A polypeptide and use thereof in biopharmaceuticals

Abstract

The application discloses a polypeptide and application thereof in biopharmaceuticals, and relates to a polypeptide with an amino acid sequence of HGAFTTES (SEQ ID NO. 1), which is reported for the first time. Activity research shows that the polypeptide can effectively inhibit paralysis of Caenorhabditis elegans, an Alzheimer's disease model, and has significant anti-Alzheimer's disease activity. In addition, the polypeptide has obvious inhibitory activity on gastric cancer, breast cancer, lung cancer, esophageal cancer and liver cancer cells, and has extremely strong inhibitory activity on gastric cancer and liver cancer cells in particular. Therefore, the polypeptide with the amino acid sequence of HGAFTTES has the prospect of development into an anti-Alzheimer's disease or anti-tumor drug.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of polypeptides, and relates to a novel polypeptide and application, in particular to a polypeptide and application in biopharmaceuticals. BACKGROUND

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

[0003] Dementia is a syndrome with cognitive function decline as the core, mainly manifested as the gradual decline of memory, thinking, language and social ability, affecting the daily life ability of patients. Common types include Alzheimer's disease, vascular dementia and Lewy body dementia, and the global cases of dementia have exceeded 50 million. Alzheimer's disease (AD) is related to the deposition of amyloid plaques (Amyloid β-protein, Aβ) and neurofibrillary tangles, loss of neurons and neuroinflammation. Vascular dementia is mainly caused by cerebrovascular diseases, leading to insufficient blood supply to the brain and affecting cognitive function. Although the current treatment mainly focuses 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 stop the progression of the disease, and there are individual differences in efficacy and side effects, which brings great challenges to the treatment of dementia, and new effective treatment strategies are urgently needed.

[0004] Tumors are formed due to abnormal proliferation of cells, which can be divided into benign tumors and malignant tumors (cancers), the latter of which has invasiveness and metastasis, seriously threatening human health. The occurrence of cancer is usually related to gene mutation, environmental factors and lifestyle choices. In clinical practice, 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, and the damage to normal cells by chemotherapy and radiotherapy and the heterogeneity and drug resistance of tumors make a single treatment regimen often ineffective. In addition, radiotherapy kills cancer cells by high-energy rays, which often needs to be combined with other treatment methods. Targeted therapy is designed according to the specific molecular characteristics of cancer cells, although it improves the accuracy of treatment, but some patients may still face drug resistance due to changes in tumor characteristics. In recent years, immunotherapy has made some significant progress, which activates the patient's own immune system to recognize and attack cancer cells. The success of this method has significantly improved the survival rate of patients with some types of cancer, but not all patients can benefit from it, and still face the problems of side effects and uneven efficacy.

[0005] The applicant has found a new type of polypeptide with excellent activity, and specifically proposes the present invention. SUMMARY

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

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

[0008] A polypeptide with an amino acid sequence as shown in SEQ ID NO. 1.

[0009] The use of the polypeptide as shown in SEQ ID NO. 1 in the preparation of a medicament for treating Alzheimer's disease.

[0010] Preferably, the medicament takes the polypeptide as shown in SEQ ID NO. 1 as the active ingredient, and is prepared into a pharmaceutically acceptable dosage form by a pharmaceutically acceptable carrier or excipient.

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

[0012] More preferably, the dosage form is a tablet, a capsule or an injection.

[0013] The polypeptide shown in SEQ ID NO. 1 is used for preparing a medicine 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 medicine takes the polypeptide shown in SEQ ID NO. 1 as an active ingredient, and is prepared into a pharmaceutically acceptable dosage form by 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 application discloses a polypeptide with an amino acid sequence of HGAFTTES (SEQ ID NO. 1), which is reported for the first time. Activity research shows that the polypeptide can effectively inhibit paralysis of Caenorhabditis elegans model of Alzheimer's disease, and has significant activity of resisting Alzheimer's disease. In addition, the polypeptide has obvious inhibitory activity on gastric cancer, breast cancer, lung cancer, esophageal cancer and liver cancer cells, and has extremely strong inhibitory activity on gastric cancer and liver cancer cells. Therefore, the polypeptide with the amino acid sequence of HGAFTTES has the prospect of developing into an anti-Alzheimer's disease or anti-tumor medicine. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The mass spectrum detection diagram of the polypeptide of SEQ ID NO. 1 sequence. DETAILED DESCRIPTION

[0021] The substantial content of the present application will be specifically introduced below in combination with examples, but the protection scope of the present application is not limited by this.

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

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

[0024] I. Experimental materials

[0025] MgSO4 was purchased from Shanghai Maikelin Biotechnology Co., Ltd.; Agar powder, yeast powder, cholesterol, tryptone, NaCl, CaCl2 were purchased from Beijing Solabio Science and Technology Co., Ltd.; Na2HPO4·12H2O, KH2PO4 were purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.; Transgenic AD model C. elegans strain CL4176 [dvIs27 [myo-3p::A-Beta(1-42)::let-8513'UTR) + rol-6(su1006)] X] and uracil-deficient E. coli OP50 (Escherichia Coli OP50) were purchased from the Caenorhabditis Genetic Center, USA.

[0026] II. Preparation of solution

[0027] M9 buffer solution: 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), and dissolve in 100 mL of deionized water. After high-temperature sterilization, it is ready for use.

