Alpha2delta1 targeted degradation molecule alpha2delta1-LYTAC as well as derivative and application thereof

Abstract

The invention discloses an alpha2delta1 targeted degradation molecule alpha2delta1-LYTAC as well as a derivative and application thereof. The alpha2delta1 targeted degradation molecule alpha2delta1-LYTAC comprises an alpha2delta1 antagonistic polypeptide with an amino acid sequence as shown in SEQ ID No: 1 and a medicine. The alpha2delta1-LYTAC can be combined with alpha2delta1, the alpha2delta1 protein is degraded by combining with the alpha2delta1, and a downstream signal channel of the alpha2delta1 protein is blocked, so that liver cancer cell proliferation is inhibited, liver cancer cell apoptosis is promoted, especially tumor drug-resistant cells are promoted, effective small molecule drugs for treating liver cancer and the like are provided, and the alpha2delta1-LYTAC 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, and more specifically, to an α2δ1-targeted degradation of the small molecule α2δ1-LYTAC and its derivatives and applications. Background Technology

[0002] In recent years, significant progress has been made in liver cancer diagnosis technology, 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), greatly improving the detection rate of early-stage and small hepatocellular carcinomas. 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 diagnostic sensitivity and specificity. Furthermore, artificial intelligence-assisted image analysis systems are gradually being applied clinically, facilitating automatic lesion identification and grading, and promoting the standardization and efficiency improvement of the diagnostic process.

[0003] Liver cancer treatment has entered an era of multidisciplinary comprehensive treatment and precision medicine. Early-stage liver cancer is primarily treated with surgical resection, liver transplantation, and local ablation, with laparoscopic and robot-assisted surgery further reducing trauma and accelerating recovery. For patients with intermediate or advanced-stage liver cancer, local treatments such as transarterial chemoembolization (TACE) and radioembolization combined with systemic drug therapy have become the standard strategy. Breakthroughs in systemic therapy have been particularly significant. The application of molecularly targeted drugs (such as lenvatinib and donafenib) and immune checkpoint inhibitors (such as pembrolizumab and atezolizumab) has significantly prolonged patient survival; targeted-immunotherapy combinations (such as the "T+A" regimen) have become an important first-line treatment option. Furthermore, emerging strategies such as genotyping-based personalized treatment, bispecific antibodies, and cell therapy are in clinical research stages, bringing new hope for improving the prognosis of liver cancer.

[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] Traditional targeted drug development faces the following challenges: (1) inability to develop "undruggable" targets; (2) susceptibility to off-target effects and adverse reactions; and (3) susceptibility to drug resistance with long-term use. Protein degradation technology (PDT) utilizes various protein degradation mechanisms in eukaryotic cells to target and degrade target proteins and block downstream signaling pathways to achieve disease treatment. LYTAC technology primarily degrades intracellular proteins but cannot degrade membrane or extracellular proteins, while most tumor targets are located in the cell membrane and extracellular matrix. Lysosome-targeting chimera (LYTAC) technology uses antibodies, peptides, and small molecules of the target protein to bind to lysosomal targeting receptors on the cell surface via chemical bonds to form an "LTR-LYTAC-target protein" ternary complex, thereby activating lysosomes to achieve target protein degradation. To further improve cell specificity, Bertozzi's research group used GalNAc to target the liver-specific lysosomal targeting receptor—Asialoglycoprotein receptor (ASGPR)—which can specifically degrade EGFR and HER2 proteins on the liver cancer cell membrane. The Apt-LYTAC molecule, constructed based on nucleic acid aptamers, has advantages such as easy synthesis and small molecular weight, and can rapidly and efficiently degrade the exocrine protein PDGF and the cell membrane protein PTK7 of liver cancer cells. However, current antibody-based LYTAC molecules suffer from problems such as difficult synthesis, low targeting, and poor internalization ability due to their large molecular weight. This invention uses phage display technology to screen binding peptides targeting α2δ1 for the construction of α2δ1... LYTAC is a targeted degradation drug with a smaller molecular weight, higher targeting specificity, and stronger internalization ability, which can significantly improve the treatment effect of liver cancer. Summary of the Invention

