A hbb-derived peptide and its use in the manufacture of a medicament for treating a tumor

Abstract

This invention discloses an HBB-derived peptide and its use in the preparation of drugs for treating tumors. The HBB-derived peptide comprises a combination peptide consisting of a polypeptide with amino acids 30-40 at the N-terminus of HBB (as shown in SEQ ID NO:2) linked to a transmembrane peptide TAT (as shown in SEQ ID NO:3). The combination peptide is formed by modifying its N-terminus or C-terminus, through amino acid deletion, substitution, cyclization, or chiral conversion. Specifically, the amino acid sequence of this HBB-derived peptide, as shown in SEQ ID NO:1, is composed of a polypeptide with amino acids 30-40 at the N-terminus of HBB linked to a transmembrane peptide TAT. It can specifically block the binding of HBB to NDUFAF5, inhibit the proliferation of chemotherapy-resistant lung cancer cells and tumor growth, and reverse chemotherapy resistance, thus possessing significant clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to an HBB-derived peptide and its uses, specifically to an HBB-derived peptide and its use in the preparation of drugs for treating tumors. Background Technology

[0002] Lung cancer is the leading cause of cancer-related morbidity and mortality worldwide. Chemotherapy, as a first-line treatment, can significantly improve the survival prognosis of lung cancer. However, due to tumor heterogeneity, 36%-72% of stage I-III lung cancer patients develop chemotherapy resistance, leading to tumor recurrence. Treatment options after chemotherapy resistance are very limited. Switching to other types of chemotherapy drugs has some efficacy, but the prognosis remains poor. Combined immunotherapy or targeted therapy also has some effect, but individual heterogeneity is high, the prognosis is difficult to achieve as expected, and adverse reactions significantly affect the patient's quality of life. Anti-angiogenic drugs, immunosuppressants, and antibody-drug conjugates are also current research hotspots, but few have achieved significant clinical benefits. Therefore, discovering new targets and proposing effective intervention strategies has become an important scientific problem that urgently needs to be solved in the field of lung cancer treatment.

[0003] Studies show that chemotherapy resistance is caused by multiple factors, and recent research indicates that protein-protein interactions can promote tumor growth and progression, which is a significant contributing factor to chemotherapy resistance. Therefore, designing small molecule drugs or short peptides to interfere with protein-protein interactions is of great importance for anti-tumor therapy. Previous studies reported that hemoglobin subunit β (HBB) can specifically bind to nicotinamide adenine dinucleotide ubiquinone oxidoreductase complex assembly factor 5 (NDUFAF5) in chemotherapy-resistant lung cancer cells, promoting chemotherapy resistance in lung cancer. This suggests that specifically interfering with this binding has potential therapeutic value in reversing chemotherapy resistance in lung cancer. However, the specific amino acid sequence of this binding is unclear, and no drugs specifically blocking this binding have yet been developed. Summary of the Invention

[0004] To address the aforementioned problems in existing technologies, the main objective of this invention is to provide an HBB-derived peptide. Based on previous research demonstrating that the binding of HBB to NDUFAF5 can promote the growth and progression of chemotherapy-resistant lung cancer, this invention further clarifies that HBB binds to NDUFAF5 through the 30th-40th amino acid position at the N-terminus to obtain a specific amino acid sequence. This invention proposes an HBB-derived peptide comprising a combination peptide composed of a polypeptide formed by linking a polypeptide with the transmembrane peptide TAT at the 30th-40th amino acid position of the N-terminus of HBB. This peptide can be taken up by chemotherapy-resistant lung cancer cells, specifically blocking the binding of HBB to NDUFAF5, inhibiting the proliferation of chemotherapy-resistant lung cancer cells and tumor growth, thereby reversing chemotherapy resistance in lung cancer.

