Alpha2delta1 antagonistic polypeptide and application thereof in resisting tumor drug resistance

Abstract

The invention relates to an alpha2delta1 antagonistic polypeptide and application of the alpha2delta1 antagonistic polypeptide in resisting tumor drug resistance, and particularly discloses the alpha2delta1 antagonistic polypeptide. The amino acid residue sequence of the alpha2delta1 antagonistic polypeptide DL12-A alphaP2 is as shown in SEQ ID No: 1. The binding peptide and the derivative thereof can bind alpha2delta1 in vitro, and block a downstream signal channel through binding with alpha2delta1, so that proliferation of liver cancer drug-resistant cells is inhibited, apoptosis of the liver cancer drug-resistant cells is promoted, effective small molecule drugs for treating liver cancer drug resistance and the like are provided, and the binding peptide and the derivative thereof can be widely applied to the fields of medicine and biology.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and biomedicine. Specifically, this invention relates to the liver cancer drug resistance target α2δ1 receptor antagonist polypeptide DL12-AαP2 and its derivatives and applications. Background Technology

[0002] Liver cancer is one of the most common malignant tumors worldwide, with persistently high incidence and mortality rates. According to the latest statistics from the World Health Organization, there are approximately 900,000 new cases of liver cancer annually, and about 830,000 deaths, with the mortality rate approaching the incidence rate, reflecting the grim reality of its poor prognosis. In recent years, liver cancer diagnostic technology has made significant progress, gradually moving towards precision and minimally invasive techniques. Imaging examinations remain the cornerstone, with multimodal fusion technologies of ultrasound, CT, and MRI, along with functional imaging (such as diffusion-weighted imaging), significantly improving the detection rate of early-stage and small liver cancers. Regarding serum biomarkers, in addition to alpha-fetoprotein, the application of novel biomarkers such as abnormal prothrombin, microRNA combinations, and circulating tumor DNA has enhanced the sensitivity and specificity of diagnosis. Furthermore, artificial intelligence-assisted image analysis systems are gradually being applied clinically, helping to achieve automatic lesion identification and grading, and promoting the standardization and efficiency improvement of the diagnostic process.

[0003] Research progress on lenvatinib resistance in liver cancer mainly revolves around its complex mechanisms and clinical management strategies. The resistance mechanisms involve multiple levels: first, activation of tumor angiogenesis pathways; after drug inhibition of major pathways such as VEGFR, tumors maintain blood supply by upregulating angiopoietin-2 or activating alternative pathways such as c-MET; second, adaptive changes in tumor cell signaling, such as feedback activation of EGFR / HER3 and its downstream PI3K / AKT survival signals, or enhanced FGF expression to counteract FGFR inhibition; third, tumor microenvironment remodeling, including increased infiltration of immunosuppressive cells such as myeloid-derived suppressor cells and M2 macrophages, and extracellular matrix fibrosis forming a drug delivery barrier; in addition, individual pharmacokinetic differences may also lead to insufficient effective blood drug concentrations. Future research focuses on identifying predictive biomarkers (such as dynamic monitoring via ctDNA), using single-cell technology to analyze the dynamic evolution of the microenvironment, developing novel drugs (such as bispecific antibodies or ADC drugs), and exploring more precise combination therapies based on mechanisms (such as targeted combination cell therapy), with the ultimate goal of achieving personalized long-term control.

[0004] α2δ1 (alpha-2-delta-1) protein, as an accessory subunit of voltage-gated calcium channels, has traditionally been considered primarily involved in calcium ion influx and signal transduction in the nervous system. However, recent studies have shown that it is abnormally overexpressed in various solid tumors and plays an important role in promoting cancer, making it an emerging target in tumor biology research. Research has confirmed that α2δ1 is not only a functional membrane protein but also a cell surface marker, enriched in tumor-initiating cells or stem cell-like cell subpopulations of various cancers, including liver cancer, breast cancer, and lung cancer. Its overexpression is closely related to tumor cell proliferation, invasion, metastasis, and treatment resistance. For example, in liver cancer, α2δ1-positive cells exhibit stronger self-renewal capacity and tumorigenicity, and are a potential source of tumor recurrence and metastasis.

