Targeted CANX and SIRT7 combined polypeptide inhibitor and application thereof in lung adenocarcinoma radiation resistance treatment

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a targeted CANX and SIRT7 combined polypeptide inhibitor and application thereof in lung adenocarcinoma radiation resistance treatment. The polypeptide has a sequence TKPPSRRPKL (SEQ ID NO: 1), and can be competitively combined with CANX protein and block succinylation modification of SIRT7 on the CANX protein, so that the localization and function of CANX in MAM are recovered, mitochondrial metabolism reprogramming is reversed, and the radiosensitivity of lung adenocarcinoma cells is enhanced. Experiments show that the polypeptide can significantly inhibit the combination of CANX and SIRT7 in vitro and improve the radiosensitivity of radiation-resistant lung adenocarcinoma cells. The invention provides a new targeting strategy and drug candidate for treatment of lung adenocarcinoma radiation resistance.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polypeptide inhibitor that targets the binding of calcinoxane (CANX) to desuccinylase SIRT7, and its application in reversing radioresistance in lung adenocarcinoma and enhancing radiotherapy sensitivity. Background Technology

[0002] Lung adenocarcinoma is the major subtype of non-small cell lung cancer, and radiotherapy is a crucial treatment for patients with inoperable locally advanced disease. However, radioresistance of tumor cells is a major cause of treatment failure. Studies have shown that tumor microenvironment hypoxia can upregulate the expression of desuccinylation enzyme SIRT7 by activating hypoxia-inducible factor-2α (HIF-2α). SIRT7 specifically removes the succinylation modification of lysine (K137) at position 137 of the endoplasmic reticulum molecular chaperone CANX protein, a modification that is key to driving CANX localization to the mitochondrial-associated endoplasmic reticulum membrane (MAM). CANX MAM localization is essential for maintaining calcium ion signaling and cellular metabolic homeostasis between the endoplasmic reticulum and mitochondria. Therefore, aberrant activation of the HIF-2α / SIRT7 / CANX signaling axis leads to MAM dysfunction, metabolic reprogramming, and ultimately, radioresistance.

[0003] Currently, intervention strategies for radioresistance in lung adenocarcinoma mostly focus on upstream targets, such as developing small-molecule inhibitors of HIF-2α or using broad-spectrum Sirtuin enzyme activity inhibitors. However, these strategies have significant limitations: HIF-2α inhibitors may affect numerous downstream genes they regulate, producing unpredictable off-target effects; while pan-Sirtuin inhibitors lack specificity for SIRT7, potentially interfering with the normal physiological functions of other Sirtuin family members, leading to significant toxic side effects. More importantly, existing technologies lack intervention tools that can directly and precisely disrupt the protein-protein interaction (PPI) between CANX and SIRT7. Directly targeting this PPI could block the aforementioned pathogenic signaling axis at a core functional level, potentially providing a new anti-radiation resistance strategy with higher specificity and fewer side effects.

[0004] Therefore, developing a novel inhibitor that can efficiently and specifically inhibit the binding of CANX and SIRT7 is of great clinical significance and application value for overcoming radioresistance in lung adenocarcinoma. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a peptide inhibitor that targets the binding of calcium conjugate protein (CANX) to desuccinylation enzyme SIRT7. The novel inhibitor provided by this invention can directly and precisely block the protein-protein interaction (PPI) between CANX and SIRT7. Based on a peptide molecule designed with the SIRT7 binding interface, this molecule can competitively bind to the CANX protein, thereby blocking SIRT7's desuccinylation modification of CANX, restoring the succinylation level of CANX and its correct location and function in the MAM, thus reversing downstream calcium homeostasis imbalance and the Warburg effect, ultimately achieving the goal of sensitizing lung adenocarcinoma cells for radiotherapy and overcoming radioresistance.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0007] The present invention discloses a polypeptide, characterized in that the polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 1.

[0008] Furthermore, the polypeptide also includes a cell-penetrating peptide sequence linked to a cell-penetrating peptide sequence.

[0009] The present invention also discloses the use of any one of the above-mentioned polypeptides in the preparation of a medicament for treating radiation resistance in lung adenocarcinoma.

[0010] The present invention also discloses a radiosensitizer, characterized in that it comprises the polypeptide described in any one of the above-mentioned examples.

[0011] The present invention also discloses the use of the sensitizer described above in the preparation of a medicament for treating radiation resistance in lung adenocarcinoma.

[0012] Furthermore, the sensitizer enhances the sensitivity of lung adenocarcinoma cells to radiotherapy, overcoming radioresistance.