[0005] Another object of the present invention is to provide the use of the HBB-derived peptide in the preparation of a medicament for treating tumors.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides an HBB-derived peptide comprising a polypeptide of amino acids at positions 30-40 of the N-terminus of HBB as shown in SEQ ID NO:2 and a transmembrane peptide TAT as shown in SEQ ID NO:3, wherein the peptide is formed by modifying the N-terminus or C-terminus of the peptide, or by the deletion, substitution, cyclization or chiral conversion of amino acids.

[0008] Preferably, the HBB-derived peptide is formed by linking the N-terminal 30-40 amino acid polypeptide (GRLLVVYPWTQ) of HBB as shown in SEQ ID NO:2 with the membrane-penetrating peptide TAT (YGKKRRQRRG) as shown in SEQ ID NO:3, which can specifically block the binding of HBB to NDUFAF5.

[0009] More preferably, the amino acid sequence of the HBB-derived peptide is shown in SEQ ID NO:1 (YGKKRRQRRGGRLLVVYPWTQ), which can specifically block the binding of HBB to NDUFAF5.

[0010] A second aspect of the invention provides the use of the HBB-derived peptide in the preparation of a medicament for treating tumors.

[0011] Preferably, the tumor type is a chemotherapy-resistant lung tumor.

[0012] A third aspect of the present invention provides a pharmaceutical composition comprising the HBB-derived peptide, and wherein the HBB-derived peptide is used as an active ingredient.

[0013] A fourth aspect of the invention provides the use of the HBB-derived peptide in the preparation of a biological agent that inhibits the binding of HBB to NDUFAF5.

[0014] In a fifth aspect, the present invention provides a biological agent that inhibits the binding of HBB to NDUFAF5, comprising the HBB-derived peptide and using it as an active ingredient.

[0015] Based on previous research findings that the interaction between HBB and NDUFAF5 promotes chemotherapy resistance in chemotherapy-resistant lung cancer cells, this invention, through plasmid construction and molecular biology experiments, clarifies that the N-terminal 30-40 amino acid region of HBB is a key NDUFAF5 binding region. This region can bind to the transmembrane peptide TAT to enter chemotherapy-resistant lung cancer cells, inhibiting the binding of intracellular HBB to NDUFAF5. This inhibits tumor cell proliferation in vitro and suppresses tumor growth in mice in vivo, thus achieving the therapeutic effect of chemotherapy-resistant lung cancer.

[0016] Compared with the prior art, the present invention creatively proposes an HBB-derived peptide and its application. The HBB-derived peptide is a polypeptide composed of a polypeptide of amino acids at the N-terminus of HBB at positions 30-40 and a membrane-penetrating peptide TAT. It is named HBB-derived peptide. It can be taken up by chemotherapy-resistant lung cancer cells, specifically blocking the binding of HBB to NDUFAF5, inhibiting the proliferation of chemotherapy-resistant lung cancer cells and tumor growth, and reversing chemotherapy resistance. It has important clinical application value. Attached Figure Description

[0017] Figure 1 The following are the verification results of HBB binding to NDUFAF5 via its N-terminal 30-40 amino acids in the examples: A: Schematic diagram of constructing a Flag-tagged HBB deletion mutant, where D77-147 represent the deletion of amino acids 77-147 of HBB, D1-76 represent the deletion of amino acids 1-76 of HBB, D1-19 represent the deletion of amino acids 1-19 of HBB, D20-40 represent the deletion of amino acids 20-40 of HBB, D30-40 represent the deletion of amino acids 30-40 of HBB, and D41-76 represent the deletion of amino acids 41-76 of HBB; B: Immunoprecipitation assay to detect the binding results of HBB D1-76 to NDUFAF5 in Figure A; C: Immunoprecipitation assay to detect the binding results of HBB D77-147 to NDUFAF5 in Figure A; D: Immunoprecipitation assay to detect the binding results of HBB in Figure A. The binding results of D1-19 and NDUFAF5; E: The binding results of HBB D20-40 and NDUFAF5 in Figure A by immunoprecipitation assay; F: The binding results of HBB D30-40 and NDUFAF5 in Figure A by immunoprecipitation assay; G: The binding results of HBB D41-76 and NDUFAF5 in Figure A by immunoprecipitation assay.