[0007] To address the problems mentioned in the background art, the present invention aims to provide an α2δ1-targeted degradation method for small molecules α2δ1. LYTAC and its derivatives and applications. This application focuses on the α2δ1-targeted degradation of small molecules α2δ1. LYTAC (derivative) includes the α2δ1 antagonistic peptide (derivative) and drug provided in this application. The antagonistic peptide (derivative) has a specific high affinity for the target α2δ1, and when α2δ1 targets and degrades small molecule α2δ1... LYTAC (derived from LYTAC) can prevent α2δ1 from binding to other proteins or genes and induce α2δ1 protein degradation, thereby affecting downstream related genes and signaling pathways. This α2δ1 targets and degrades small molecule α2δ1. LYTAC (derived from LYTAC) plays an important role in targeting and inhibiting the proliferation of liver cancer cells and promoting apoptosis of liver cancer cells, and it has great application value in targeted therapy for liver cancer.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: On the one hand, the present invention provides an α2δ1 antagonistic polypeptide, the amino acid sequence of which is shown in SEQ ID No:1.

[0009] On the other hand, the present invention provides a derivative of an α2δ1 antagonistic peptide, wherein 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 described above, and / or the amino terminus or carboxyl terminus of the α2δ1 antagonistic peptide fragment described above. Alternatively, it could be a product obtained by attaching a tag for peptide or protein detection or purification to the α2δ1 antagonistic peptide described above.

[0010] Furthermore, the conventional modifications include amylation, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation, cyclization, biotinylation, fluorescent group modification, polyethylene glycol (PEG) modification, or immobilization modification; Preferably, the tag includes His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c My c or ProfinityeXact.

[0011] Furthermore, the derivative of the α2δ1 antagonistic peptide is a product obtained by end-amidation modification of the aforementioned α2δ1 antagonistic peptide.

[0012] Furthermore, the α2δ1 antagonistic polypeptide and its derivatives can be obtained using known methods in the prior art, either through chemical synthesis using an automated polypeptide synthesizer; by deducing the nucleotide sequence from the short peptide sequence and then cloning it into a vector for biosynthesis; or by large-scale extraction and purification from existing organisms.

[0013] Furthermore, the α2δ1 antagonistic peptide and its derivatives may be derived from mammals or birds, such as primates (humans); rodents, including mice, rats, hamsters, rabbits, horses, cattle, dogs, cats, etc.

[0014] On the other hand, the present invention provides an α2δ1-targeted degradation small molecule lysosome-targeting chimera, wherein the lysosome-targeting chimera is a conjugate of an α2δ1 binding receptor and a lysosome-targeting receptor. The α2δ1 binding receptor is a polypeptide as shown in SEQ ID NO:1; The lysosomal targeting receptor contains a group capable of binding to the sialic acid glycoprotein receptor (ASGPR) or CI-M6PR.

[0015] Furthermore, the group capable of binding to the sialic acid glycoprotein receptor (ASGPR) is N-acetylgalactosamine (GalNAc). Furthermore, the group capable of binding to CI-M6PR is mannose-6-phosphate (M6P) or its oligosaccharide / glycopeptide derivative.

[0016] Furthermore, the lysosomal targeting receptor is a self-assembled microsphere, with the following structural formula: It is coupled to the α2δ1 binding receptor via the amino group of the lysine side chain.

[0017] Furthermore, the α2δ1 binding receptor and the lysosomal targeting receptor are linked by a linker group, or the α2δ1 binding receptor and the lysosomal targeting receptor are directly linked.

[0018] In another aspect, the present invention provides a polynucleotide characterized in that it encodes the aforementioned α2δ1 antagonistic polypeptide or a derivative thereof.

