A polypeptide and uses thereof
By preparing the peptide TCL6148, the problem of limited efficacy in existing treatments for renal cell carcinoma was solved, and the proliferation and migration of renal cell carcinoma cells were inhibited, providing a new therapeutic target and drug strategy.
Patent Information
- Application Number
- CN202510244226.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-03
- Publication Date
- 2025-12-12
- Estimated Expiration
- 2045-03-03
AI Technical Summary
Current treatments for renal cell carcinoma have limited effectiveness, particularly in addressing its tendency to metastasize, develop drug resistance, and relapse, where effective treatment strategies are lacking.
The polypeptide TCL6148, prepared by solid-phase synthesis, was used to prepare an anti-renal cell carcinoma drug to inhibit the proliferation and migration of renal cell carcinoma cells.
The peptide TCL6148 can significantly inhibit the proliferation and migration of renal cell carcinoma cells, providing a new target and drug strategy for the treatment of renal cell carcinoma, with low toxicity and side effects.
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Figure CN119978073B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biological medicine, and particularly relates to a polypeptide and application thereof. BACKGROUND
[0002] Renal cell carcinoma is the most common primary renal malignancy, accounting for about 2-3% of all malignant tumors worldwide, and is also one of the most common and deadly urogenital cancers, with a high incidence and mortality. In China, there are about 70,000 new cases of renal cell carcinoma and 20,000 deaths related to renal cell carcinoma each year. At present, the treatment strategies for renal cell carcinoma mainly include surgical treatment, tyrosine kinase inhibitor targeted therapy, immune checkpoint inhibitor immunotherapy and cell therapy. However, the characteristics of RCC such as metastasis, drug resistance and recurrence limit the effectiveness of these treatments. Therefore, it is urgent to explore new treatment strategies to alleviate the malignant progression of patients with advanced renal cell carcinoma.
[0003] As a new biological treatment strategy, polypeptide drugs have high specificity and targeting, and can target receptors and molecules specific to cancer cells. The advantages of polypeptide drugs include relatively low toxicity, side effects and good biocompatibility. Polypeptides can inhibit tumor cell proliferation, promote apoptosis and regulate tumor microenvironment, thereby exerting anticancer effects. Therefore, developing polypeptide drugs for renal cell carcinoma has become an important research direction. SUMMARY
[0004] The present application aims at the deficiencies in the prior art, and provides a polypeptide and application thereof.
[0005] To achieve the above object, the technical scheme adopted by the present application is as follows:
[0006] The first aspect is to provide a polypeptide TCL6148, which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1.
[0007] RKKRRQRRR-MCLSFRFSFELAVTPLQFLH (SEQ ID NO: 1).
[0008] Further, the polypeptide TCL6148 is prepared by solid-phase synthesis.
[0009] The second aspect is to provide the application of the above polypeptide TCL6148 in the preparation of an anti-renal cell carcinoma drug.
[0010] Further, the anti-renal cell carcinoma drug inhibits the proliferation and migration of renal cell carcinoma cells; the renal cell carcinoma cells are human renal carcinoma cells 786-O.
[0011] A third aspect is to provide a pharmaceutical composition, wherein the effective component contains the polypeptide TCL6148 as described above.
[0012] Compared with the prior art, the polypeptide TCL6148 has the following technical effects:
[0013] The polypeptide TCL6148 is prepared by the solid-phase synthesis method, and it is first proved that the polypeptide TCL6148 can inhibit the proliferation and migration of renal cell carcinoma cells, thereby providing a new target and a new drug strategy for treating renal cell carcinoma. BRIEF DESCRIPTION OF DRAWINGS
[0014] Figure 1 The HPLC and protein spectrum detection maps of the polypeptide TCL6148 are shown.
[0015] Figure 2 The effect of the polypeptide TCL6148 on the cell viability of renal cancer cells 786-O after 24h of treatment is shown.
[0016] Figure 3 The effect of the polypeptide TCL6148 on the cell proliferation of renal cancer cells 786-O after 24h of treatment is shown.
[0017] Figure 4 The effect of the polypeptide TCL6148 on the cell migration of renal cancer cells 786-O after 24h of treatment is shown. DETAILED DESCRIPTION
[0018] The present application will be further described below in combination with the drawings and specific examples, but is not limited by the examples and the features in the examples can be combined with each other without conflict.