[0018] Figure 2 The following are experimental results from the examples demonstrating the in vitro inhibitory effect of HBB-derived peptides on chemotherapy resistance in lung cancer: A: Microscopic examination of HBB-derived peptide uptake (marked in red) in chemotherapy-resistant lung cancer cells (A549 / DDP cells, nuclei are marked in blue); B: Immunoprecipitation assay to detect the interference of HBB-derived peptides on the binding of HBB to NDUFAF5 in chemotherapy-resistant lung cancer cells; C: MTS assay to detect the inhibitory effect of HBB-derived peptides on the proliferation of chemotherapy-resistant lung cancer cells; D: Colony formation assay to detect the inhibitory effect of HBB-derived peptides on the proliferation of chemotherapy-resistant lung cancer cells; E: Schematic diagram of the statistical results in Figure D.

[0019] Figure 3The following are experimental results from the examples demonstrating the effect of HBB-derived peptides on inhibiting chemotherapy resistance in lung cancer in vivo: A: Schematic diagram of tumors showing the inhibitory effect of HBB-derived peptides on the growth of chemotherapy-resistant lung cancer; B: Statistical graph of tumor weight showing the inhibitory effect of HBB-derived peptides on the growth of chemotherapy-resistant lung cancer; C: Graph of tumor volume showing the inhibitory effect of HBB-derived peptides on the growth of chemotherapy-resistant lung cancer.

[0020] Figure 4 The following are experimental results for detecting the toxic side effects of HBB-derived peptides on mice in the examples; A: Schematic diagram of the effect of HBB-derived peptides on the body weight of tumor-bearing nude mice; B: Schematic diagram of the effect of HBB-derived peptides on the organ / organ ratio of tumor-bearing nude mice. Detailed Implementation

[0021] To more fully understand and demonstrate the technical solutions, objectives, and advantages of the present invention, the technical effects produced by the present invention will be further described in detail and completely below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. It should be noted that other embodiments obtained by those skilled in the art without departing from the concept of the present invention are all within the protection scope of the present invention.

[0022] Unless otherwise specified, all reagents used in the following examples are of analytical grade and can be obtained from commercial sources.

[0023] Materials used in the following embodiments:

[0024] 1. The human chemotherapy-resistant lung cancer cell line A549 / DDP is a commercial cell line purchased from Qingqi (Shanghai) Biotechnology Development Co., Ltd.

[0025] 2. The HBB deletion mutant plasmid was prepared by Beijing Maijin Biotechnology Co., Ltd., and a Flag tag was attached.

[0026] 3. SPF grade BALB / c nude mice, 4 weeks old, female, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0027] 4. DMEM medium was purchased from Beijing Solarbio Science & Technology Co., Ltd., product number 11995; FBS was purchased from Ecosai Biotechnology (Taicang) Co., Ltd., product number FSP500; penicillin-streptomycin mixture (100×) was purchased from Beijing Solarbio Science & Technology Co., Ltd., product number P1400; 5 mL of penicillin-streptomycin mixture (100×) and 50 mL of FBS were added to 500 mL of DMEM medium to make complete DMEM medium.

[0028] 5. Opti-MEM™ serum-depleted culture medium was purchased from Thermo Fisher Scientific, product number 31985070.

[0029] 6. The transfection reagent Lipofectamine 2000 Reagent (Lipofectamine 2000) was purchased from Thermo Fisher Scientific, product number 11668030.

[0030] 7. HBB-derived peptides were synthesized by GenScript, linked with red fluorescent labels, and had a purity greater than 95%.

[0031] 8. The HBB-derived peptide transfection reagent, Protein Transfection Reagent, was purchased from MedChemExpress, product number HY-K2016.

[0032] 9. Hochest 33342, a nuclear dye, was purchased from Beyotime Biotechnology Co., Ltd., product number C1025.