[0019] In another aspect, the present invention provides the application of the above-mentioned α2δ1-targeting degradation small molecule lysosome-targeting chimera in the preparation of a drug for the prevention and / or treatment of tumors that highly express α2δ1.

[0020] In another aspect, the present invention provides the use of the above-mentioned α2δ1-targeted degradation small molecule lysosome-targeting chimera, α2δ1 antagonistic peptide or derivative of α2δ1 antagonistic peptide in a drug for inhibiting tumor cell proliferation, promoting tumor cell apoptosis, preventing or treating tumor diseases, and inhibiting tumor metastasis.

[0021] 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.

[0022] Furthermore, the tumor cells are drug-resistant tumor cells.

[0023] 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.

[0024] 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 antitumor drugs sorafenib, lenvatinib, regorafenib, cabozantinib, ramucirumab, ramucirumab, sunitinib, pazopanib, axitinib, vandetanib, donafenib, bevacizumab, dabrafenib, and trametinib.

[0025] 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.

[0026] In another aspect, the present invention provides a pharmaceutical composition or detection reagent comprising the above-mentioned α2δ1-targeted degradation small molecule lysosomal targeting chimera, α2δ1 antagonistic peptide or derivative of α2δ1 antagonistic peptide.

[0027] Furthermore, the pharmaceutical composition contains one or more pharmaceutically acceptable carriers.

[0028] Furthermore, the pharmaceutically acceptable carriers include diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, adsorbents, surfactants, or lubricants, etc.

[0029] Furthermore, the pharmaceutical composition can be formulated into various forms such as tablets, granules, capsules, oral liquids, or injections, and the various dosage forms of the drug can be prepared according to conventional methods in the pharmaceutical field.

[0030] The beneficial effects of this invention are: (1) This invention provides an α2δ1-targeted degradation drug for small molecules α2δ1. LYTAC and its derivatives, the α2δ1 LYTAC and its derivatives can bind specifically to α2δ1.

[0031] (2) The α2δ1 targeted degradation small molecule drug α2δ1 provided by the present invention LYTAC and its derivatives can inhibit cancer cell proliferation and promote apoptosis by blocking the α2δ1 signaling pathway. They can serve as biodegradable small molecule drugs with α2δ1 binding sites and can be used to prepare drugs for the prevention and / or treatment of tumors. They have the potential for wide application in the medical and biological fields, generating significant social and economic benefits. Attached Figure Description

[0032] Figure 1 Screening and molecular docking data of α2δ1 antagonistic peptides. A: Screening and enrichment data of DL12-AαP5 peptide; B: Docking of DL12-AαP5 peptide with α2δ1 molecules; Figure 2 : Validation data for the α2δ1 antagonistic peptide. A: High-performance liquid chromatography (HPLC) of the DL12-AαP5 peptide; B: LC-MS identification of the DL12-AαP5 peptide.

[0033] Figure 3 : The binding ability of AαP5 bacteriophage to α2δ1.

[0034] Figure 4 DL12-AαP5 inhibits the proliferation of drug-resistant hepatocellular carcinoma cells. A: DL12-AαP5 inhibits the proliferation of drug-resistant hepatocellular carcinoma cells HepG2-DR; B: DL12-AαP5 inhibits the proliferation of drug-resistant hepatocellular carcinoma cells HCCLM3-DR.

[0035] Figure 5 : GalNAc peptide validation data. A: GalNAc peptide high performance liquid chromatography (HPLC); B: GalNAc peptide LC-MS identification.

[0036] Figure 6 : α2δ1 LYTAC molecules can significantly reduce α2δ1 protein expression; Figure 7 : α2δ1 LYTAC molecules can significantly reverse lenvatinib resistance in liver cancer and reduce stem cell spheroidization ability. A: α2δ1 LYTAC molecules can significantly reverse lenvatinib resistance in liver cancer; B: α2δ1 LYTAC molecules can significantly reduce the ability of drug-resistant liver cancer stem cells to form spheroids.