[0005] At the mechanistic level, the pro-cancer function of α2δ1 far exceeds its classic role in regulating calcium channels. Studies have found that it can drive tumor progression through multiple signaling pathways. First, it can activate key pro-survival and proliferative pathways, such as the PI3K / AKT and Ras / MAPK pathways, independently of calcium channel function. Second, α2δ1 can act as a receptor for extracellular matrix proteins (such as laminin), mediating the interaction between tumor cells and the microenvironment, thereby enhancing cell adhesion, migration, and invasion. Third, in models of liver cancer and colorectal cancer, α2δ1 has been shown to promote epithelial-mesenchymal transition and chemotherapy resistance by stabilizing growth factor receptors such as EGFR or activating integrin-related signaling. Particularly noteworthy is that in liver cancer, α2δ1 expression is associated with resistance to targeted drugs such as sorafenib, suggesting it may be a crucial breakthrough in overcoming treatment resistance.

[0006] Phage display techniques (PDT) were proposed by Smith et al. in 1985; the first successful construction of a phage display peptide library was achieved in 1988; and since 1990, phage display peptide libraries have experienced rapid development and application. The principle of phage display technology is to clone foreign DNA into a suitable phage vector using genetic engineering techniques, causing the expression product corresponding to the foreign DNA fragment to fuse with the capsid protein of the phage, forming a fusion protein displayed on the phage surface. The displayed peptide or protein maintains its relative spatial structure and biological activity. Then, using a target molecule and appropriate washing methods, non-specifically binding phages are removed, and finally, target phages that can bind to the target molecule are screened from the phage library. The foreign peptide or protein is expressed on the surface of the phage, and its encoding gene, as part of the phage genome, can be sequenced through phage DNA sequencing. A significant feature of this technology is the establishment of a correspondence between genotype and phenotype. Phage display technology is suitable for the preparation of fully human antibody drugs. Humira, an anti-tumor necrosis factor-alpha (TNF-α) antibody for treating rheumatoid arthritis, was the first fully human antibody drug produced using phage display technology and approved by the U.S. Food and Drug Administration (FDA). As of 2014, the FDA had approved six antibodies produced using phage display technology, and more than 30 related drugs were in clinical trials. Besides screening preparative antibodies, phage display technology can also be used to screen corresponding antigens. Therefore, phage display antibody libraries, due to their advantages of high capacity, high efficiency, convenience, and flexible screening, are widely used in many fields of life sciences, especially in tumor diagnosis and tumor antibody drug preparation, where they are receiving increasing attention and can serve as a valuable tool for screening antibodies targeting non-small cell lung cancer surface antigens. Summary of the Invention

[0007] To overcome the shortcomings and disadvantages of the prior art, the primary objective of this invention is to provide an α2δ1 antagonistic polypeptide. This antagonistic polypeptide has a specific high affinity for the receptor α2δ1 and can block the α2δ1 signaling pathway by binding to α2δ1. This demonstrates that the polypeptide plays an important role in targeting and inhibiting the proliferation of drug-resistant liver cancer cells and promoting apoptosis of drug-resistant liver cancer cells, and has great application value in targeted therapy for drug-resistant liver cancer.

[0008] Another object of the present invention is to provide a derivative of the above-mentioned α2δ1 receptor antagonistic peptide, which also has a specific high affinity for the α2δ1 receptor and specifically binds to α2δ1.

[0009] Another object of the present invention is to provide the application of the above-mentioned α2δ1 antagonistic polypeptide and its derivatives.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: One aspect of the present invention provides an α2δ1 antagonistic polypeptide, the amino acid sequence of which is shown in SEQ ID No:1.

[0011] Asp-Ser-Ser-Arg-Val-Pro-Tyr-Met-Trp-Ser-Gly-Pro (abbreviated as: DSSRVPYMWSGP) SEQ ID No:1.

[0012] Another aspect of the present invention provides a derivative of an α2δ1 antagonistic peptide, wherein the derivative of the α2δ1 antagonistic peptide is a product obtained by conventional modification of the amino acid side chain group of the α2δ1 antagonistic peptide, the amino terminus or carboxyl terminus of the α2δ1 antagonistic peptide fragment, or a product obtained by attaching a tag for peptide or protein detection or purification to the α2δ1 antagonistic peptide.

