.

[0010] Preferably, the drug is made into a pharmaceutically acceptable dosage form with the polypeptide shown in SEQ ID NO.1 as the active ingredient and through pharmaceutically acceptable carriers or excipients.

[0011] More preferably, the carrier or excipient is solid, liquid or semi-solid.

[0012] More preferably, the dosage form is tablets, capsules or injections.

[0013] Use of the polypeptide shown in SEQ ID NO.1 in the preparation of a medicament for treating cancer; wherein, the cancer is gastric cancer, breast cancer, lung cancer, esophageal cancer or liver cancer.

[0014] Preferably, the cancer is gastric cancer or liver cancer.

[0015] More preferably, the medicament uses the polypeptide shown in SEQ ID NO.1 as the active ingredient and is made into a pharmaceutically acceptable dosage form through a pharmaceutically acceptable carrier or excipient.

[0016] More preferably, the carrier or excipient is solid, liquid or semi-solid.

[0017] More preferably, the dosage form is tablet, capsule or injection.

[0018] Beneficial effects:

[0019] The present invention discloses a polypeptide with the amino acid sequence HGAFTTES (SEQ ID NO.1), and this sequence of polypeptide is reported for the first time. Activity research shows that this polypeptide can effectively inhibit the paralysis of Caenorhabditis elegans in the Alzheimer's disease model and has significant anti-Alzheimer's disease activity. In addition, this polypeptide has obvious inhibitory activity against gastric cancer, breast cancer, lung cancer, esophageal cancer and liver cancer cells, especially extremely strong inhibitory activity against gastric cancer and liver cancer cells. Therefore, the polypeptide with the amino acid sequence HGAFTTES has the prospect of being developed into an anti-Alzheimer's disease or anti-tumor medicament. Description of the drawings

[0020] Figure 1 It is the mass spectrometry detection chart of the polypeptide of SEQ ID NO.1 sequence. Specific implementation manners

[0021] The following specifically introduces the substantial content of the present invention in combination with examples, but does not limit the protection scope of the present invention thereto.

[0022] The polypeptide with the amino acid sequence HGAFTTES (SEQ ID NO.1) was synthesized by Zhengzhou Huazhan Pharmaceutical Technology Co., Ltd. using the conventional solid-phase synthesis method. The theoretical molecular weight is 848.858, and the measured [M+H] by mass spectrometry identification + is 849.90, which is consistent with the theory; the polypeptide purity ≥95% (HPLC method). The polypeptides tested in Example 1 and Example 2 are both this polypeptide.

[0023] Example 1: Influence of the polypeptide on the CL4176 nematode paralysis model

[0024] I. Experimental materials

[0025] MgSO4 was purchased from Shanghai Macklin Biochemical Co., Ltd.; agar powder, yeast powder, cholesterol, tryptone, NaCl, and CaCl2 were all purchased from Beijing Solarbio Science & Technology Co., Ltd.; Na2HPO4·12H2O and KH2PO4 were both purchased from Sinopharm Chemical Reagent Co., Ltd.; the transgenic AD model Caenorhabditis elegans strain CL4176 [dvIs27[myo-3p::A-Beta(1-42)::let-85 1 3'UTR)+rol-6(su1006)]X] and the uracil-deficient Escherichia coli OP50 were both purchased from the Caenorhabditis elegans Genetics Center of the United States.

[0026] II. Solution Preparation

[0027] M9 buffer: Weigh 0.6 g of disodium hydrogen phosphate, 0.3 g of potassium dihydrogen phosphate, 0.5 g of sodium chloride, and 0.025 g of magnesium sulfate (anhydrous), dissolve them in 100 mL of deionized water, and sterilize at high temperature for later use.

[0028] NGM culture plate: Weigh 3.0 g of sodium chloride, 2.5 g of tryptone, and 17 g of agar and place them in a conical flask, add 975 mL of deionized water, and sterilize them together under high pressure. When the temperature of the sterilized solution drops to about 55 °C, add 0.5 mL of 1 M calcium chloride solution, 1 mL of 5 mg / mL cholesterol solution, 1 mL of 1 M magnesium sulfate solution, and 25 mL of potassium phosphate buffer in sequence. Use a pipette to add the prepared culture medium liquid to the corresponding petri dishes or well plates respectively, and let it cool and solidify overnight.