[0013] The present invention also discloses a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the polypeptide described in any one of the above-mentioned examples, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0014] Furthermore, the dosage form of the pharmaceutical composition is a pharmaceutically acceptable dosage form.

[0015] The present invention also discloses the use of any of the above-described pharmaceutical compositions in the preparation of a medicament for treating radiation resistance in lung adenocarcinoma.

[0016] The present invention also discloses a reagent for inhibiting or detecting the interaction between CANX and SIRT7 protein in vitro, characterized in that it comprises the polypeptide described in any one of the above.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0018] Compared with currently researched HIF-2α small molecule inhibitors or pan-Sirtuin inhibitors, this invention has the following significant advancements and outstanding advantages: Novel and groundbreaking mechanism of action: This is the first reported inhibitor specifically targeting CANX-SIRT7 PPI. It regulates post-translational modifications and subcellular organelle functions from a completely new perspective (intervening in protein-protein interactions), providing an original strategy for overcoming radioresistance.

[0019] Precise targeting and high specificity: Compared to upstream HIF-2α inhibitors (which may affect multiple downstream pathways) or SIRT7 enzyme activity inhibitors (which may affect all their substrates), this peptide inhibitor precisely targets a specific pair of protein interactions, theoretically resulting in a lower risk of off-target effects, less interference with normal cell function, and superior safety potential.

[0020] Targeting the core link with high efficiency: This peptide acts directly on the key link of signal transduction (SIRT7-CANX binding), avoiding the compensatory or feedback activation that may be caused by intervention from further upstream, and is expected to more efficiently reverse downstream pathological phenotypes.

[0021] A clear chain of evidence demonstrating clinical translational potential: This invention not only has rigorous structural biology evidence (MOE docking) and biochemical validation (Co-IP), but also demonstrates clear functional effects in disease models: In a radioresistant lung adenocarcinoma cancer model, this peptide effectively enhances radiosensitivity and inhibits tumor growth. This lays a solid experimental foundation for its development as a novel radiosensitizer. Attached Figure Description

[0022] Figure 1 Schematic diagram of the interaction interface and molecular docking interaction between CANX and SIRT7 protein. A represents the CANX-SIRT7 protein interaction structure; B represents the docking interaction between the molecule and the target site.

[0023] Figure 2 Figure showing the protein-protein interaction results between CANX and SIRT7.

[0024] Figure 3 CANX and SIRT7 immunofluorescence co-localization map.

[0025] Figure 4 The inhibitory effect of L-17 on lung adenocarcinoma cells A549.

[0026] Figure 5 L-17 survival rate of lung adenocarcinoma cells (bar chart).

[0027] Figure 6 Co-IP verification of the inhibitory effect of L-17 on the interaction between CANX and SIRT7 proteins.

[0028] Figure 7 Image showing the radiosensitizing effect of L-17 on A549 lung adenocarcinoma cells. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0030] Unless otherwise specified, all reagents and materials used in this invention are commercially available.

[0031] I. Experimental Materials.

[0032] 1. Cell line: Human lung adenocarcinoma cell line A549.

[0033] 2. Experimental reagents: DMEM medium, trypsin, fetal bovine serum, CCK8 reagent, antibodies, etc.

[0034] II. Experimental Methods.

[0035] 1. Use MOE software for drug screening.

[0036] First, the protein structures of CANX and SIRT7 were modeled using MOE homology. After structural optimization, molecular docking was performed, and finally, the peptide inhibitor sequence with the best score was selected: TKPPSRRPKL (synthesized by WuXi AppTec).

[0037] Peptide sequence: The peptide inhibitor of the present invention has the following amino acid sequence: Thr-Lys-Pro-Pro-Ser-Arg-Arg-Pro-Lys-Leu (TKPPSRRPKL), as shown in SEQ ID NO: 1.

[0038] Synthesis and modification of the peptide: To enhance its cell penetration, the N-terminus of the peptide was acetylated and the C-terminus was amidated. The peptide was prepared using conventional solid-phase peptide synthesis methods in the art and purified by high-performance liquid chromatography (HPLC) to a purity >95%. Its molecular weight was verified by mass spectrometry.

[0039] Acquisition of the polypeptide: The polypeptide can be synthesized by a commercial polypeptide synthesis company (such as WuXi AppTec) according to the above sequence and modification requirements. In the specific experiments of this invention, the polypeptide used was obtained in this manner.

[0040] 2. CCK8 cell proliferation assay.