[0033] 10. 0.25% trypsin solution was purchased from Wuhan Pronosai Life Technology Co., Ltd., product number PB180227.

[0034] 11. The hemocytometer was purchased from Hangzhou Xiaoyou Biotechnology Co., Ltd., product number 02-671-5.

[0035] 12. The matrix adhesive was purchased from Beijing Solarbio Technology Co., Ltd., product number 356234.

[0036] 13. The MTS reagent was purchased from Bio-Labs, product number KFS322.

[0037] 14. Crystal violet staining solution was purchased from Beyotime Biotechnology Co., Ltd., product number C0121.

[0038] Example 1: It was determined that HBB binds to NDUFAF5 via its N-terminal 30-40 amino acid.

[0039] The constructed HBB deletion mutant, such as Figure 1 As shown in A, HBB contains 147 amino acids, including the N-terminus and C-terminus. D77-147, D1-76, D1-19, D20-40, D30-40, and D41-76 are HBB mutants with deletions of amino acids at positions 77-147, 1-76, 1-19, 20-40, 30-40, and 41-76, respectively. The red text indicates a flag tag. The above HBB and related mutant plasmids were prepared by Beijing Maijin Biotechnology Co., Ltd.

[0040] The HBB deletion mutant that binds to NDUFAF5 in chemotherapy-resistant lung cancer cells A549 / DDP were detected by immunoprecipitation assay, as follows:

[0041] 1. Cell Plating: Take A549 / DDP cells in logarithmic growth phase, digest them with 1 mL of 0.25% trypsin solution for 1 minute, then add 1 mL of DMEM complete medium to neutralize, disperse the cells, calculate the cell concentration using a hemocytometer, and seed the cells into six-well plates. Add DMEM complete medium to a final volume of 2 mL and culture. When the cell density reaches approximately 40%-50%, wash the cells twice with PBS buffer, and then perform cell transfection.

[0042] 2. Cell Transfection: Add 1.5 mL of Opti-MEM™ serum-depleted culture medium to each well of a six-well plate. The following is the preparation of the transfection solution required for one well of a six-well plate: Take a 1.5 mL RNase-free EP tube, dilute 2 μg of HBB deletion mutant plasmid with 250 μL of Opti-MEM™ serum-depleted medium and let stand for 5 minutes; take another 1.5 mL RNase-free EP tube, dilute 5 μL of Lipofectamine 2000 with 250 μL of Opti-MEM™ serum-depleted medium and let stand for 5 minutes, then mix the two tubes to form the transfection solution, let stand for 20 minutes, and finally add the entire transfection solution to one well of a six-well plate. Incubate in a CO2 incubator for 6 hours, then replace the transfection solution with DMEM complete medium and continue culturing for 48 hours. Cells were collected after 48 hours, and protein was extracted by adding an appropriate amount of NP-40 lysis buffer. 50 μL of protein lysate was added to 30 μL of Protein A / G beads and 2 μg of Flag antibody and incubated at 4°C overnight. The beads were collected, and 30 μL of 1×SDS loading buffer was added. The mixture was boiled for 5 min and then subjected to SDS-PAGE electrophoresis to detect the co-precipitated NDUFAF5.

[0043] The results are as follows Figure 1 As shown in BG, Figure 1 B shows that HBB D1-76 does not bind to NDUFAF5, indicating that HBB binds to NDUFAF5 through amino acids at positions 1-76; Figure 1 C shows that HBB D77-147 binds to NDUFAF5, indicating that the HBB sequence that binds to NDUFAF5 is not located at amino acids 77-147. Figure 1 The D-scan shows that HBB D1-19 binds to NDUFAF5, indicating that the HBB sequence that binds to NDUFAF5 is not located at amino acids 1-19. Figure 1E shows that HBB D20-40 does not bind to NDUFAF5, indicating that HBB binds to NDUFAF5 through amino acids at positions 20-40; Figure 1 F shows that HBB D30-40 does not bind to NDUFAF5, indicating that HBB binds to NDUFAF5 through amino acids at positions 30-40. Figure 1 G shows that HBB D41-76 binds to NDUFAF5, indicating that the HBB sequence that binds to NDUFAF5 is not at amino acid positions 41-76.