[0028] NGM culture plate: weigh 3.0 g of sodium chloride, 2.5 g of tryptone, and 17 g of agar in a conical flask, add 975 mL of deionized water, and sterilize together under high pressure. When the temperature of the sterilized solution decreases to about 55°C, add 0.5 mL of 1M calcium chloride solution, 1 mL of 5 mg / mL cholesterol solution, 1 mL of 1M magnesium sulfate solution, and 25 mL of potassium phosphate buffer solution in sequence. The prepared medium liquid is added to the corresponding flat plate or well plate using a pipette, and cooled and solidified overnight.

[0029] Polypeptide stock solution to be tested: weigh an appropriate amount of polypeptide and dissolve in DMSO to prepare a 10 mM solution.

[0030] III. Experimental method

[0031] 1. Modeling and drug administration

[0032] After the polypeptide stock solution was diluted 20 times with M9 buffer solution, 500 μL was uniformly coated on a freshly prepared NGM plate at room temperature. 50 μL of OP50 bacterial solution was added to the center of the plate and dried overnight. The synchronized C. elegans was inoculated into the drug-containing plate. At the same time, a group without polypeptide was set as the model group.

[0033] The synchronized and treated C. elegans was cultured at 16°C for 48 h, i.e., the C. elegans grew to the L3 stage, and the culture plate was transferred to 25°C to induce the transcription and expression of the Aβ gene.

[0034] The plates are transferred to a 25℃ incubation condition, and the number of paralyzed C. elegans is recorded at 36h. Among them, the paralyzed C. elegans individuals exhibit trunk rigidity, leading to motor impairment or even loss of motor function, and the head can be twisted.

[0035] 2. Detection index

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

[0037] 3. Statistical analysis

[0038] The data are expressed as mean ± SD, and t test is used for comparison between groups, P < 0.05 represents a significant difference.

[0039] Four, experimental results

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

[0041] Table 1: Nematode paralysis rate of each group

[0042] Test sample Paralysis rate (%) Model group 93.96±2.98 Polypeptide group 61.21 ± 3.95 ** ]]

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

[0044] Example 2: Anti-tumor activity test

[0045] I. Drugs and cells

[0046] Polypeptide stock solution: The polypeptide shown in SEQ ID NO. 1 is weighed and prepared into a solution with a concentration of 20mM with DMSO, and stored at 4℃. During the experiment, it is diluted with DMSO according to the required concentration.

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

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

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

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

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

[0052] II. Experimental method

[0053] Take the tumor cells in the logarithmic growth phase, count after digestion, adjust the cell density with the medium, inoculate 4000-8000 cells / well into a 96-well plate, 100 μL per well, culture for 24 h, discard the culture medium, add the polypeptide drugs diluted with the culture medium, set 3 replicate wells for each concentration, and set a blank control group. After 72 h of drug action, add 20 μL of MTT solution to each well, continue to culture for 4 h, remove the liquid, add 150 μL of DMSO, shake uniformly, detect the absorbance value at 490 nm on the microplate reader, calculate the inhibition rate of different concentrations of polypeptides on different tumor cells, and the calculation formula is as follows:

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

[0055] III. Experimental results

[0056] According to the inhibition rate of different concentrations of polypeptides on different tumor cells, the IC 50 values of polypeptides on different tumor cells were calculated by SPSS software. The experimental results are shown in Table 2.

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

[0058] Tumor cell line Tumor type 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 Hepatocellular carcinoma 2.21±0.95

[0059] The results show that the polypeptide described in SEQ ID NO. 1 has obvious inhibitory activity on gastric cancer, breast cancer, lung cancer, esophageal cancer and hepatocellular carcinoma cells, especially on gastric cancer and hepatocellular carcinoma cells, and the inhibition on hepatocellular carcinoma cells is the strongest, with an IC 50The value is only about 2 μM. Therefore, the polypeptide described by SEQ ID NO. 1 has the prospect of developing into an anti-gastric cancer, breast cancer, lung cancer, esophageal cancer and liver cancer drug.

[0060] From the above, it can be seen that:

[0061] The application discloses a polypeptide with an amino acid sequence of HGAFTTES (SEQ ID NO. 1), which is reported for the first time. Activity research shows that the polypeptide can effectively inhibit paralysis of a Caenorhabditis elegans model of Alzheimer's disease, and has significant anti-Alzheimer's disease activity. In addition, the polypeptide has obvious inhibitory activity on gastric cancer, breast cancer, lung cancer, esophageal cancer and liver cancer cells, and has extremely strong inhibitory activity on gastric cancer and liver cancer cells in particular. Therefore, the polypeptide with the amino acid sequence of HGAFTTES has the prospect of developing into an anti-Alzheimer's disease or anti-tumor drug.

[0062] The above examples serve to specifically introduce the essential content of the application, but those skilled in the art should know that the protection scope of the application should not be limited to the specific examples.

Claims

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

1.

2. The use of the polypeptide according to claim 1 in the preparation of a medicament for treating cancer; wherein, The cancers mentioned are stomach cancer, breast cancer, lung cancer, esophageal cancer, or liver cancer.

3. The application according to claim 2, characterized in that: The cancer in question is either stomach cancer or liver cancer.

4. The application according to claim 2 or 3, characterized in that: The drug uses the polypeptide of claim 1 as the active ingredient and is formulated into a pharmaceutically acceptable dosage form using a pharmaceutically acceptable carrier or excipient.

5. The application according to claim 4, characterized in that: The carrier or auxiliary material is a solid, liquid or semi-solid.

6. The application according to claim 4, characterized in that: The dosage form is tablet, capsule, or injection.

Images