[0029] Mother solution of the polypeptide to be tested: Weigh an appropriate amount of the polypeptide and dissolve it in DMSO to prepare a 10 mM solution.

[0030] III. Experimental Methods

[0031] 1. Modeling and Drug Administration

[0032] Take the mother solution of the polypeptide, dilute it 20 times with M9 buffer, then take 500 μL and evenly coat it on a freshly prepared NGM plate, and let it dry naturally at room temperature. Drop 50 μL of OP50 bacterial solution at the center of the plate and dry it overnight. Inoculate the synchronized Caenorhabditis elegans into the drug-containing plate. At the same time, set up a group without the polypeptide as the model group.

[0033] Cultivate the synchronized Caenorhabditis elegans at 16 °C for 48 h, that is, when the Caenorhabditis elegans grows to the L3 stage, transfer the culture plate to 25 °C to induce the transcriptional expression of the Aβ gene.

[0034] The culture plates were transferred to a culture condition of 25 °C, and the number of paralyzed Caenorhabditis elegans was recorded at 36 h. Among them, the paralyzed Caenorhabditis elegans individuals showed rigidity in the trunk part, resulting in movement disorders or even loss of motor function, and the head could twist.

[0035] 2. Detection indicators

[0036] In the paralysis rate detection experiment, the number of nematode individuals in each group was not less than 30. Each experiment was repeated 3 times. Calculate the nematode paralysis rate at 36 h: nematode paralysis rate % at 36 h = number of paralyzed nematodes counted / total number of nematodes × 100%.

[0037] 3. Statistical analysis

[0038] The data were expressed as mean ± SD, and the t-test was used for comparison between groups. P < 0.05 represented significant difference.

[0039] IV. Experimental results

[0040] The results of the nematode paralysis rate detection at 36 h are shown in Table 1 ( * indicating compared with the model group, ** P < 0.01).

[0041] Table 1 Paralysis rate of nematodes in each group

[0042] sample to be tested Paralysis rate (%) model group 93.96±2.98 polypeptide group <![CDATA[61.21±3.95 ** >

[0043] CL4176 is a strain of nematode that can induce the expression of human Aβ1-42 in muscles and produce a paralyzed phenotype, and is widely used in AD models. The above results show that the polypeptide described in SEQ ID NO.1 can effectively inhibit the paralysis of Caenorhabditis elegans in the AD model, and the paralysis rate is significantly lower than that of the model group, indicating that the polypeptide provided by the present invention has significant anti-AD activity.

[0044] Example 2: Antitumor activity test

[0045] I. Drugs and cells

[0046] Polypeptide stock solution: Weigh the polypeptide shown in SEQ ID NO.1 and prepare a solution with a concentration of 20 mM in DMSO, store it at 4 °C, and dilute it with DMSO according to the required concentration during the experiment.

[0047] Gastric cancer SGC-7901 cells: Cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture at 37 °C and 5% CO2, and logarithmic-phase cells were used for the experiment.

[0048] Breast cancer MCF-7 cells: Cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture at 37 °C and 5% CO2, and logarithmic-phase cells were used for the experiment.

[0049] Lung cancer H460 cells: Cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture at 37°C and 5% CO2. Cells in the logarithmic growth phase were used for experiments.

[0050] Esophageal cancer EC-109 cells: Cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture at 37°C and 5% CO2. Cells in the logarithmic growth phase were used for experiments.

[0051] Hepatocellular carcinoma HepG2 cells: Cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture at 37°C and 5% CO2. Cells in the logarithmic growth phase were used for experiments.