[0044] comprehensive Figure 1 The results from BG indicate that HBB binds to NDUFAF5 via amino acids at positions 30-40, with the specific sequence being GRLLVVYPWTQ.

[0045] Example 2: Detection of the effect of HBB-derived peptides on inhibiting chemotherapy resistance in lung cancer in vitro

[0046] (1) Microscopic detection of HBB-derived peptide uptake (marked in red) in chemotherapy-resistant lung cancer cells A549 / DDP (nuclei are labeled in blue):

[0047] HBB-derived peptide synthesis: Based on the results in Example 1, it was determined that amino acids 30-40 of HBB bind to NDUFAF5, with the sequence GRLLVVYPWTQ, i.e., Gly-Arg-Leu-Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln. The HBB-derived peptide is formed by linking amino acids 30-40 of HBB with the membrane-penetrating peptide TAT (sequence YGKKRRQRRG, whose function is to promote cellular uptake). The sequence following the linking of the membrane-penetrating peptide TAT is as follows: YGKKRRQRRGGRLLVVYPWTQ, i.e., Tyr-Gly-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Gly-Gly-Arg-Leu-Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln. A red fluorescent label was attached to visualize the HBB-derived peptide. The control peptide sequence is YGKKRRQRRG. Both the HBB-derived peptide and the control peptide were synthesized by GenScript with a purity greater than 95%.

[0048] Cell seeding: Select A549 / DDP cells that are in good growth condition, in the exponential growth phase, and have a viability > 90% for transfection. Taking a 24-well plate as an example, the day before transfection, divide the digested cells into 1-3 × 10⁶ cells per well. 5 Each cell is plated to ensure a density of 50%-100% during transfection.

[0049] Prepare the transfection reagent-protein complex: Add 1-10 μg of HBB-derived peptide to a 1.5 mL centrifuge tube, add 1-3 μL of MCE Protein Transfection Reagent, mix gently and incubate at room temperature for 3 min.

[0050] Cell transfection: During incubation of the transfection reagent-protein complex, discard the cell culture medium and wash 2-3 times with preheated serum-free medium (37°C) or 1×PBS. Gently add the transfection reagent-protein complex to each well, mix gently, and continue incubation.

[0051] Assay for HBB-derived peptide uptake by A549 / DDP cells: Six hours after transfection, the transfection reagent-protein complex was removed, and the cells were washed 2-3 times with pre-warmed serum-free medium (37°C) or 1×PBS. Hoechst 33342 nuclear dye was diluted to 1× with serum-free DMEM and added to the cells. The cells were incubated for half an hour. Finally, the cells were washed again with pre-warmed serum-free medium (37°C), and 1 mL of complete DMEM was added. The uptake of HBB-derived peptides by A549 / DDP cells was detected under a microscope.

[0052] The results are as follows Figure 2 As shown in Figure A, the nuclei of A549 / DDP cells, stained with Hoechst 33342, appear blue and are round or nearly round. HBB-derived peptides, carrying a red fluorescent label, show fluorescence surrounding the cell nucleus, indicating that A549 / DDP cells successfully took up HBB-derived peptides.

[0053] (2) The interference of HBB-derived peptides on the binding of HBB to NDUFAF5 in chemotherapy-resistant lung cancer cells A549 / DDP was detected by immunoprecipitation experiment: The cell plating, transfection of HBB-derived peptides and control peptides were the same as in (1), the immunoprecipitation detection method was the same as in Example 1, and finally the changes in the binding of HBB to NDUFAF5 were detected by SDS-PAGE electrophoresis.

[0054] The results are as follows Figure 2 As shown in Figure B, compared with the control peptide treatment group, the binding of HBB to NDUFAF5 was significantly reduced in the HBB-derived peptide treatment group, indicating that HBB-derived peptides can interfere with the binding of HBB to NDUFAF5.