[0019] Example 1
[0020] The present embodiment provides a preparation method of the polypeptide TCL6148, and the specific operation steps are as follows:
[0021] 1) Take 10 grams of chloromethyl resin and place it in a polypeptide synthesis tube, then add 100 milliliters of dichloromethane (DCM) for 60 minutes of swelling treatment; after the swelling is completed, perform suction filtration to remove the solvent, then wash with 100 milliliters of N,N-dimethylformamide (DMF) three times, and finally perform suction filtration and dry.
[0022] 2) Measure L-histidine (His) to ensure that the molar ratio of His to resin is 6:1, and dissolve it in DMF; at the same time, weigh diisopropylethylamine (DIEA) to make the molar ratio of DIEA to resin 8:1; add the two substances to the polypeptide synthesis tube and shake overnight.
[0023] 3) After the reaction overnight, the system was washed with 100 ml of DMF for three times; then, methanol was added to close the active site of the chloromethyl resin.
[0024] 4) After the completion of the closing step, the system was washed with 100 ml of DMF for four times, and Fmoc-protecting group was added in two of the washing processes to play a protective role.
[0025] 5) After the removal of the protecting group, leucine (Leu) was weighed and the molar mass ratio of leucine to resin was 6:1; at the same time, the condensing reagent was also weighed and the molar mass ratio of the condensing reagent to resin was 6:1; both were dissolved in DMF and DIEA was added, and the reaction was carried out in the synthesis tube for 40 minutes; then, the system was washed with 100 ml of DMF for three times and dried by suction filtration.
[0026] 6) The operation procedures of steps 4) and 5) were repeated until the last amino acid (Met) was added; then, the protecting group Fmoc was removed, the system was washed with 100 ml of DMF for three times and dried by suction filtration to obtain the dried peptide resin.
[0027] 7) 20 ml of cleavage reagent was added to each gram of dried peptide resin, and the reaction was carried out at room temperature for 3 hours.
[0028] 8) After the reaction was completed, the system was suction filtered, the filtrate was collected and transferred to an EP tube; then, 10 times the volume of the filtrate of ether was added to precipitate the polypeptide; the precipitate was separated by centrifugation and the supernatant was discarded; this process was repeated three times to obtain the crude polypeptide.
[0029] 9) The crude polypeptide was purified by high performance liquid chromatography (HPLC), and then freeze-dried and weighed.
[0030] The purity of the anti-renal cell carcinoma polypeptide TCL6148 was more than 95% by HPLC analysis, and the structure was identified by electrospray ionization mass spectrometry (ESI-MS), and the results are shown in Figure 1 .
[0031] Example 2
[0032] This example explores the effect of polypeptide TCL6148 on the viability of human renal cell carcinoma 786-O cells, and the specific experimental steps and results are as follows:
[0033] First, the 786-O cells in the logarithmic growth phase were digested with trypsin. After the cells were separated from the bottom of the culture dish, the digestion process was stopped by adding culture medium. Then, the digested cell suspension was collected into a centrifuge tube and centrifuged at 1000 rpm for 3 minutes. After centrifugation, the supernatant was removed, the cell pellet was resuspended with culture medium, and the cell density was diluted to 5*10 3ml. Subsequently, the cell suspension was dispensed into a 96-well plate at 100 μl per well, and incubated for 24 hours in a constant temperature incubator at 37°C and 5% CO2.
[0034] After the incubation, the old culture medium in the 96-well plate was removed and replaced with fresh culture medium containing different concentrations of polypeptide TCL6148, and the 786-O cells were further incubated for 24 hours.
[0035] After the incubation, 10 μl of CCK8 reagent was added to each well of the 96-well plate, and incubated at 37°C for 1 hour.
[0036] The absorbance value of each well was measured at a wavelength of 450 nm using a microplate reader, and the data was analyzed to calculate the cell survival rate of the renal cell carcinoma cells.
[0037] The results, as shown in Figure 2 , showed that as the concentration of polypeptide TCL6148 increased, the cell survival rate gradually decreased, indicating that polypeptide TCL6148 had a significant inhibitory effect on the cell viability of renal cell carcinoma cells.