[0013] Furthermore, the conventional modifications are preferably amylation, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation, cyclization, biotinylation, fluorescent group modification, polyethylene glycol (PEG) modification, or immobilization modification, etc. The preferred tags are His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, or ProfinityeXact. Furthermore, the derivative of the α2δ1 antagonistic peptide is preferably an amidation modification of the end of the α2δ1 antagonistic peptide.

[0014] Furthermore, the α2δ1 antagonistic peptide and its derivatives are derived from mammals or birds, such as primates (humans); rodents, including mice, rats, hamsters, rabbits, horses, cattle, dogs, cats, etc.

[0015] Furthermore, DL12-AαP2 is a hydrophilic polypeptide.

[0016] In another aspect, the present invention provides a polynucleotide encoding the polypeptide described in SEQ ID No. 1.

[0017] In another aspect, the present invention provides a carrier comprising the aforementioned polynucleotide.

[0018] Furthermore, the polynucleotide is linked to the promoter sequence using gene technology.

[0019] In another aspect, the present invention provides a host cell transfected with the vector described herein.

[0020] In another aspect, the present invention provides a pharmaceutical composition comprising an α2δ1 antagonistic peptide or a derivative thereof.

[0021] Furthermore, the α2δ1 antagonistic peptide and / or derivatives of the α2δ1 receptor antagonistic peptide in the pharmaceutical composition are the sole active ingredient, or are combined with other active ingredients.

[0022] Furthermore, the other active ingredients are active ingredients with anti-tumor effects, such as sorafenib, lenvatinib, regorafenib, cabozantinib, ramuximab, ramuximab, sunitinib, pazopanib, axitinib, vandetanib, donafenib, bevacizumab, dabrafenib, and trametinib-resistant cells.

[0023] In the technical solution of the present invention, the drug contains one or more pharmaceutically acceptable carriers; In the technical solution of the present invention, the pharmaceutically acceptable carrier is preferably a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption promoter, adsorbent carrier, surfactant or lubricant, etc. In the technical solution of the present invention, the drug is prepared in the form of tablets, granules, capsules, oral liquids or injections.

[0024] In another aspect, the present invention provides a detection reagent comprising an α2δ1 antagonistic peptide and / or a derivative of an α2δ1 receptor antagonistic peptide.

[0025] In another aspect, the present invention provides an antibody against the aforementioned α2δ1 antagonistic peptide or a derivative thereof.

[0026] In another aspect, the present invention provides the use of the α2δ1 antagonistic peptide and its derivatives in the preparation of medicaments for inhibiting tumor cell proliferation, promoting tumor cell apoptosis, preventing or treating tumor diseases, and inhibiting tumor metastasis.

[0027] In the technical solution of the present invention, the tumor cells are tumor cells that express α2δ1 on their cell surface, and are further selected from liver cancer cells, lung cancer cells, and breast cancer cells.

[0028] Furthermore, the tumor cells are drug-resistant tumor cells.

[0029] In the technical solution of the present invention, the tumor disease is selected from tumor diseases mediated by tumor cells that express α2δ1 on the surface, preferably liver cancer, lung cancer, and breast cancer.

[0030] Furthermore, the tumor cells expressing α2δ1 on their surface are drug-resistant tumor cells. Furthermore, the drug-resistant tumor cells are cells resistant to the targeted anti-tumor drugs sorafenib, lenvatinib, regorafenib, cabozantinib, ramuximab, ramuximab, sunitinib, pazopanib, axitinib, vandetanib, donafenib, bevacizumab, dabrafenib, and trametinib.

[0031] In another aspect, the present invention provides an in vitro method for screening or inhibiting cells for non-diagnostic and therapeutic purposes, the method comprising the step of co-incubating the above-mentioned α2δ1 antagonistic peptide or α2δ1 antagonistic peptide derivative with cells.

[0032] Beneficial effects (1) The present invention provides an α2δ1 antagonistic peptide DL12-AαP2 and its derivatives, wherein the antagonistic peptide and its derivatives can specifically bind to α2δ1 and specifically bind to α2δ1 to inhibit the α2δ1 signaling pathway.