[0037] Figure 8 : α2δ1 LYTAC molecules can significantly inhibit the migration and invasion of lenvatinib-resistant liver cancer cells. A: α2δ1 LYTAC molecules can significantly inhibit the migration of lenvatinib-resistant liver cancer cells; B: α2δ1 LYTAC molecules can significantly inhibit the invasion of lenvatinib-resistant liver cancer cells. Detailed Implementation

[0038] 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.

[0039] Example 1: Panning, amplification, purification, sequencing and synthesis of the α2δ1 antagonistic peptide DL12-AαP5.

[0040] 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-AαP5 is synthesized.

[0041] 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.

[0042] 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.

[0043] The specific primer sequences used are as follows: Primer sequences: 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.

[0044] 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).

[0045] 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 conventional 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 titer again. The amplified product was stored at 4 °C 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 °C.

[0046] 2.3 Determination of phage titers: 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 °C. Prepare one LB / IPTG / Xgal plate for each phage dilution, preheated in a 37 °C 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.

[0047] 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 storage at -20 °C with 50% glycerol. 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 at 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.

[0048] 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), 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.

[0049] 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 target peptide sample sequence was Asp-Ala-Gln-Met-His-Pro-Pro-Ser-Tyr-Phe-Gln-Ser SEQ ID NO:1, represented as DL12-AαP5. The final short peptide was obtained from Hefei Guotai Biotechnology Co., Ltd.

[0050] Figure 1 A represents phage screening and enrichment data; Figure 1 B shows the docking diagram of the DL12-AαP5 peptide and the target protein α2δ1, proving that the DL12-AαP5 peptide and α2δ1 bind effectively.

[0051] Figure 2 A represents the high-performance liquid chromatography (HPLC) of the DL12-AαP5 peptide. Figure 2 B is the DL12-AαP5 polypeptide identified by LC-MS.

[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. 3 Cells 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 are as follows Figure 3 Compared to 293T wild-type cells that do not express α2δ1, DL12-AαP5 phage showed significantly higher expression of α2δ1 compared to 293T wild-type cells. + / + Cells, HepG2-DR cells, and HCCLM3-DR cells showed high binding capacity, with significant differences. *** P < 0.001.

[0054] Example 3: DL12-AαP5 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 reaction at a concentration of 5 × 10⁻⁶ cells / year. 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] In previous work, the inventor's laboratory successfully constructed lenvatinib-resistant liver cancer cells HepG2-DR and HCCLM3-DR, and detected high expression of α2δ1 in the lenvatinib-resistant liver cancer cells HepG2-DR and HCCLM3-DR by q-PCR.

[0057] Figure 4 The results showed that different concentrations of DL12-AαP5 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 α2δ1 Synthesis of LYTAC The lysosomal targeting receptor Lauryl-P3GKS (GalNAc) polypeptide was further synthesized using a solid-phase peptide synthesis method, with the following structural formula: . P3GKS is a six-amino acid peptide (PPPGKS SEQ ID NO.4), where Lauryl is lauroyl and GalNAc is N-acetylgalactosamine. The Lauryl-P3GKS (GalNAc) peptide JUYOU is amphiphilic (hydrophobic Lauryl tail + hydrophilic peptide chain), allowing it to self-assemble into uniform nanospheres in aqueous solution. These nanospheres serve as drug delivery platforms, with GalNAc surface modification for targeting hepatocyte ASGPR.

[0059] All the above peptides were purified by high performance liquid chromatography (HPLC) and their structures were characterized by LC-MS, which was performed by Hefei Guotai Biotechnology Co., Ltd.

[0060] The lysine (K) in the P3GKS peptide chain provides a free amino group, which is linked to the α2δ1 targeting peptide via a coupling agent (EDC / NHS).

