Anti-tumor polypeptide HDP21, and preparation method and application thereof

Abstract

The application provides an antitumor polypeptide HDP21 and a preparation method and application thereof. The antitumor polypeptide is selected from the following: (1) a polypeptide having the amino acid sequence shown in SEQ ID No. 1; and (2) a polypeptide having at least 70% homology with the amino acid sequence shown in SEQ ID No. 1 and having the same or similar function. The novel polypeptide HDP21 provided by the application can efficiently penetrate cells, induce renal tumor cell death, has less toxicity to normal cells, can obviously inhibit the proliferation and migration ability of tumor cells, and can inhibit the growth of tumors, thereby providing a new strategy for preparing a drug for treating tumors, in particular, renal tumors.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an anti-tumor polypeptide HDP21, its preparation method, and its applications. Background Technology

[0002] Renal cell carcinoma (RCC) is a heterogeneous tumor originating from renal tubular epithelial cells. The male-to-female ratio is approximately 2:1, with a peak age of onset between 60 and 70 years. Globally, nearly 295,000 new cases of RCC are diagnosed annually, accounting for about 2% of all cancers. The main subtypes of RCC include clear cell renal cell carcinoma, papillary renal cell carcinoma, chromophobe renal cell carcinoma, and collecting duct carcinoma. Clear cell renal cell carcinoma is the most common histological subtype of RCC, predominating in both in situ and metastatic RCC. Approximately 30% of patients with clear cell renal cell carcinoma experience recurrence and metastasis after nephrectomy, eventually leading to disease progression. Advanced and metastatic RCC has a high mortality rate, imposing a heavy psychological and economic burden on patients and their families.

[0003] Research on peptide drugs has a 100-year history, dating back to the first medical use of insulin in 1922. In 1963, the invention of solid-phase peptide synthesis technology, coupled with the development of purification techniques such as high-performance liquid chromatography (HPLC), propelled peptide research onto a fast track. From initial hormone agonist peptides to bioprobes, peptides with diverse and rich biological functions have been discovered or invented and ultimately applied clinically. As a highly efficient, low-toxicity, and highly selective biological medium, peptides have broad application prospects. Currently, there are more than 80 peptide drugs on the global market, and over 400 peptides are in preclinical research stages. Through breakthroughs and innovations in several key technologies, peptide research and development has formed a mature system. For example, gene recombination technology can regulate the absorption, distribution, metabolism, and excretion characteristics of peptides by altering individual amino acids, thereby improving the efficacy of peptide drugs. Animal venom contains abundant bioactive peptides. Since animals have many receptors similar to those in humans, some animal-derived bioactive peptides have higher selectivity, stronger efficacy, and in vivo stability. Integrative venomomics has emerged to address this need. By using liquid chromatography-tandem mass spectrometry, peptide data from venom can be collected rapidly and efficiently, forming a vast natural peptide library, which provides a foundation for subsequent peptide research and development.

[0004] Peptides possess attractive pharmacological effects and have been extensively studied in cancer treatment. Peptides can promote biological functions crucial for cancer therapy, including targeted delivery, enhanced tumor penetration, endogenous enzyme reactivity, and biocatalysis. Therapeutic peptides can exert specific effects, such as targeting different cell types, signaling pathways, or tumor suppressor proteins, killing discrete cell populations while minimizing damage to healthy cells. Other peptides can induce cell death through membrane-degrading activity. In addition to therapeutic peptides, peptide antigen vaccines can also induce antigen-specific T lymphocyte responses, targeting and destroying tumor cells, representing an effective strategy for cancer immunotherapy. For example, the peptide Aurein 1.2 (GLFDIIKKIAESF) is a peptide isolated from the granular dorsal gland of the green bell frog and the golden bell frog. It is both an antibacterial peptide with broad antibacterial activity and an antitumor peptide, exhibiting high activity against a wide range of cancer cell lines in vitro and low cytotoxicity against normal cells. Scientists from the Korea Advanced Institute of Science and Technology (KAIST) have developed an antitumor peptide, Buforin IIb, derived from histone H2A. Through interaction with surface gangliosides, it crosses the cell membrane without compromising cell membrane integrity, inducing mitochondrial-dependent apoptosis and showing selective cytotoxicity against 62 cancer cell lines. The high selectivity and potency of peptides make them superior antitumor drugs compared to traditional chemotherapeutic agents, possessing significant value for clinical cancer research and promising broad application prospects.