[0033] (2) The α2δ1 antagonistic peptides and their derivatives obtained by screening in this invention can inhibit the proliferation of lenvatinib-resistant liver cancer cells by blocking the α2δ1 signaling pathway. They can be used as biological peptide drugs with α2δ1 binding sites and can be used to prepare drugs for the prevention and / or treatment of drug-resistant liver cancer. They can be widely used in the fields of medicine and biology and generate huge social and economic benefits. Attached Figure Description

[0034] Figure 1 : Validation data for the synthesis of the α2δ1 antagonistic peptide. A: High-performance liquid chromatography (HPLC) of the DL12-AαP2 peptide; B: LC-MS identification of the DL12-AαP2 peptide.

[0035] Figure 2 The binding affinity of AαP2 bacteriophage to α2δ1; Figure 3 Construction of lenvatinib-resistant hepatocellular carcinoma cells. A: Construction of HepG2 lenvatinib-resistant cells; B: Construction of HCCLM3 lenvatinib-resistant cells.

[0036] Figure 4 DL12-AαP2 inhibits the proliferation of drug-resistant hepatocellular carcinoma cells. A: DL12-AαP2 inhibits the proliferation of drug-resistant hepatocellular carcinoma cells HepG2-DR; B: DL12-AαP2 inhibits the proliferation of drug-resistant hepatocellular carcinoma cells HCCLM3-DR.

[0037] Figure 5DL12-AαP2 inhibits the proliferation of drug-resistant HCCLM3-DR hepatocellular carcinoma cells. A: Photograph of clone formation; B: Statistical analysis of data on the inhibition of HCCLM3-DR hepatocellular carcinoma cell proliferation by DL12-AαP2.

[0038] Figure 6 DL12-AαP2 inhibits the migration of drug-resistant hepatocellular carcinoma cells HCCLM3-DR. A: Scratch assay image; B: Statistical analysis of data on the inhibition of HCCLM3-DR migration by DL12-AαP2. Detailed Implementation

[0039] To better understand the present invention, it is now further described with reference to the following embodiments and accompanying drawings. The embodiments are for illustrative purposes only and do not limit the invention in any way. In the embodiments, all original reagents and materials are commercially available, and experimental methods not specifically specified are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0040] Example 1: Panning, amplification, purification, sequencing and synthesis of the α2δ1 antagonistic peptide DL12-AαP2.

[0041] This embodiment is mainly for screening to obtain positive phages that specifically bind to α2δ1. Then, the positive phages are amplified, purified, and single-stranded DNA (ssDNA) of the phages is extracted and sequenced. The obtained sequences are analyzed and compared, and finally, a high-purity antagonistic polypeptide DL12-AαP2 is synthesized.

[0042] Specifically as follows: 1. Establishment of a 293T cell line that permanently overexpresses α2δ1: 293T-α2δ1 + / + 1.1 Select vigorous, luminescent human 293T cells, and administer them at a dose of 5 × 10⁻⁶ cells per cell line the day before transfection. 5 Cells were seeded per well in 6-well plates, and after culturing for two days, the cell confluence was 60%. 1.2 On the second day, transfection was performed. Using one well of a 6-well plate as a unit, 3 μg of plasmid was diluted with 200 μL of Opti-MEM medium, and 6 μL of liposome Lipofectamine 2000 was diluted with 200 μL of Opti-MEM medium. After gently mixing, the mixture was incubated at room temperature for 5 minutes. 1.3 Gently mix the two dilutions, let stand at room temperature for 20 minutes, and then gently add 600 μL of opti-MEM medium to the mixed dilution; 1.4 Gently rinse the cells to be transfected once with PBS, then gently add the mixed dilution to the culture wells and incubate in a carbon dioxide incubator; 1.5 After culturing for 4-6 hours, discard the transfection medium and add 3 mL of complete culture medium to the wells; 1.6 After 48 hours, the cells were screened using a medium containing 1 μg / mL puromycin; once cell death ceased, a stable 293T cell line expressing α2δ1 was obtained.

[0043] 1.7 Total RNA was extracted using TRIzol, and 2 μg of RNA was quantified for reverse transcription (reverse transcription kit, purchased from Promega), followed by qPCR using specific primer sequences.

[0044] The specific primer sequence used is the Hu-α2δ1 primer sequence: Fw 5'-GAGCTAAGGGAAGGGCGG-3' SEQ ID No: 2 Rv 5'-ATAAACAGCAGCCCCAGGAC-3' SEQ ID No: 3 1.8 Compared with that transfected with pSM2c-Hu-scramble RNA, the high expression level of α2δ1 was detected and named: 293T-α2δ1 + / + This means it can be used for positive phage screening.

