Active polypeptide as well as preparation method and application thereof
The active peptides prepared by solid-phase synthesis target and inhibit the growth of renal cell carcinoma cells, solving the problems of drug resistance and limited efficacy of existing renal cell carcinoma treatments, and achieving a specific inhibitory effect on renal cell carcinoma cells.
Patent Information
- Application Number
- CN202511327929.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-17
- Publication Date
- 2025-12-12
AI Technical Summary
Current treatment options for kidney cancer suffer from problems such as the tendency for targeted drugs to induce drug resistance, low response rates to immunotherapy, and limited efficacy against certain subtypes of kidney cancer, resulting in a lack of targeted treatment methods.
A bioactive peptide was developed and prepared by solid-phase synthesis with the amino acid sequence NVNSIGLSYTLQGTKKLGPI. This peptide was used to target and inhibit the growth of renal cell carcinoma cells and was prepared into dosage forms such as oral liquids, capsules, and pills for application in pharmaceuticals, food, and daily chemical products.
It significantly inhibits the growth of human kidney tumor-associated cells, has a therapeutic effect on kidney cancer, especially on human kidney cancer cells and human kidney clear cell adenocarcinoma cells, and has no effect on normal cells.
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Figure CN121108261A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of protein technology, and particularly relates to an active polypeptide and a preparation method and application thereof. BACKGROUND
[0002] Renal cancer is a malignant tumor originating from the renal tubular epithelial system of the kidney, also known as renal cell carcinoma, and is one of the most common malignant tumors in the urinary system, with an increasing trend in the incidence worldwide. According to statistics, renal cancer accounts for about 2%-3% of adult malignant tumors, and clear cell renal carcinoma (ccRCC) is the most common pathological subtype, accounting for about 70%-80% of all renal cancer cases.
[0003] At present, the clinical treatment methods for renal cancer mainly include surgical resection, targeted therapy, immunotherapy and chemotherapy. For early-stage localized renal cancer, surgical resection is the preferred treatment option, but the postoperative recurrence rate is high, and about 30% of patients have distant metastasis at the time of initial diagnosis, losing the opportunity for radical surgery. For advanced or metastatic renal cancer, targeted therapy (such as inhibitors targeting VEGF / VEGFR pathway, mTOR inhibitors, etc.) and immune checkpoint inhibitors (such as PD-1 / PD-L1 antibodies) have become first-line treatment options, significantly improving patient survival. However, there are still many problems to be solved in clinical practice: (1) Targeted drugs are prone to cause drug resistance, and most patients develop disease progression after 6-12 months of treatment; (2) The response rate of immunotherapy is only 20%-30%, and there is a risk of immune-related adverse reactions; (3) The existing treatment options have limited efficacy for some special subtypes of renal cancer (such as collecting duct carcinoma and chromophobe carcinoma), and lack of targeted treatment options.
[0004] Therefore, it is an urgent problem for those skilled in the art to develop a new polypeptide that can inhibit the regeneration of renal cancer cells. SUMMARY
[0005] The present application aims to overcome the shortcomings of the prior art and provide an active polypeptide that can inhibit the growth of renal cancer cells, as well as a preparation method and application thereof.
[0006] To achieve the above-mentioned purpose, the technical solution adopted by the present application is as follows:
[0007] In a first aspect, the present application provides an active polypeptide, the amino acid sequence of which is shown in SEQ ID NO. 1. The present application has been confirmed by experiments that the active polypeptide can target the kidney organ and significantly inhibit tumor cell growth, indicating that the active polypeptide can be applied in the development of drugs for preventing or treating renal cancer.
[0008] As a preferred embodiment of the active polypeptide of the present application, the active polypeptide is mainly prepared by solid-phase synthesis.
[0009] As a preferred embodiment of the active polypeptide of the present application, the solid phase synthesis method comprises the following steps:
[0010] 1. Taking chlorotrityl chloride resin as a starting material, taking 9-fluorenylmethoxycarbonyl-protected amino acid as a monomer, and sequentially condensing amino acids according to the amino acid sequence of SEQ ID NO. 1 from the C-terminal to the N-terminal to synthesize polypeptides;
[0011] 2. Adding a cleavage reagent to the system after the synthesis in step 1 is completed, so as to cleave the polypeptide from the resin to obtain a crude polypeptide;
[0012] 3. Precipitating or nitrogen blowing the crude polypeptide in step 2 with ether, and then purifying the active polypeptide by preparative HPLC.
[0013] In a second aspect, the present application provides an application of the active polypeptide in the preparation of a product for preventing and / or treating kidney cancer.