[0041] The Cell Counting Kit-8 (CCK-8) reagent is a simple and accurate method for analyzing cell proliferation and cytotoxicity, serving as an alternative to the MTT assay. Its basic principle is based on WST (water-soluble tetrazolium salt, chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt), a rapid and highly sensitive assay widely used for cell proliferation and cytotoxicity. It can be used for drug screening, cell proliferation assays, cytotoxicity assays, and tumor drug sensitivity testing. In this experiment, logarithmic growth phase lung adenocarcinoma cells A549 were seeded at a density of 8 × 10³ cells / well in a 96-well plate. The cells were then treated with 3.61 nM for 24 hours, followed by replacement with 100 µL of fresh culture medium, and then 10 µL of CCK-8 (Dojindo Molecular Technologies Inc., Japan) was added to each well for 1 hour. The absorbance at 450 nm was measured using a multi-mode microplate reader (LD942, Beijing, China). Each group had 6 replicates, with an equal volume of culture medium added to the control group, and blank wells containing neither cells nor the test substance were also included.

[0042] Cell viability = (OD value of experimental wells - OD value of blank wells) / (OD value of control group - OD value of blank wells) × 100%.

[0043] 3. Cell transfection and Western blotting.

[0044] Select an appropriate density for seeding A549 cells in a six-well plate based on their growth rate. Before transfection, ensure the cells reach 60-70% confluence. Prepare the transfection reagent in a 1mL system as follows: 500µL serum-free medium + 2µL lipo, mix gently, and incubate at room temperature for 5 min. Then, add 1000ng plasmid to 500µL serum-free medium, mix gently, and incubate at room temperature for 5 min. Combine the two tubes, mix well, and incubate for 20 min. The transfection reagent is now ready. Aspirate the original medium, wash twice with 1×PBS, add the prepared transfection reagent, and gently shake the cell culture plate. Extract protein from the cultured cells, quantify the protein, denature the protein, prepare a gel, load the sample, perform electrophoresis, transfer to a membrane, block, and incubate with primary and secondary antibodies. Finally, perform chemiluminescence.

[0045] 4. Co-IP experiments were used to detect the interaction between CANX and SIRT7.

[0046] Protein samples were prepared from A549 lung adenocarcinoma cells: Protein A / G Sepharose was added and incubated at 4°C for half an hour. The antibody to be tested was added to the supernatant sample and incubated at 4°C for two hours. After incubation, Protein A / G Sepharose was added and incubated at 4°C overnight for Western blotting experiments.

[0047] 5. Immunofluorescence detection of co-localization of CANX and SIRT7.

[0048] (1) Preparation and treatment of cell spreaders: Place clean coverslips in 24-well plates and seed each group of cells at a density of 5×10⁴ / well. When the cells have adhered and grown to about 70% confluence, perform the appropriate treatments according to the groups (such as transfection, drug addition). After treatment, aspirate the culture medium and gently wash the cells twice with pre-cooled PBS.

[0049] (2) Fixation and permeabilization: Add 1 mL of 4% PFA to each well and fix for 20 minutes at room temperature. Discard the fixative and wash three times with PBS for 5 minutes each time. Add 1 mL of 0.5% Triton X-100 to each well and permeabilize for 15 minutes at room temperature. Wash three times with PBS for 5 minutes each time.

[0050] (3) Blocking and non-specific binding inhibition: Add 1 mL of 5% BSA to each well and block at room temperature for 1 hour. Discard the blocking solution; no rinsing is required.

[0051] (4) Primary antibody incubation: Dilute the primary antibody with 5% BSA according to the recommended ratio in the instructions (CANX 1:200, SIRT7 1:100 is recommended).

[0052] Add 50-100 μL of the prepared primary antibody mixture (containing rabbit anti-CANX and mouse anti-SIRT7) to the cell surface of the coverslip, ensuring complete coverage of the cells. Place the 24-well plate in a humidified chamber and incubate overnight (approximately 16 hours) at 4°C.

[0053] (5) Secondary antibody incubation: Recover the primary antibody and wash the cells three times with PBS-T (PBS containing 0.1% Tween-20), 10 minutes each time. Dilute the fluorescent secondary antibody (Alexa Fluor 488 goat anti-rabbit, Alexa Fluor 594 goat anti-mouse, recommended dilution 1:500) with 5% BSA. Under light-protected conditions, add the secondary antibody mixture to cover the cells and incubate at room temperature for 1 hour. Discard the secondary antibody and wash the cells three times with PBS-T, 10 minutes each time.