[0045] 2. Panning, amplification, purification, sequencing, and synthesis of α2δ1 antagonistic peptides. 2.1 ER2738 Preparation of host bacterial culture: Aseptic technique was used. First, 200 μL of LB-Tet liquid culture medium was transferred to a 1.5 mL sterile centrifuge tube, then... E. coli Take 0.2 μL of bacterial suspension from the glycerol cryopreservation of ER2738, mix thoroughly, and spread the entire amount onto LB-Tet plates. Label the plates, incubate at room temperature for 3 min, and then incubate overnight in an inverted 37 ℃ incubator. Observe the next day; after colonies have grown, seal with sealing film and store at 4 ℃ protected from light for later use. Aseptically pick single colonies using a sterile pipette tip and place them into a 10 mL sterile centrifuge tube pre-added with 3 mL of LB-Tet liquid medium. Label the tube and incubate overnight on a shaker at 37 ℃ with shaking at 300 rpm / min. The next day, store the bacterial amplification solution at 4 ℃ for later use. Take a 10 mL sterile centrifuge tube, aseptically add 3 mL of LB-Tet liquid medium, inoculate with 30 μL of overnight cultured bacteria, and incubate on a shaker at 37 ℃ with shaking at 300 rpm / min for 2–3 h. The bacteria are in the exponential growth phase and appear hazy to the naked eye (OD). 600 ~0.5).

[0046] 2.2 Selection of α2δ1 antagonistic peptides: Cells expressing high levels of α2δ1 were selected at a ratio of 10... 5 Individuals were inoculated per culture dish into 60×15 mm plates pre-coated with poly-L-lysine. 2 In culture dishes, cells were cultured to a density of 80%–90% using standard methods. The eluent was then used for washing (with a cell line that does not express α2δ1 as a blank control). 1 μL of the eluent was taken from each round of elution to measure the titer. The remainder was added to 20 mL of LB medium for amplification, followed by purification and measurement of the amplified titer. The amplified product was stored at 4 ℃ for a short period, and an equal order of magnitude was used for the next round of washing. The remaining amplified product was stored in 50% glycerol at -20 ℃.

[0047] 2.3 Phage Titer Determination: Take four sterile 10 mL centrifuge tubes, one for each phage dilution. Melt the top agarose in a microwave oven, add 3 mL of top agarose to each tube, and incubate at 45 ℃. Prepare one LB / IPTG / Xgal plate for each phage dilution, preheated in a 37 ℃ incubator. OD... 600 ~0.5 E. coli ER2738 *E. coli* was aliquoted at 200 μL / tube and stored at 4 °C for later use. Four sterile 1.5 mL centrifuge tubes were prepared, each containing 100 μL, 90 μL, 90 μL, and 90 μL of LB-Tet medium, respectively. One μL of the phage to be tested was added to 100 μL of LB-Tet medium, and the tubes were serially diluted 10-fold, labeled as 10, 10, and 10, respectively. -1 10 -2 10 -3 10 -4 Gently vortex to mix each dilution, then centrifuge briefly. Take 10 μL of the phage to be titrated at each dilution and mix with 200 μL of... E. coli Mix ER2738, gently vortex to mix, centrifuge briefly, and incubate at room temperature for 5 min. Quickly add the mixed bacterial culture to the top agar, rapidly vortex to mix, and immediately pour into a preheated LB / IPTG / Xgal plate, spread it evenly, cool at room temperature for 5 min, and incubate overnight in a 37 ℃ inverted plate incubator.