[0005] Currently, targeted therapy combined with immunotherapy is the main treatment for metastatic clear cell renal cell carcinoma because it exhibits high tolerance to conventional chemotherapy and radiotherapy. Given the highly vascular nature of renal cell carcinoma, targeted therapies primarily target the VEGF, PDGFR, and mTOR signaling axes. However, these therapies are accompanied by certain side effects; for example, sunitinib can cause diarrhea, hand-foot syndrome, and hypertension, while bevacizumab is associated with proteinuria, hypertension, and bleeding. Although increased target specificity reduces the risk of drug-related toxicities, resistance to targeted therapies in renal cell carcinoma remains a problem. In the treatment of clear cell renal cell carcinoma, limited target availability leaves unresponsive patients with virtually no other treatment options. Therefore, the search for and development of safe, effective drugs with novel mechanisms of action is a major research direction and hot topic for the future.

[0006] Peptides possess immense therapeutic potential as anti-tumor drugs, but they also face certain limitations. Peptides have poor membrane permeability, hindering their ability to enter cells and exert their biological functions. While most anti-tumor peptides exhibit membrane-dissolving properties, capable of disrupting tumor cell membranes, high doses of these membrane-dissolving anti-tumor peptides may be toxic to healthy mammalian cells, with some also exhibiting severe hemolytic side effects. Therefore, there is a need to construct novel peptides that can efficiently enter cells, inhibit tumor cells, and exhibit low toxicity to normal cells. Summary of the Invention

[0007] To overcome the shortcomings of the prior art, the present invention provides an anti-tumor polypeptide HDP21, its preparation method, and its application.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] The first aspect of the present invention is to provide an antitumor polypeptide HDP21, selected from:

[0010] (1) A polypeptide having the amino acid sequence shown in SEQ ID No. 1; and

[0011] (2) A polypeptide that has at least 70% homology with the amino acid sequence shown in SEQ ID No.1 and has the same or similar function.

[0012] Furthermore, the aforementioned antitumor polypeptide is a polypeptide that has at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% homology with the amino acid sequence shown in SEQ ID No. 1, and has the same or similar function.

[0013] Furthermore, the amino acid sequence of the above-mentioned antitumor polypeptide is SEQ ID No. 1.

[0014] A second aspect of the present invention is to provide a method for preparing the above-mentioned antitumor polypeptide, comprising the step of synthesizing the antitumor polypeptide using a solid-phase synthesis method.

[0015] Furthermore, the above preparation method includes the following steps:

[0016] (1) Resin swelling: weigh the chlorinated resin, add DMF / DCM to swell for 30-60 minutes, and wash with DMF;

[0017] (2) To add the first amino acid: Dissolve 4-6 times the molar excess of Ala in DMF, transfer 8-10 times the molar excess of DIEA to the polypeptide synthesis tube, and shake to react overnight;

[0018] (3) Blocking: After the reaction overnight, wash with DMF and add methanol to block the resin;

[0019] (4) Deprotection: After sealing the resin, wash with DMF, add piperidine to deprotect twice, and wash with DMF in between;

[0020] (5) Next amino acid Ser: After deprotection, weigh 4-6 times the molar excess of Ser and 4-6 times the molar excess of condensation reagent, dissolve them in DMF, and add 8-10 times the molar excess of DIEA to react;

[0021] (6) Remaining amino acid coupling: Repeat steps (4) and (5) until the last amino acid is coupled and deprotected;

[0022] (7) Crude peptides are cut, chromatographically analyzed and purified.

[0023] A third aspect of the present invention is to provide the use of the above-mentioned antitumor polypeptide in the preparation of an antitumor drug, the drug comprising the above-mentioned antitumor polypeptide and a pharmaceutically acceptable carrier or excipient thereof.

[0024] Furthermore, the aforementioned tumor is a kidney tumor.

[0025] A fourth aspect of the present invention is to provide an antitumor pharmaceutical composition comprising the above-described antitumor polypeptide and a pharmaceutically acceptable carrier or excipient thereof.

[0026] Furthermore, the aforementioned tumor is a kidney tumor.

[0027] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0028] The novel polypeptide HDP21 provided by this invention can efficiently penetrate cells, induce kidney tumor cell death, and has low toxicity to normal cells. It can also significantly inhibit the proliferation and migration of tumor cells and suppress tumor growth, providing a new strategy for the preparation of drugs to treat tumors, especially kidney tumors. Attached Figure Description

[0029] Figure 1 The HPLC chromatogram of the polypeptide HDP21 is shown below.

[0030] Figure 2 The protein spectrum of the polypeptide HDP21 is shown.

[0031] Figure 3 Cell viability curves measured by CCK-8 assay after treating human renal cell carcinoma cells 786-O (Figure A) and normal human renal tubular epithelial cells HKC (Figure B) with different concentrations of the peptide HDP21 for 24 hours.

[0032] Figure 4 Bright field images of human renal cell carcinoma cells 786-O after treatment with different concentrations of the peptide HDP21. In Figure A, the blank control group is shown, and in Figure B, the treatment group with an HDP21 concentration of 80 μg / ml is shown.