[0061] EDC was added to the α2δ1 targeting peptide, followed by the addition of sulfonyl-NHS and reaction at room temperature for 15 min. EDC was then inactivated using 2-mercaptoethanol. The lysosome-targeting receptor peptide was then thoroughly mixed with the α2δ1 targeting peptide solution and reacted at room temperature for 2 h. Finally, the product was filtered and centrifuged using a PVDF membrane to obtain the α2δ1-targeted degradation small molecule lysosome-targeting chimera.

[0062] Figure 5 A represents the high-performance liquid chromatography (HPLC) of GalNAc peptide; Figure 5 B is the GalNAc polypeptide identified by LC-MS.

[0063] Example 5 α2δ1 LYTAC can induce the degradation of FOXM1 in HCCLM3 cells. HCCLM3-DR liver cancer cells resistant to lenvatinib were used at a dose of 5×10⁻⁶. 5 Cells were seeded per well in 6-well cell culture plates, with 2 mL of culture medium per well, and cultured for 24 h. Different concentration gradients (0 μM, 4 μM, 8 μM, 16 μM, 32 μM) of α2δ1 were then added. LYTAC cells were cultured for 16 hours (6 days for drug-resistant organoids from liver cancer), and cells were collected. Lysis buffer was added to collect proteins. 10% SDS was prepared using the Yaxin gel preparation kit. PAGE gel. Add an appropriate amount of SDS to the collected protein sample. PAGE protein loading buffer is mixed, and an appropriate amount of B is added. Mercaptoethanol. Mix protein sample with buffer, centrifuge for 10 seconds at 12,000 rpm, and set at 100°C for 5 minutes. Denature the protein for 10 minutes. Briefly centrifuge at 12000 rpm and cool. After cooling to room temperature, load the protein sample directly onto SDS-PAGE. Add the sample and pre-stained protein marker to the wells of the PAGE gel, following the designed order and aligning them with the sample loading slots. Electrophoresis is usually stopped when the bromophenol blue reaches near the bottom of the gel, or it can be stopped once the target protein is expected to be properly separated based on the electrophoresis results using the pre-stained protein molecular weight standard. Prepare the box and fill it with methanol and transfer buffer. Remove the gel glass plate from the electrophoresis apparatus. Remove the gel and use a blade to cut off the stacking gel and the bottom bromophenol blue. Measure the length and width of the gel accurately. Cut two sheets of filter paper and one PVDF membrane of the same size. Treat the membrane with methanol and shake for 1 minute, then wash it 5 times with RO water, 1 minute each time. Soak it in transfer buffer for at least 10 minutes. Also soak the filter paper and gel in transfer buffer. Use this time to clean the cotton swab inside the transfer apparatus, dry it, and then moisten it with transfer buffer. With the transfer apparatus blackboard facing you, assemble it in the following order: sponge—filter paper—gel—PVDF membrane—filter paper—sponge. Place the membranes in the correct order, ensuring there are no air bubbles on the contact surfaces between the gel and the membrane. There should also be no air bubbles between the membrane and the filter paper, or between the filter paper and the gel. You can remove air bubbles by gently rolling a spreader or a 15mL glass centrifuge tube back and forth on the surface. Using two layers of filter paper will yield better results. Incubate at 260mA for 100 minutes. Remove the membrane with tweezers, protein side up, and block it in 5% skim milk blocking buffer. 1.5h. Dilute the primary antibody with diluent according to the appropriate ratio, referring to the primary antibody's instructions. Incubate the primary antibody overnight. The next day, wash 3 times with TBST, 5 min each time, gently shaking on a shaker. Dilute the secondary antibody with diluent according to the appropriate ratio, referring to the secondary antibody's instructions. Incubate for 1 hour with gentle shaking on a shaker. Wash 5 times with TBST, 5 min each time, gently shaking on a shaker. Use ECL Western fluorometric assay kit to detect the protein.