[0054] (6) Nuclear staining and mounting: Dilute DAPI to the working concentration (1 μg / mL) with PBS, add it to cover the cells, and incubate at room temperature in the dark for 5 minutes. Discard the DAPI and rinse 3 times with PBS in the dark. Use tweezers to remove the coverslip, cell side down, add one drop of anti-fluorescence quenching mounting medium to the slide, gently cover and avoid air bubbles.

[0055] (7) Image acquisition and analysis: The prepared slides were stored at 4°C in the dark and observed under a laser confocal microscope within 24 hours.

[0056] Image acquisition: Using a 63x or 100x oil immersion lens, images of the same field of view were acquired sequentially using laser channels at 405nm (DAPI, blue), 488nm (Alexa Fluor 488, green), and 561nm (Alexa Fluor 594, red), respectively. At least 10 fields of view were randomly acquired for each group.

[0057] 6. The plate cloning experiment uses a single-machine multi-target model to detect radiosensitivity.

[0058] (1) Cell preparation and seeding: A549 cells in the logarithmic growth phase were digested with trypsin to prepare a single-cell suspension. The cells were resuspended in complete culture medium and accurately counted using a cell counter.

[0059] Based on the cell seeding density determined in the preliminary experiments, cells were seeded at 5000 cells per well in 6-well plates. Key point: The seeding density should ensure approximately 50-150 morphologically sound single clones in the unirradiated group (0 Gy), while still maintaining a countable number of clones in the highest dose group (8 Gy). Typically, A549 cells are seeded in gradient experiments at 500-5000 cells per well. Gently shake to ensure even cell distribution, and incubate at 37°C, 5% CO2 for 12-24 hours to allow for full cell adhesion and cell cycle initiation.

[0060] (2) X-ray irradiation treatment: Well-adhered cells were removed from the incubator and labeled into groups. Irradiation was performed using 6MV X-rays. Irradiation dose groups were set up as follows: 0 Gy (sham control), 2 Gy, 4 Gy, 6 Gy, and 8 Gy. Each dose group had at least three replicates. Cells were placed on the linear accelerator treatment bed, ensuring uniform dose distribution. The 0 Gy control group underwent the same treatment process as the irradiated groups (e.g., transport to the radiotherapy room), but was not irradiated.

[0061] After irradiation, the cells were immediately returned to the incubator to continue culturing.

[0062] (3) Clonal Culture and Fixation / Staining: Cells are cultured continuously in an incubator for 10-14 days, with regular observation and replacement of fresh complete culture medium every 2-3 days. Culture is terminated when a clear clone (usually consisting of more than 50 cells) is visible in the 6-well plate and the background is clean. Discard the culture medium and gently wash the cells 1-2 times with PBS. Add 2 mL of methanol to each well and fix at room temperature for 15 minutes. Discard the fixative and allow the plate to air dry naturally or gently aspirate. Add an appropriate amount of crystal violet staining solution (approximately 1 mL) to each well and stain at room temperature for 20-30 minutes. Discard the staining solution and rinse gently with running tap water until the background is clean. Invert the plate to air dry.

[0063] (4) Clone counting and data calculation: The clones formed in each well are counted using the naked eye or a microscope. A cell cluster with ≥ 50 cells is usually defined as a countable clone.

[0064] Calculate the plating efficiency (PE) and survival fraction (SF): PE (%) = (Average number of clones in the 0 Gy group / Number of cells seeded in the 0 Gy group) × 100% SF = (Average clone count in the dose irradiation group) / (Average clone count in the 0 Gy group) Note: Inoculation efficiency needs to be considered here, but relative survival is usually calculated directly from the observed number of clones.

[0065] III. Experimental Results.

[0066] (a) Validation results of the interaction between CANX and SIRT7 protein.

[0067] 1. Computational simulation of the CANX-SIRT7 interface and design of peptide inhibitors ( Figure 1 ).

[0068] To target the protein-protein interaction (PPI) between CANX and SIRT7, homology modeling and optimization of the protein structures of CANX and SIRT7 were first performed using MOE software. Molecular docking analysis predicted that the amino acid sequence at positions 285-294 of the SIRT7 protein (TKPPSRRPKL) constitutes the key interface for its binding with CANX. This sequence is located within the NAD+ binding domain of SIRT7, and we hypothesized that it could act as a "competitive peptide," mimicking the local structure of SIRT7 to occupy the binding site on CANX, thereby blocking their binding. Based on this, a peptide inhibitor with the sequence TKPPSRRPKL was designed and synthesized, named L-17.