[0055] (3) The effect of HBB-derived peptide on inhibiting the proliferation of chemotherapy-resistant lung cancer cells was detected by MTS assay: 3000 A549 / DDP cells were plated in 96-well plates according to method (1), and transfected with HBB-derived peptide and control peptide for 0, 24, 48, and 72 h according to method (1). The MTS solution was diluted with serum-free DMEM medium (volume ratio 1:9) to prepare MTS working solution. The medium was removed from the 96-well plates, and 100 μL of MTS working solution was added to each well. The cells were incubated in a cell culture incubator for 1-2 h. The OD value at 490 nm was detected using an enzyme-linked immunosorbent assay (ELISA) reader. The difference in cell activity was evaluated based on the difference in OD value compared with the control peptide group.

[0056] The results are as follows Figure 2 As shown in Figure C, the experiment was independently repeated three times. Statistical analysis was performed using Student's t-test to assess the significance of the effects of HBB-derived peptides and control peptides on cell proliferation. A p-value indicates statistical significance, with a p-value less than 0.05 indicating statistical significance. ns indicates no statistical significance, suggesting no difference between the two groups; ** and *** indicate p-values ​​less than 0.01 and 0.001, respectively. The results showed that compared to the control peptide treatment group, cell viability was not different at 0 h, but significantly decreased at 24, 48, and 72 h in the HBB-derived peptide treatment group, indicating that HBB-derived peptides can significantly inhibit the proliferation of chemotherapy-resistant lung cancer cells.

[0057] (4) The effect of HBB-derived peptide on inhibiting the proliferation of chemotherapy-resistant lung cancer cells was detected by colony formation assay: 1000 A549 / DDP cells were seeded in 6-well plates according to method (1), and transfected with HBB-derived peptide and control peptide. The transfection was repeated once after seven days. After 14 days, the culture medium was removed, and the cells were fixed with paraformaldehyde for 15 min. Then, 0.5 mL of crystal violet staining solution was added to stain the cells for 15 min. Finally, the crystal violet solution was removed, the cells were rinsed under tap water, air-dried, and clones with more than 50 cells were counted.

[0058] The results are as follows Figure 2 As shown in Figure D, the number of cell clones in the HBB-derived peptide treatment group was less than that in the control group, indicating that HBB-derived peptides inhibited the proliferation of chemotherapy-resistant lung cancer cell A549 / DDP clones.

[0059] (5) Statistical analysis: The statistical results of the three independent repeated experiments in (4) are shown in the figure below. Figure 2As shown in Figure E, the statistical significance of the effects of HBB-derived peptides and control peptides on clonal proliferation was assessed using Student's t-test. P-values ​​were used to indicate statistical significance, with a value less than 0.05 indicating statistical significance. ** indicates a P-value less than 0.01. The results showed that clonal proliferation in the HBB-derived peptide treatment group was significantly lower than that in the control peptide treatment group, indicating that HBB-derived peptides can significantly inhibit the clonal proliferation of chemotherapy-resistant lung cancer cells.

[0060] from Figure 2 As can be seen from CE, HBB-derived peptides significantly inhibit the proliferation and colony formation of chemotherapy-resistant lung cancer cells.

[0061] Example 3: Detection of the effect of HBB-derived peptides in inhibiting chemotherapy resistance in lung cancer in vivo

[0062] (1) Growth inhibitory effect of HBB-derived peptides on chemotherapy-resistant lung cancer (tumor illustration):

[0063] 1. Subcutaneous xenograft construction and grouping

[0064] (1) Subcutaneous xenograft construction: Ten 4-week-old female BALB / c nude mice were used. A549 / DDP cells in the logarithmic growth phase were digested and collected by centrifugation. The cells were washed twice with PBS buffer and resuspended in PBS buffer. The cells were counted using a hemocytometer and the cell density was adjusted to 1×10⁻⁶. 6 50 μL. 50 μL contains 1×10 6 A PBS suspension of A549 / DDP cells was mixed with matrix gel at a volume ratio of 1:1 and then injected into the subcutaneous area of ​​the right rib of nude mice.