[0038] Example 3
[0039] This example investigates the effect of polypeptide TCL6148 on the proliferation of human renal cell carcinoma cells 786-O cells. The specific experimental steps and results are as follows:
[0040] First, the 786-O cells in the logarithmic growth phase were digested with trypsin. After the cells were separated from the bottom of the culture dish, medium was immediately added to stop the digestion process. Then, the digested cell suspension was collected into a centrifuge tube and centrifuged at 1000 rpm for 3 minutes. After centrifugation, the supernatant was removed, the cell pellet was resuspended with medium, and the cell density was diluted to 1*10 3 ml. Subsequently, the cell suspension was dispensed into a 96-well plate at 100 μl per well, and incubated for 24 hours in a constant temperature incubator at 37°C and 5% CO2.
[0041] After the incubation, the old culture medium in the 96-well plate was removed and replaced with fresh culture medium containing different concentrations of polypeptide TCL6148, and the 786-O cells were further incubated for 24 hours.
[0042] After the incubation, the old culture medium in the 96-well plate was removed and replaced with fresh culture medium containing 10 μM EdU reagent, and the 786-O cells were incubated for 6 hours.
[0043] 6 hours later, remove the culture medium, fix the cells with 4% paraformaldehyde for 15 minutes, then wash the cells with PBS solution containing 3% BSA for 3 times. Permeabilize the cells with Triton X-100 for 10 minutes, then wash the cells with PBS solution containing 3% BSA for 3 times.
[0044] Incubate the cells with EdU staining reagent at room temperature for 30 minutes in the dark. After 30 minutes, wash the cells with PBS solution containing 3% BSA for 3 times.
[0045] Incubate the cells with DAPI for 5 minutes, then wash the cells with PBS for 3 times.
[0046] Take pictures of blue light (DAPI) and green light (EdU) respectively using a fluorescence microscope, then analyze the data and calculate the cell proliferation rate of renal cell carcinoma cells.
[0047] The results are shown in Figure 3 As the concentration of polypeptide TCL6148 increases, the cell proliferation rate gradually decreases. This indicates that polypeptide TCL6148 has an inhibitory effect on the proliferation of renal cell carcinoma cells.
[0048] Example 4
[0049] This example investigates the effect of polypeptide TCL6148 on the migration of human renal cell carcinoma 786-O cells. The specific experimental steps and results are as follows:
[0050] First, the 786-O cells in the logarithmic growth phase are digested with trypsin. After the cells are separated from the bottom of the culture dish, add culture medium to stop the digestion process. Then, collect the digested cell suspension into a centrifuge tube and centrifuge at 1000 rpm for 3 minutes. After centrifugation, remove the supernatant, resuspend the cell pellet with culture medium, and distribute the cell suspension into 6-well plates. Incubate in a constant temperature incubator at 37°C, 5% CO2 for 24 hours.
[0051] When the cell density just covers the bottom of the well, remove the old culture medium from the 6-well plate, draw a vertical line on the bottom of the well with a 200 μl sterile gun tip, then wash the detached cell debris with PBS, add culture medium containing different concentrations of polypeptide TCL6148 to the 6-well plate, and take pictures of the cell scratch using a microscope.
[0052] After 24 hours, remove the old culture medium from the 6-well plate, wash the detached cell debris with PBS, and take pictures of the cell scratch again using a microscope. Analyze the changes in the scratch between the two photographs, and calculate the migration rate of the cells.
[0053] The results are shown in Figure 4As shown, with the increase of the concentration of polypeptide TCL6148, the cell scratch healing area gradually decreased. It showed that polypeptide TCL6148 had inhibitory effect on the migration ability of renal cell carcinoma cells.
[0054] The above merely describes preferred embodiments of the present application, and is not intended to limit the embodiments and protection scope of the present application. It should be noted by those skilled in the art that any equivalent substitution and obvious change made according to the content of the present application and the drawings should be included in the protection scope of the present application.
Claims
1. A polypeptide TCL6148 characterized in that, The polypeptide TCL6148 is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:
1.
2. The polypeptide TCL6148 according to claim 1, characterized in that, The polypeptide TCL6148 is prepared by solid phase synthesis.
3. Use of the polypeptide TCL6148 according to any one of claims 1-2 in the preparation of a drug for treating renal cell carcinoma.
4. Use according to claim 3, characterized in that, The drug for treating renal cell carcinoma inhibits the proliferation and migration of renal cell carcinoma cells.
5. Use according to claim 4, characterized in that, The renal cell carcinoma cells are human renal carcinoma cells 786-O.
6. A pharmaceutical composition, characterized by, The effective component in the pharmaceutical composition comprises the polypeptide TCL6148 according to claim 1.