[0048] 2.4 Amplification and purification of eluted phage: Take a 250 mL Erlenmeyer flask and add the overnight cultured ER2738 host bacteria at a 1:100 ratio to 20 mL of LB liquid medium. Incubate at 37 °C with vigorous shaking at 250 rpm for 2 h. Then, add the phage to be amplified to the Erlenmeyer flask and incubate at 37 °C with vigorous shaking at 250 rpm for 4.5 h. Transfer the culture to a 50 mL centrifuge tube and centrifuge at 4 °C and 10,000 rpm for 10 min. Transfer the supernatant to another clean centrifuge tube and centrifuge again at 4 °C and 10,000 rpm for 10 min. Take 80% of the supernatant and transfer it to another clean centrifuge tube. Add 1 / 4 volume of PEG / NaCl, mix by inversion, and incubate at 4 °C overnight to precipitate. The next day, centrifuge the precipitate at 4 °C and 12,000 rpm for 20 min. Carefully aspirate the supernatant with a clean pipette tip, then centrifuge at 12,000 rpm for 1 min at 4 °C to remove residual supernatant. Resuspend the precipitate in 1 mL TBS, gently pipetting 100 times. Transfer the suspension to a 2 mL centrifuge tube and centrifuge at 10,000 rpm for 5 min at 4 °C to remove residual cells. Add 1 / 4 volume of PEG / NaCl to the supernatant and incubate on ice for 60 min to reprecipitate. Remove the centrifuge tube and centrifuge at 12,000 rpm for 20 min at 4 °C to remove the supernatant. Resuspend the precipitate in 200 μL TBS and centrifuge at 10,000 rpm for 1 min at 4 °C. Transfer the supernatant to another centrifuge tube. Store at 4 °C for short periods, or long-term with 50% glycerol at -20 °C. The amplification of monoclonal bacteriophages included: (1) adding overnight cultured ER2738 host bacteria at a ratio of 1:100 to 2 mL of LB liquid medium and incubating at 37°C with vigorous shaking at 250 rpm for 2 h; using a sterile toothpick, selecting plates with fewer than 100 plaques from the fourth-round titration plates, picking well-separated blue plaques, and adding them to culture tubes, incubating at 37°C with vigorous shaking at 250 rpm for 4.5 h; then transferring the culture to fresh centrifuge tubes and centrifuging at 4°C and 10,000 rpm for 30 sec. The supernatant was transferred to fresh tubes and centrifuged again; 80% of the supernatant was transferred to fresh centrifuge tubes and stored at 4°C, or it could be stored long-term at -20°C using 50% glycerol.

[0049] 2.5 Identification of M13 phage ssDNA by agarose gel electrophoresis: Place the gel mold horizontally, place the selected comb, leaving a 1 mm gap between the bottom of the comb and the mold; weigh 1 g of agarose for DNA electrophoresis into a 250 mL Erlenmeyer flask, add 100 mL of 1×TAE buffer, mix well, place the flask in a microwave oven, heat to boiling until the agarose is completely dissolved; turn off the microwave oven, remove the Erlenmeyer flask, and cool it to room temperature (the flask should be tolerable to hold by hand), then add 5 μL of ethidium bromide, mix well, and then pour the gel solution onto a gel plate. The gel plate used in this experiment requires approximately 100 mL of gel solution. Allow the gel to solidify completely at room temperature, which takes about 30 minutes. Remove the comb teeth and place the gel plate into the electrophoresis tank. Add 1×TAE buffer to the electrophoresis tank, ensuring it is 2 mm above the gel surface. Dilute the sample with loading buffer and add it to the gel plate, ensuring the pipette tip is positioned precisely in the gel well, avoiding puncturing the gel and preventing sample overflow. Turn on the power, adjust the voltage to 50 volts, and electrophoresis for 90 minutes. Remove the gel plate and observe the results under UV light.

[0050] 2.6 ssDNA Sequencing and Sequence Analysis: The extracted M13 phage ssDNA was sent to Shanghai Ingenic Biotechnology Co., Ltd. for DNA sequencing. Sequence analysis was performed using Bioedit software. The analysis results showed that the sample sequence was SEQ ID NO:1 Asp-Ser-Ser-Arg-Val-Pro-Tyr-Met-Trp-Ser-Gly-Pro, represented as DL12-AαP2. The final short peptide was obtained from Hefei Guotai Biotechnology Co., Ltd.

[0051] Figure 1 Data for the synthesis and validation of the α2δ1 antagonistic peptide. A: High-performance liquid chromatography (HPLC) of the DL12-AαP2 peptide; B: LC-MS identification of the DL12-AαP2 peptide.