[0014] As a preferred embodiment of the application, the dosage form of the product includes but is not limited to at least one of oral liquid, capsule, pill, decoction, tablet, soft capsule, granule and powder.
[0015] As a preferred embodiment of the application, the product includes but is not limited to at least one of a pharmaceutical product, a food product and a daily chemical product.
[0016] In a third aspect, the present application provides an application of the active polypeptide in the preparation of a pharmaceutical product for inhibiting the growth of kidney cancer cells.
[0017] As a preferred embodiment of the application, the kidney cancer cells include but are not limited to human kidney cancer cells and / or human renal clear cell adenocarcinoma cells.
[0018] As a preferred embodiment of the application, the human kidney cancer cells include A-498 cells and / or SN12C cells, and the human renal clear cell adenocarcinoma cells include 786-O cells.
[0019] Compared with the prior art, the present application has the following beneficial effects:
[0020] The present application proves by cell experiments that the active polypeptide has the effect of inhibiting the growth of human kidney tumor-related cells, thereby playing the effect of treating kidney cancer. BRIEF DESCRIPTION OF DRAWINGS
[0021] Figure 1 It is a liquid chromatogram of the active polypeptide in Example 1 of the present application;
[0022] Figure 2 It is a mass spectrum of the active polypeptide in Example 1 of the present application;
[0023] Figure 3 The test results of the influence of the active polypeptide in the effect example 1 of the present application on the growth of different cells are shown in the table, wherein "*" indicates that there is a significant difference between the two groups (P<0.05), and "nd" indicates that there is no significant difference between the two groups (P>0.05). DETAILED DESCRIPTION
[0024] For the purpose of better illustrating the object, technical scheme and advantages of the present application, the present application will be further described in combination with specific examples.
[0025] The other materials, reagents and the like used in the examples, comparative examples and effect examples can be obtained from commercial channels unless otherwise specified.
[0026] In the following examples and effect examples, DCM is dichloromethane; HBTU is benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate; DIEA is N,N-diisopropyl ethylamine; DMF is N,N-dimethylformamide; TFA is trifluoroacetic acid; EDT is 1,2-ethanedithiol; Fmoc is 9-fluorenylmethoxycarbonyl; and TIS is triisopropylsilane.
[0027] The cutting solution in the following example 1 includes the following reagents in mass percentage: TFA 95%, water 1%, EDT 2% and TIS 2%.
[0028] Example 1
[0029] Example 1 provides an active polypeptide and a preparation method thereof, wherein the amino acid sequence of the active polypeptide is NVNSIGLSYTLQGTKKLGPI (SEQ ID NO. 1), and the preparation method comprises the following steps:
[0030] S1, 3g of chlorotrityl chloride resin (degree of substitution is 1.03mmol / g) is placed in a reaction tube, 45mL of DCM is added, and shaken for 30min;
[0031] S2, the solvent is removed by sand core suction filtration, 10mmol of Fmoc-Gly amino acid is added, dissolved in DMF, and then 30mmol of DIEA is added, and shaken for 60min;
[0032] S3, remove the DMF, add 45mL of 20% piperidine DMF solution, react for 5min; remove and add 20% piperidine DMF solution again, for 15min;
[0033] S4, wash twice with 30mL of DMF, then wash twice with 30mL of DCM, and finally wash twice with 30mL of DMF;
[0034] S5, protecting amino acid 12 mmol, HBTU 13 mmol, dissolved in 15 mL of DMF, added to the reaction tube, and immediately added DIEA 13 mmol, reacted for 30 min, washed twice according to the operation of step S4;
[0035] S6, repeat steps S3-S5, sequentially connect the amino acids in the sequence described in SEQ ID NO. 1 from right to left until the end;
[0036] S7, wash twice with 30 mL of DMF, wash twice with 30 mL of methanol, wash twice with 30 mL of DMF; wash twice with 30 mL of DCM, dry for 10 min;
[0037] S8, add 30 cleavage solution and cut for 120 min, dry the cleaved solution with nitrogen, wash with ether 6 times, dry at room temperature, purify the crude product with preparative HPLC chromatography to obtain the active polypeptide.
[0038] The active polypeptide obtained above is quantitatively and qualitatively analyzed by liquid chromatography and mass spectrometry:
[0039] 1. The conditions of liquid chromatography are as follows:
[0040] Mobile phase A: 0.1% TFA aqueous solution; mobile phase B: 0.1% TFA acetonitrile solution;
[0041] Gradient: 20%-35% mobile phase B, 10 min;
[0042] Chromatographic column: Kromasil 100-5 C18 4.6x300mm (5um);
[0043] Wavelength: 220 nm;
[0044] Flow rate: 1.0 mL / min;
[0045] The obtained liquid chromatogram is shown in Figure 1 , and the purity of the active polypeptide is measured to be 98.42%.