[0065] (2) Nude mouse grouping and treatment: When the tumor reaches a size of approximately 100 mm 3 Nude mice were randomly divided into a control peptide treatment group and an HBB-derived peptide treatment group. 200 μL of the control peptide and HBB-derived peptide were injected intraperitoneally once a day for a total of 10 times.

[0066] 2. Tumor illustration: Mice were euthanized after treatment, tumor tissues were removed, arranged in groups and photographed to compare the effects of HBB-derived peptides and control peptides on the growth of chemotherapy-resistant lung cancer.

[0067] HBB-derived peptides inhibit the growth of chemotherapy-resistant lung cancer tumors such as Figure 3 As shown in Figure A, it is visible to the naked eye that the tumors in the HBB-derived peptide-treated group were smaller than those in the control peptide-treated group, indicating that HBB-derived peptides have a significant inhibitory effect on the growth of chemotherapy-resistant lung cancer.

[0068] (2) Growth inhibitory effect of HBB-derived peptides on chemotherapy-resistant lung cancer: Tumor weight statistics: Figure 3 The tumors in group A were weighed, and a statistical test using Student's t was performed to assess the significance of the effects of HBB-derived peptides and control peptides on tumor growth. The p-value represents statistical significance, with a p-value less than 0.05 indicating statistical significance. **** indicates a p-value less than 0.0001. Results are as follows... Figure 3 As shown in Figure B, the tumor weight in the HBB-derived peptide treatment group was significantly lower than that in the control peptide treatment group, indicating that HBB-derived peptides can significantly inhibit the growth of chemotherapy-resistant lung cancer.

[0069] (3) Growth inhibitory effect of HBB-derived peptides on chemotherapy-resistant lung cancer: tumor volume statistics: Figure 3 The tumor in A was measured for its major and minor diameters using the formula: Volume = 1 / 2 × Shortest Diameter. 2 Tumor volume was calculated using the longest diameter. Statistical analysis was performed using Student's t-test, with a p-value less than 0.05 indicating statistical significance. **** indicates a p-value less than 0.0001. Results are as follows... Figure 3 As shown in Figure C, HBB-derived peptides significantly inhibited tumor volume, which was statistically significant, indicating that HBB-derived peptides have an extremely significant effect on inhibiting tumor growth.

[0070] from Figure 3 AC results show that HBB-derived peptides significantly inhibit the growth of chemotherapy-resistant lung cancer tumors.

[0071] Example 4: Detection of the toxic side effects of HBB-derived peptides on mice

[0072] (1) Effect of HBB-derived peptides on body weight in tumor-bearing nude mice: Drug treatment may produce side effects such as vomiting, weight loss, bone marrow suppression, and liver and kidney toxicity. Among these, body weight is the most convenient indicator to monitor throughout the treatment process and will not cause adverse reactions in nude mice. The absence of weight loss indicates that the drug is essentially non-toxic and will not cause treatment side effects. For animal experiments, assessing the ratio of vital organs to body weight can also indirectly reflect the drug's toxic side effects and help determine them. In this embodiment, drug side effects were assessed by monitoring changes in mouse body weight during administration and the ratio of vital organs (heart, spleen, lung, kidney, liver) to body weight after treatment.

[0073] The body weight of nude mice in each group was monitored from the start to the end of treatment, and a line graph of body weight growth was plotted using GraphPad Prism 8.0 software. Statistical analysis was performed using Student's t-test, with a p-value less than 0.05 indicating statistical significance. ns indicates a p-value greater than 0.05, meaning no significant difference in the indicators between the two groups. Results are as follows... Figure 4As shown in Figure A, it can be seen that HBB-derived peptides did not cause a decrease in body weight in nude mice.