[0033] Figure 5 Figures show the colony formation experiments of human renal cell carcinoma 786-O after treatment with different concentrations of the peptide HDP21. Figure A is the blank control group, and Figure B is the treatment group with an HDP21 concentration of 80 μg / ml.

[0034] Figure 6Figures show the scratch assay results of human renal cell carcinoma cells 786-O after treatment with different concentrations of the peptide HDP21. Figure A is the blank control group, and Figure B is the treatment group with an HDP21 concentration of 30 μg / ml.

[0035] Figure 7 The images show the morphology of the tumor after treatment with the peptide HDP21 (Figure A) and the tumor doubling rate (Figure B). Detailed Implementation

[0036] This invention provides an antitumor polypeptide HDP21, its preparation method, and its applications. The amino acid sequence of this antitumor polypeptide is RKKRRQRRR-AGQAFRKFLPLFDRVLVERSA (SEQ ID No. 1). The invention will be described in detail below with reference to specific embodiments and accompanying drawings to provide a better understanding of it; however, these embodiments do not limit the scope of the invention.

[0037] Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared according to conventional methods, unless otherwise specified.

[0038] Example 1

[0039] This embodiment provides an anti-tumor polypeptide HDP21, and the specific preparation method is as follows:

[0040] (1) Weigh 10g of chlorinated resin, add it to the polypeptide synthesis tube, add 100ml of LDCM to swell for 60min, dry it after swelling, add 100ml of LDMF to wash 3 times, and finally dry it.

[0041] (2) Weigh Ala, with a molar ratio of 6:1 to the resin, and dissolve it in DMF; weigh DIEA, with a molar ratio of 8:1 to the resin, and add the two materials to the peptide synthesis tube and shake to react overnight.

[0042] (3) After the reaction was carried out overnight, 100 mL of DMF was added to wash three times, and then methanol was added to block the chlorine resin.

[0043] (4) After sealing, add 100mL of DMF and wash 4 times. Fmoc is added during the two middle washes to provide protection.

[0044] (5) After removing the protection, weigh Ser, and the molar mass ratio of it to the resin is 6:1; weigh the condensation reagent, and the molar mass ratio of it to the resin is 6:1. Dissolve it with DMF, then add DIEA, react in the synthesis tube for 40 min, add 100 mL of DMF to wash 3 times and then dry it.

[0045] (6) Repeat steps (4) and (5) until the last amino acid (Arg) is added, remove the protecting group Fmoc, add 100ml LDM, wash 3 times and then dry to obtain dried peptide resin.

[0046] (7) Add 20 mL of cleavage reagent to each gram of dry peptide resin and react at room temperature in a sealed environment for 3 h.

[0047] (8) After filtration, the filtrate is transferred to an EP tube and 10 times the volume of diethyl ether is added. After the polypeptide precipitates, centrifugation is performed to obtain the precipitate. The supernatant is discarded and the process is repeated 3 times to obtain crude polypeptide.

[0048] (9) The peptide was purified by high performance liquid chromatography (HPLC), then lyophilized and weighed.

[0049] The antitumor polypeptide HDP21 was analyzed by HPLC and its purity reached over 95%. Figure 1 The structure was identified by ESI-MS as follows. Figure 2 As shown.

[0050] Example 2

[0051] This embodiment investigates the effect of peptide HDP21 on the activity of human renal cell carcinoma 786-O and normal human renal epithelial cells HKC. The specific experimental steps and results are as follows:

[0052] Adherent 786-O and HKC cells in the logarithmic growth phase were digested with trypsin. Once most cells detached, culture medium was added to stop the digestion. The cells were collected and transferred to centrifuge tubes, centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended in complete culture medium, counted, and the cell density adjusted to 5 x 10⁻⁶ cells / mL. 4 Cells were seeded at a density of 100 μl per well in 96-well plates and incubated overnight at 37°C with 5% CO2. The next day, HDP21 peptide at final concentrations of 0 μg / ml, 20 μg / ml, 40 μg / ml, 60 μg / ml, 80 μg / ml, and 100 μg / ml were added, respectively. Cells were then returned to the incubator and incubated for another 22 hours. Afterward, 10 μl of CCK-8 reagent was added to each well, and the cells were incubated for another 2 hours. The absorbance at 450 nm was then measured using a microplate reader. The data were integrated, and the cell viability of each group was calculated.

[0053] The results are as follows Figure 3 As shown, the peptide HDP21 exhibited significant inhibitory effects on human renal cell carcinoma 786-O in a gradient-dependent manner within a concentration range of 0-100 μg / ml, while having no effect on normal human renal tubular epithelial cells HKC.