[0064] Depend on Figure 6 It can be seen that α2δ1 LYTAC's degradation effect on α2δ1 protein is concentration-dependent. With increasing α2δ1... Increased LYTAC concentration leads to greater degradation of α2δ1 protein.

[0065] Example 6 α2δ1 LYTAC can significantly inhibit the proliferation of drug-resistant liver cancer cells. HCCLM3-DR, a drug-resistant liver cancer cell line, was used at a concentration of 5 × 10⁻⁶. 3 Cells / well were seeded into 96-well cell culture plates, with 200 μL of culture medium per well, and cultured for 24 h, followed by overnight starvation. Different concentration gradients of α2δ1 were added (100 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM). LYTAC cells were cultured for 24 hours (6 days for drug-resistant hepatocellular carcinoma organoids). 20 μL of MTT working solution was added to each well (the drug-killing effect of drug-resistant hepatocellular carcinoma organoids was detected using a CTG assay kit), and the cells were incubated in a CO2 incubator for another 4 hours. The supernatant in the culture plate was discarded, and 150 μL of DMSO (dimethyl sulfoxide) was added. The plate was shaken for 10 minutes, and the cells were detected at 490 nm using a microplate reader to plot the cell growth curve.

[0066] Figure 7 A study showed that α2δ1-LYTAC molecules can significantly reduce the IC50 of lenvatinib in drug-resistant organoids and cells. 50 The results confirmed that α2δ1-LYTAC molecules can reverse the resistance of liver cancer cells to lenvatinib. **** P <0.0001.

[0067] Example 7 α2δ1 LYTAC can significantly inhibit the spheroidization ability of drug-resistant liver cancer stem cells. First, HCCLM3-DR hepatocellular carcinoma cells were digested and prepared into single-cell suspensions. The cells were resuspended in serum-free DMEM / F12 spheroidizing medium containing B27 additive, epidermal growth factor (20 ng / mL), basic fibroblast growth factor (20 ng / mL), and insulin (4 μg / mL). The cell density was adjusted to 5 × 10³ cells / mL by cell counting. The cell suspension was then seeded into 6-well ultra-low adsorption plates, with 2 mL added to each well. α2δ1-LYTAC molecular solution was immediately added to the wells of the experimental group. The culture plates were placed in a 37°C, 5% CO2 incubator for static culture. During this period, half of the spheroidizing medium containing the appropriate drug concentration was replaced every 2 to 3 days. After 7 to 14 days of culture, at least 5 non-overlapping fields of view were randomly selected under an inverted microscope to count cell spheroids with a diameter greater than 50 μm.

[0068] Figure 7 B showed that α2δ1-LYTAC molecules can significantly inhibit the spheroidization ability of drug-resistant liver cancer stem cells, confirming that α2δ1-LYTAC molecules can inhibit the stemness of drug-resistant liver cancer cells. *** P <0.001.

[0069] Example 8 α2δ1 LYTAC can significantly inhibit the migration and invasion of drug-resistant liver cancer cells. Transwell assays were used to assess cell migration and invasion. For the migration assay, drug-resistant hepatocellular carcinoma cells HCCLM3-DR were cultured at 1 × 10⁶ cells / well. 5Cells were seeded at a density of 1,000 μL in the upper cavity of the Transwell insert, with different concentrations of α2δ1-LYTAC molecules added to serum-free medium, while the lower cavity was filled with medium containing 20% ​​FBS as a chemical inducer (600 μL). L For invasion assays, a high concentration of Matrigel basement membrane matrix was pre-diluted with serum-free culture medium at a volume ratio of 1:8. 100 μl of the diluted solution was evenly spread onto the surface of a Transwell polycarbonate membrane and cured at 37°C for 30 minutes. Subsequent steps were the same as for cell migration assays. Cells were treated with α2δ1-LYTAC molecules for 24 hours. After removing the upper chamber fluid, cells that had not penetrated the matrix and residual Matrigel were gently removed using pre-cooled PBS-moistened swabs. Cells were then fixed and stained with 4% PFA and 0.5% crystal violet for 30 minutes, respectively. The number of migrating or invading cells was quantified using ImageJ software after image acquisition.