[0046] 2. The mass spectrum of electrospray mass spectrometry of the above active polypeptide is shown in Figure 2 , the molecular ion peak of the obtained active polypeptide is 2103.47 m / z, which is close to the predicted molecular weight, indicating that the amino acid sequence of the active polypeptide prepared in Example 1 is the same as the sequence described in SEQ ID NO. 1.
[0047] Effect example 1
[0048] In order to verify the efficacy of the active polypeptide of the application in treating kidney cancer, the active polypeptide obtained in Example 1 is subjected to cell experiment, and the specific scheme is as follows:
[0049] 1. The effect of the active polypeptide on the growth of human renal clear cell adenocarcinoma cell 786-O (hereinafter referred to as 786-O cell).
[0050] The active polypeptide obtained in Example 1 was dissolved in RPMI-1640 culture solution to obtain a solution containing 100 μg of the active polypeptide per 1 mL. The 786-O cells were cultured in 10 v / v% calf serum culture solution to the logarithmic growth phase, and the logarithmic growth phase 786-O cells were digested with 0.25% trypsin-EDTA disodium solution and diluted to contain 2.5 x 10 4 ~ 5 x 10 4 ~ 6 cells per 1 mL with 10 v / v% calf serum culture solution, and the cell suspension was plated on a 96-well cell plate at 100 μL per well.
[0051] The test sample group was added with 100 μL of the test sample solution per well, and 3 wells were prepared for each batch of the test sample. The cell control group was added with 100 μL of RPMI-1640 culture solution per well, and was placed in a 37°C incubator containing 5% saturated carbon dioxide for 48 h. Four hours before the end of the culture, the culture plate was taken out, and the culture solution was aspirated. Each well was washed once with 0.01 mol / L phosphate buffer (pH = 7.3), and then 100 μL of the above phosphate buffer (pH 7.3) and 20 μL of MTT solution were added to each well, and the culture was continued.
[0052] After the culture was completed, the culture solution was aspirated, 100 μL of dimethyl sulfoxide was added to each well, and the mixture was shaken. The absorbance was measured at 550 nm on an enzyme marker instrument, and the results are shown in Figure 3 .
[0053] 2. The effect of the active polypeptide on the growth of human embryonic kidney cell 293T, human gastric cancer cell NCI-N87 and breast cell MCF-10A, and the test method was the same as that of the above 786-O cell, and the results are shown in Figure 3 .
[0054] 3. The effect of the active polypeptide on the growth of human renal cancer cell A498 and SN12C, and the test method was the same as that of the above 786-O cell, and the results are shown in Figure 3 .
[0055] As shown in Figure 3 , the active polypeptide of the present application can inhibit the growth of human renal clear cell adenocarcinoma cells and human renal cancer cells, and has no effect on the growth of normal kidney, breast cells and human gastric cancer cells, indicating that the active polypeptide of the present application has the potential to target and inhibit renal cancer, and can specifically target renal cancer cells to inhibit their growth.
[0056] Finally, it should be noted that the above examples are merely intended to illustrate the technical solutions of the present application and not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present application.
Claims
1. An active polypeptide, characterized in that, The amino acid sequence of the active polypeptide is shown in SEQ ID NO.
1.
2. The active polypeptide according to claim 1, characterized in that, The active polypeptides are mainly prepared by solid-phase synthesis.
3. The use of the active polypeptide as described in claim 1 in the preparation of products for the prevention and / or treatment of renal cancer.
4. The application as described in claim 3, characterized in that, The dosage form of the product includes at least one of oral liquid, capsule, pill, decoction, tablet, soft capsule, granule and powder.
5. The application as described in claim 3, characterized in that, The product includes at least one of pharmaceuticals, food, and daily chemical products.
6. The use of the active polypeptide as described in claim 1 in the preparation of a drug for inhibiting the growth of renal cell carcinoma cells.
7. The application as described in claim 6, characterized in that, The renal cell carcinomas include human renal cell carcinomas and / or human clear cell adenocarcinomas.
8. The application as described in claim 7, characterized in that, The human renal cell carcinoma cells include A-498 cells and / or SN12C cells, and the human renal clear cell adenocarcinoma cells include 786-O cells.