[0069] 2. Co-IP verification of the interaction between CANX and SIRT7 protein ( Figure 2 ).

[0070] The endogenous interaction between CANX and SIRT7 in A549 lung adenocarcinoma cells was detected using a co-immunoprecipitation (Co-IP) assay. In the cell group overexpressing the Flag tag CANX, a distinct SIRT7 protein band was detected by Western blotting after immunoprecipitation with an anti-Flag antibody. No specific band was detected in the control group (Vector) or the negative control group using standard IgG. These results confirm the existence of a specific physical interaction between CANX and SIRT7 in A549 cells.

[0071] 3. Validation of intracellular co-localization of CANX and SIRT7 ( Figure 3 ).

[0072] Using immunofluorescence colocalization, CANX (green fluorescence) and SIRT7 (red fluorescence) were labeled in A549 cells, respectively. Confocal microscopy images showed extensive overlap of their fluorescence signals in the cytoplasm (exhibiting yellow fluorescence), indicating a high degree of consistency in the intracellular spatial distribution of CANX and SIRT7, further supporting their interaction at the subcellular level.

[0073] (II) Results of the validation of the inhibitory activity and mechanism of action of the peptide inhibitor L-17 on A549 cells.

[0074] 1. Effects of L-17 on A549 cell viability and IC50 assay ( Figure 4 , 5 ).

[0075] The inhibitory effect of L-17 on the proliferation of A549 cells was detected using the CCK-8 assay. The results showed that L-17 significantly inhibited cell viability in a dose-dependent manner. The half-maximal inhibitory concentration (IC50) of L-17 on A549 cells was calculated to be 3.61 nM by fitting the dose-response curve. In the time-effect assay, after treatment with the IC50 concentration for 24 h, 48 h, and 72 h, cell viability decreased significantly in a time-dependent manner, indicating that L-17 has a sustained and potent inhibitory effect on cell proliferation.

[0076] 2. Verification of L-17's specific blocking of CANX-SIRT7 interaction ( Figure 6 ).

[0077] To clarify the mechanism of action of L-17, a co-IP experiment was performed in A549 cells overexpressing CANX-Flag and SIRT7-HA. The results showed that in the untreated group, a strong CANX-Flag signal was detected after SIRT7 precipitation with an anti-HA antibody; however, this binding signal was significantly weakened in the L-17-treated group. Conversely, when CANX was precipitated with an anti-Flag antibody, L-17 treatment also significantly reduced the co-precipitated SIRT7-HA signal. This experiment directly demonstrates that L-17 can specifically and efficiently block the protein-protein interaction between CANX and SIRT7.

[0078] (III) Verification of the radiosensitizing effect of L-17 on A549 cells ( Figure 7 ).

[0079] The radiosensitizing effect of L-17 was evaluated using a plate colony formation assay. A549 cells were pretreated with L-17 or DMSO and then irradiated with different doses (0-8 Gy) of X-rays. The results showed that, at all irradiation doses, the colony formation number in the L-17-treated groups was significantly lower than that in the control group. By fitting cell survival curves and calculating the radiosensitization ratio (SER), the SER value of L-17 was found to be 1.68, indicating that L-17 effectively enhances the radiosensitivity of A549 cells.

[0080] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A polypeptide, characterized in that, The polypeptide contains an amino acid sequence as shown in SEQ ID NO:

1.

2. The polypeptide according to claim 1, characterized in that, The polypeptide also includes a cell-penetrating peptide sequence.

3. The use of any one of the polypeptides according to claims 1-2 in the preparation of a medicament for treating radioresistance in lung adenocarcinoma.

4. A radiosensitizer, characterized in that, The polypeptide comprising any one of claims 1-2.

5. The use of the sensitizer of claim 4 in the preparation of a medicament for treating radioresistance in lung adenocarcinoma.

6. The application as described in claim 5, characterized in that, The sensitizer enhances the sensitivity of lung adenocarcinoma cells to radiotherapy and overcomes radioresistance.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the polypeptide of any one of claims 1-2, and one or more pharmaceutically acceptable carriers, diluents or excipients.

8. The pharmaceutical composition according to claim 5, characterized in that, The dosage form of the pharmaceutical composition is pharmaceutically acceptable.

9. Use of the pharmaceutical composition of any one of claims 7-8 in the preparation of a medicament for treating radiation resistance in lung adenocarcinoma.

10. A reagent for inhibiting or detecting the interaction between CANX and SIRT7 protein in vitro, characterized in that, The polypeptide comprising any one of claims 1-2.

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