[0070] See results Figure 8 . Figure 8 A showed that α2δ1-LYTAC molecules can significantly inhibit the migration ability of drug-resistant liver cancer cells. Figure 8 B showed that α2δ1-LYTAC molecules can significantly inhibit the invasive ability of drug-resistant liver cancer cells. ** P <0.01.

Claims

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

1.

2. A derivative of an α2δ1 antagonistic polypeptide, 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 according to claim 1, and / or the amino or carboxyl terminus of the α2δ1 antagonistic peptide fragment. Alternatively, it could be a product obtained by attaching a tag for peptide or protein detection or purification to the α2δ1 antagonistic peptide. Preferably, the conventional modifications include amination, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation, cyclization, biotinylation, fluorescent group modification, polyethylene glycol (PEG) modification, or immobilization modification; Preferably, the tag includes His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c My c or ProfinityeXact.

3. A chimeric α2δ1-targeted degradation of small molecule lysosomes, characterized in that, The lysosomal targeting chimera is a conjugate of the α2δ1 binding receptor and the lysosomal targeting receptor. The α2δ1 binding receptor is a polypeptide as shown in SEQ ID NO:1; The lysosomal targeting receptor contains a group capable of binding to the sialic acid glycoprotein receptor (ASGPR) or CI-M6PR.

4. The α2δ1-targeted degradation small molecule lysosome-targeting chimera according to claim 3, characterized in that, α2δ1 binding receptors and lysosomal targeting receptors are linked via adapters or directly; Preferably, the group capable of binding to the sialic acid glycoprotein receptor (ASGPR) is N-acetylgalactosamine (GalNAc). Preferably, the group that can bind to CI-M6PR is mannose-6-phosphate (M6P) or its oligosaccharide / glycopeptide derivative.

5. The α2δ1-targeted degradation small molecule lysosome-targeting chimera according to claim 3, characterized in that, The lysosomal targeting receptor is a self-assembled microsphere with the following structural formula: It is coupled to the α2δ1 binding receptor via the amino group of the lysine side chain.

6. A polynucleotide, characterized in that, It encodes the α2δ1 antagonistic peptide of claim 1 or a derivative thereof.

7. The use of the α2δ1-targeting degradation small molecule lysosome-targeting chimera according to any one of claims 3-5 in the preparation of a medicament for the prevention and / or treatment of tumors that highly express α2δ1.

8. The use of the α2δ1-targeted degradation small molecule lysosome-targeting chimera according to any one of claims 3-5, the α2δ1 antagonistic peptide according to claim 1, or a derivative of the α2δ1 antagonistic peptide according to claim 2 in the preparation of a medicament 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, and more preferably they 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; More preferably, the tumor cells expressing α2δ1 on their surface are drug-resistant tumor cells.

9. A method for screening or inhibiting cells in vitro for non-diagnostic and non-therapeutic purposes, characterized in that, The method includes the step of co-incubating the α2δ1 antagonistic peptide of claim 1 or the α2δ1 antagonistic peptide derivative of claim 2 with cells.

10. A pharmaceutical composition or diagnostic reagent, characterized in that, It comprises the α2δ1-targeted degradation small molecule lysosome-targeting chimera according to any one of claims 3-5, the α2δ1 antagonistic peptide according to claim 1, or a derivative of the α2δ1 antagonistic peptide according to claim 2; Preferably, the pharmaceutical composition contains one or more pharmaceutically acceptable carriers; Preferably, the pharmaceutically acceptable carrier includes diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, adsorbents, surfactants, or lubricants; Furthermore, the pharmaceutical composition is formulated as tablets, granules, capsules, oral liquids, or injections.

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