[0054] Example 3

[0055] This embodiment verifies that the peptide HDP21 induces cell death in human renal cell carcinoma 786-O. The specific experimental steps and results are as follows:

[0056] 786-O adherent cells in the logarithmic growth phase were digested with trypsin. When most cells detached, culture medium was added to stop the digestion. The cells were collected and transferred to centrifuge tubes, centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended in complete culture medium, counted, and the cell density was adjusted to 2*102. 5 Cells were seeded at a density of 2 ml per well in 12-well plates and incubated overnight at 37°C with 5% CO2. After confluence, 0 μg / ml and 80 μg / ml of the peptide HDP21 were added, respectively. The plates were then returned to the incubator and incubated for another 12 hours. Bright-field images were then taken under a microscope at 200× magnification.

[0057] The results are as follows Figure 4 As shown, after treatment with high concentrations of the polypeptide HDP21, human renal cell carcinoma cells 786-O completely lost their normal cell morphology, their cell volume shrank, and their cell membranes ruptured into fragments.

[0058] Example 4

[0059] This embodiment investigates the effect of peptide HDP21 on the proliferation ability of human renal cell carcinoma cells 786-O. The specific experimental steps and results are as follows:

[0060] 786-O adherent cells in the logarithmic growth phase were digested with trypsin. Once most cells detached, culture medium was added to stop the digestion. The cells were collected and transferred to centrifuge tubes, centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were serially diluted with complete culture medium to a final cell density of 1 × 10⁻⁶ cells / mL. 4 Cells were cultured at a density of 100 μl / ml and added to 2 wells of a 6-well plate. Complete culture medium was added to a final volume of 2 ml, and the cells were shaken to disperse evenly. Cells were incubated overnight at 37°C with 5% CO2. After 24 hours of seeding and complete cell adhesion, a specific concentration of the peptide HDP21 was added to each well. After one week of incubation, the old culture medium was discarded, and the cells were fixed with methanol for 20 minutes, followed by staining with 0.2% crystal violet for 15 minutes. After washing with PBS, the cells were air-dried and photographed for cell counting.

[0061] The results are as follows Figure 5 As shown, treatment with the peptide HDP21 significantly reduced the proliferation ability of human renal cell carcinoma 786-O.

[0062] Example 5

[0063] This embodiment investigates the effect of peptide HDP21 on the migration ability of human renal cell carcinoma cells 786-O. The specific experimental steps and results are as follows:

[0064] 786-O adherent cells in the logarithmic growth phase were digested with trypsin. Once most cells detached, culture medium was added to stop the digestion. The cells were collected and transferred to centrifuge tubes, centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were serially diluted with complete culture medium to a final cell density of 3*102. 5 Cells were seeded at a density of 2 ml per well in a 12-well plate. Cells were incubated at 37°C with 5% CO2 until confluence. A vertical line was drawn perpendicular to the bottom of the plate using a 200 μl sterile yellow pipette tip. Cells were washed twice with PBS. 500 μl of complete culture medium was added to each well, and the plates were photographed and recorded under a microscope. The old culture medium was discarded, and culture medium containing different concentrations of peptides (containing 1% fetal bovine serum) was added to each well. The plates were returned to the culture medium and incubated for another 6 hours. The results were then observed and recorded under a microscope.

[0065] The results are as follows Figure 6 As shown, treatment with the peptide HDP21 significantly reduced the migration ability of human renal cell carcinoma cells 786-O.

[0066] Example 6

[0067] This embodiment investigates the effect of peptide HDP21 on tumor growth in vivo. The specific experimental steps and results are as follows:

[0068] Tumor cells were subcutaneously injected into the left back of 3-4 week old male BALB / c nude mice, with each mouse receiving 5*10 cells. 6 786-O cells were collected, and the tumor was allowed to grow to 60-70 mm. 3 The drug was administered by preparing a solution of the polypeptide HDP21 with physiological saline and injecting it intraperitoneally at a concentration of 20 mg / kg every 48 hours. The control group was injected with the same volume of physiological saline. The tumor volume was measured and recorded daily. The experiment was terminated after 24 days. The nude mice were sacrificed, the tumors were removed, and photographs were taken for recording.

[0069] The results are as follows Figure 7 As shown, treatment with the peptide HDP21 slowed the growth rate of tumors, indicating that the peptide HDP21 has an inhibitory effect on tumor growth in vivo.

[0070] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. Use of an antitumor polypeptide for the preparation of a medicament for the treatment of renal cell carcinoma, characterized in that, The amino acid sequence of the anti-tumor polypeptide is SEQ ID No. 1; the drug comprises the anti-tumor polypeptide and a pharmaceutically acceptable carrier or excipient thereof.

2. Use according to claim 1, characterized in that, The renal cell carcinoma is renal clear cell carcinoma.

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