[0052] Example 2: Enzyme-linked immunosorbent assay (ELISA) for detecting the binding of bacteriophage monoclonal cells to α2δ1. First, select a single DL12-AαP2 phage clone and inoculate it into LB medium containing ER2738. Incubate at 37°C with shaking at 230 rpm for 7 hours. Centrifuge at 5000 rpm for 15 min at room temperature, collect the supernatant, and store at 4°C. 293T wild-type cells and 293T α2δ1... + / + Cells, HCT-116 cells, and HT-29 cells were spaced at 5 x 10⁻⁶ cells per well. 3Cells were inoculated into 96-well plates and cultured for 24 hours. The next day, the cell culture medium was discarded, and blocking buffer (DMEM + 5% BSA) was added to each well for 2 hours. After discarding the blocking buffer, 100 μL of phage dilution buffer (1:100) was added to each well and incubated for 2 hours. Then, the cells were washed three times with PBST (PBS + 0.1% Tween-20) for 5 minutes each time to remove unbound phages. 100 μL of peroxidase-linked anti-M13 phage antibody (purchased from GE) diluted (1:5000) was added to each well and incubated at room temperature for 1 hour. The cells were washed three times with PBST (PBS + 0.1% Tween-20) for 5 minutes each time to remove unbound antibody. 50 μL of LTMB substrate (Sigma) was added to each well and incubated at room temperature in the dark for 30 minutes. The reaction was stopped by adding 50 μL of concentrated H2SO4 to each well, and the readings were taken at 450 nm using a microplate reader.

[0053] The results showed that, compared with 293T wild-type cells that did not express α2δ1, DL12-AαP2 phage and 293T cells that highly expressed α2δ1... + / + Cells, HepG2-DR cells, and HCCLM3-DR cells showed high binding capacity, with significant differences. *** P < 0.001), results are shown in [link to results]. Figure 2 .

[0054] Example 3: DL12-AαP2 can significantly inhibit the proliferation of lenvatinib-resistant liver cancer cells. 1. HepG2 and HCCLM3 cells were treated with lenvatinib at an initial concentration of 1 μM. The culture medium was changed every 48 h. After the cells stabilized, the concentration of lenvatinib was increased (0.5-1.0 μM / time) until a maximum concentration of 20 μM was reached. After the cells adapted to the 20 μM selection, the cell resistance characteristics were stably maintained to obtain lenvatinib-resistant liver cancer cells HepG2-DR and HCCLM3-DR. The IC50 of lenvatinib in inhibiting cell proliferation was detected by MTT assay. 50 The values ​​were used to verify the drug resistance characteristics of the cells.

[0055] 2. HepG2-DR and HCCLM3-DR hepatocellular carcinoma cells were subjected to a 5x10 ratio. 3 Cells / well were seeded into 96-well cell culture plates with 200 μL of culture medium per well, cultured for 24 h, and then starved overnight; 3. Treat with DL12-AαP2 peptides at different concentration gradients for 24 hours; 4. Add 20 μL of MTT working solution to each well and continue incubation in a CO2 incubator for 4 hours; 5. Discard the supernatant in the culture plate, add 150 μL of DMSO (dimethyl sulfoxide), vortex for 10 minutes, and detect the cells at a wavelength of 490 nm using a microplate reader to plot the cell growth curve.

[0056] Figure 3 The results showed that, compared with wild-type HepG2 and HCCLM3 cells, the IC50 of drug-resistant cells was significantly lower. 50 The significantly elevated values ​​demonstrate the successful construction of lenvatinib-resistant cells HepG2-DR and HCCLM3-DR. In previous work, the inventors' laboratory detected high expression of α2δ1 in HepG2-DR and HCCLM3-DR hepatocellular carcinoma cells using q-PCR.

[0057] Figure 4 The results showed that different concentrations of DL12-AαP2 short peptide could significantly inhibit the proliferation of lenvatinib-resistant hepatocellular carcinoma cells HepG2-DR and HCCLM3-DR, and the inhibitory effect of the short peptide on the proliferation of drug-resistant hepatocellular carcinoma cells was more significant with increasing concentration.

[0058] Example 4: DL12-AαP2 can inhibit the formation and migration of drug-resistant liver cancer cell clones. 1. HCCLM3-DR hepatocellular carcinoma cells were seeded at 100 cells / well in 6-well cell culture plates with 2 mL of culture medium per well and cultured for 24 h. Then, DL12-AαP2 and an appropriate amount of fresh culture medium were added, with the untreated group serving as a control. The culture medium and drugs were changed every two days, and the cells were cultured continuously until the cell clusters contained more than 50 cells. After fixing the cells in both experimental and control groups with methanol, they were stained with 0.1% crystal violet, and the number of colonies formed in each group was counted. The results are shown below. Figure 5 As shown, compared with the control group, the number of drug-resistant cell clones was significantly reduced after the addition of DL12-AαP2, and DL12-AαP2 can effectively inhibit the formation of drug-resistant cell clones in liver cancer.