[0052] II. Experimental methods

[0053] Tumor cells in the logarithmic growth phase were digested and counted, and then the cell density was adjusted with the medium. The cells were seeded into 96-well plates at a density of 4000 - 8000 cells / well, 100 μL per well. After culturing for 24 h, the medium was discarded, and the polypeptide drug diluted with the medium was added. Three replicates were set for each concentration, and a blank control group was also set. After the drug acted for 72 h, 20 μL of MTT solution was added to each well. After continuing to culture for 4 h, the liquid was aspirated, 150 μL of DMSO was added, and the mixture was shaken evenly. The absorbance value was measured at 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and the inhibition rate of the polypeptide at different concentrations on different tumor cells was calculated. The calculation formula is as follows:

[0054] Inhibition rate (%) = (1 - absorbance value of the polypeptide group / absorbance value of the blank group) × 100%.

[0055] III. Experimental results

[0056] Based on the inhibition rates of the polypeptide at different concentrations on different tumor cells, the IC 50 value of the polypeptide on different tumor cells was calculated using SPSS software. The experimental results are shown in Table 2.

[0057] Table 2 IC 50 values of the polypeptide on different tumor cells

[0058] tumor cell line tumor type <![CDATA[IC 50 (μM)]]> SGC-7901 gastric cancer 8.25±1.58 MCF-7 breast cancer 13.20±3.25 H460 lung cancer 31.20±4.20 EC-109 esophageal cancer 29.33±6.21 HepG2 liver cancer 2.21±0.95

[0059] The results showed that the polypeptide described in SEQ ID NO.1 had obvious inhibitory activity on gastric cancer, breast cancer, lung cancer, esophageal cancer, and hepatocellular carcinoma cells, especially strong inhibitory activity on gastric cancer and hepatocellular carcinoma cells, and the inhibitory activity on hepatocellular carcinoma cells was the strongest, with an IC 50The value is only about 2 μM. Therefore, the polypeptide described in SEQ ID NO.1 has the prospect of being developed into a drug for treating gastric cancer, breast cancer, lung cancer, esophageal cancer and liver cancer.

[0060] In summary:

[0061] The present invention discloses a polypeptide with an amino acid sequence of HGAFTTES (SEQ ID NO.1), and this sequence of polypeptide is reported for the first time. Activity studies have shown that this polypeptide can effectively inhibit the paralysis of Caenorhabditis elegans in the Alzheimer's disease model, and has significant anti-Alzheimer's disease activity. In addition, this polypeptide has obvious inhibitory activity against gastric cancer, breast cancer, lung cancer, esophageal cancer and liver cancer cells, especially extremely strong inhibitory activity against gastric cancer and liver cancer cells. Therefore, the polypeptide with the amino acid sequence of HGAFTTES has the prospect of being developed into an anti-Alzheimer's disease or anti-tumor drug.

[0062] The role of the above embodiments is to specifically introduce the substantial content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to this specific embodiment.

Claims

1. A polypeptide, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. Use of the polypeptide according to claim 1 in the preparation of a medicament for treating Alzheimer's disease.

3. The application according to claim 2, characterized in that: The medicament uses the polypeptide according to claim 1 as the active ingredient and is made into a pharmaceutically acceptable dosage form through a pharmaceutically acceptable carrier or excipient.

4. The application according to claim 3, characterized in that: The carrier or excipient is solid, liquid or semi-solid.

5. The application according to claim 3, wherein: The dosage form is tablet, capsule or injection.

6. Use of the polypeptide according to claim 1 in the preparation of a medicament for treating cancer; wherein, The cancer is gastric cancer, breast cancer, lung cancer, esophageal cancer or liver cancer.

7. The application according to claim 6, wherein: The cancer is gastric cancer or liver cancer.

8. The application according to claim 6 or 7, characterized in that: The medicament uses the polypeptide according to claim 1 as the active ingredient and is made into a pharmaceutically acceptable dosage form through a pharmaceutically acceptable carrier or excipient.

9. The application according to claim 8, wherein: The carrier or excipient is solid, liquid or semi-solid.

10. The application according to claim 8, characterized in that: The dosage form is tablet, capsule or injection.

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