[0074] (2) Effect of HBB-derived peptides on organ-to-body weight ratio in tumor-bearing nude mice: After treatment, the body weight and organ (heart, spleen, lung, kidney, liver) weights of the mice were measured, and the organ-to-body weight ratio was calculated. Graphs were plotted using GraphPad Prism 8.0 software, and statistical analysis was performed using Student's t-test. A p-value less than 0.05 indicated statistical significance. ns indicates a p-value greater than 0.05, meaning there was no significant difference in the comparative indicators between the two groups. Results are as follows: Figure 4 As shown in Figure B, it can be seen that, compared with the control group, HBB-derived peptides did not cause changes in the organ / body weight ratio in nude mice.

[0075] from Figure 4 A and Figure 4 As shown in B, HBB-derived peptides have virtually no effect on the weight and organ / body weight ratio of nude mice, indicating that HBB-derived peptides have no significant toxic side effects on nude mice.

[0076] from Figure 3 AC and Figure 4 The results from AB show that HBB-derived peptides inhibit the growth of chemotherapy-resistant lung cancer without significant toxic side effects.

[0077] In summary, based on previous research reports that HBB binding to NDUFAF5 promotes chemotherapy resistance in lung cancer, this invention firstly, through the construction of an HBB deletion mutant plasmid and combined with immunoprecipitation experiments, clarified that HBB binds to NDUFAF5 via its N-terminal 30-40 amino acids. Secondly, experiments demonstrated that the in vitro synthesized HBB N-terminal 30-40 amino acid-derived peptide can enter chemotherapy-resistant lung cancer cells via the transmembrane peptide TAT, specifically inhibiting the intracellular binding of HBB to NDUFAF5, and significantly inhibiting the proliferation of chemotherapy-resistant lung cancer cells in in vitro cell experiments, as well as inhibiting the growth of chemotherapy-resistant lung tumors in mice. Furthermore, by monitoring mouse body weight and the ratio of vital organs to body weight, this HBB-derived peptide showed no significant toxic side effects when used in vivo. This invention proposes a novel HBB-derived peptide that can be taken up by chemotherapy-resistant lung cancer cells, specifically blocking the binding of HBB to NDUFAF5, inhibiting the proliferation of chemotherapy-resistant lung cancer cells and tumor growth, thereby reversing chemotherapy resistance in lung cancer. This peptide has significant clinical application value for the preparation of drugs to treat chemotherapy-resistant lung tumors.

[0078] The above are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An HBB-derived peptide, characterized in that, It comprises a combination peptide consisting of a polypeptide with amino acids at positions 30-40 of the N-terminus of HBB as shown in SEQ ID NO:2 and a transmembrane peptide TAT as shown in SEQ ID NO:3, which is formed by modifying the N-terminus or C-terminus of the combination peptide, or by the deletion, substitution, cyclization or chiral conversion of amino acids.

2. The HBB-derived peptide of claim 1, wherein, The HBB-derived peptide is formed by linking the N-terminal 30-40 amino acid polypeptide of HBB as shown in SEQ ID NO:2 with the membrane-penetrating peptide TAT as shown in SEQ ID NO:3, and can specifically block the binding of HBB to NDUFAF5.

3. The HBB-derived peptide of claim 1, wherein, The amino acid sequence of the HBB-derived peptide is shown in SEQ ID NO:1, and it can specifically block the binding of HBB to NDUFAF5.

4. Use of the HBB-derived peptide according to any one of claims 1 to 3 in the preparation of a medicament for treating tumors.

5. Use according to claim 4, characterized in that, The tumor type is chemotherapy-resistant lung tumor.

6. A pharmaceutical composition, characterized by, It includes the HBB-derived peptide as described in any one of claims 1 to 3, and uses it as an active ingredient.

7. Use of the HBB-derived peptide according to any one of claims 1 to 3 in the preparation of a biological agent that inhibits the binding of HBB to NDUFAF5.

8. A biological agent that inhibits the binding of HBB to NDUFAF5, characterized in that, It includes the HBB-derived peptide as described in any one of claims 1 to 3, and uses it as an active ingredient.

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