[0059] 2. HCCLM3-DR hepatocellular carcinoma cells, which were confluent in 6-well plates, were seeded at a 1:2 ratio into new 6-well cell culture plates with 2 mL of culture medium per well. After culturing for 24 h until the cells were confluent, the cells were scratched with a 200 μl pipette tip, and floating cells were washed away with PBS. The scratch distance was recorded by microscopy. DL12-AαP2 and an appropriate amount of serum-free fresh culture medium were added, with the untreated group serving as a control. After 48 h, floating cells were washed away, and the scratch distance was recorded by microscopy.

[0060] The results are as follows Figure 6 As shown, compared with the control group, the addition of DL12-AαP2 resulted in a wider scratch and slower cell migration, indicating that DL12-AαP2 can effectively inhibit the migration of drug-resistant liver cancer cells.

Claims

1. An α2δ1 antagonistic polypeptide, characterized in that: The amino acid residue sequence of the α2δ1 antagonistic polypeptide is shown in SEQ ID No:

1.

2. A derivative of the α2δ1 antagonistic polypeptide according to claim 1, characterized in that: The derivative of the α2δ1 antagonistic peptide is a product obtained by conventionally modifying the amino acid side chain group of the α2δ1 antagonistic peptide of claim 1, or the amino terminus or carboxyl terminus of the α2δ1 antagonistic peptide fragment of claim 1, or a product obtained by attaching a tag for peptide or protein detection or purification to the α2δ1 antagonistic peptide of claim 1. The conventional modifications mentioned are amylation, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation, cyclization, biotinylation, fluorescent group modification, polyethylene glycol (PEG) modification, or immobilization modification; The tags mentioned are His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, or ProfinityeXact.

3. A polynucleotide, characterized in that, It encodes the α2δ1 antagonistic polypeptide of claim 1 or the derivative of claim 2.

4. A carrier, characterized in that, It contains the polynucleotide as described in claim 3.

5. A host cell, characterized in that, It was transfected with the vector described in claim 4.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the α2δ1 antagonistic polypeptide of claim 1 or the derivative of claim 2; Preferably, the α2δ1 antagonistic peptide and / or derivatives of the α2δ1 receptor antagonistic peptide in the pharmaceutical composition are the sole active ingredient, or are combined with other active ingredients; Preferably, the drug contains one or more pharmaceutically acceptable carriers; More preferably, the pharmaceutically acceptable carrier is a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, adsorbent carrier, surfactant, or lubricant.

7. A detection reagent comprising the α2δ1 antagonistic polypeptide of claim 1 or the derivative of claim 2.

8. An antibody of an α2δ1 antagonistic peptide or a derivative thereof, wherein the α2δ1 antagonistic peptide is the α2δ1 antagonistic peptide as described in claim 1, and the derivative thereof is the derivative as described in claim 2.

9. Use of the α2δ1 antagonistic peptide of claim 1 and derivatives of the α2δ1 antagonistic peptide of claim 2 in the preparation of medicaments for inhibiting tumor cell proliferation, promoting tumor cell apoptosis, preventing or treating tumor diseases, and inhibiting tumor metastasis; Preferably, the tumor cells are tumor cells that express α2δ1 on their cell surface; more preferably, the tumor cells are selected from liver cancer cells, lung cancer cells, and breast cancer cells. Preferably, the tumor cells are drug-resistant tumor cells; Preferably, the tumor disease is selected from tumor diseases mediated by tumor cells that express α2δ1 on their surface, and more preferably liver cancer, lung cancer, and breast cancer; Preferably, the tumor cells expressing α2δ1 on their surface are drug-resistant tumor cells.

10. An in vitro method for screening or inhibiting cells for non-diagnostic and non-therapeutic purposes, the method comprising the step of co-incubating cells with the α2δ1 antagonistic peptide of claim 1, a derivative of the α2δ1 antagonistic peptide of claim 2, and the cells.

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