Use of succinylated modified ATP5B and its inhibitors and detection reagents and tumor prognosis preparations

Abstract

The application discloses succinyl-modified ATP5B, application of an inhibitor and a detection reagent of the succinyl-modified ATP5B and a tumor prognosis preparation, and belongs to the technical field of tumor molecular markers and application thereof. Through clinical sample analysis, it is found that the expression of ATP5B-K259su in metastatic tumor patients is significantly higher than that in non-metastatic patients, and ATP5B-K259su can be used as a reliable marker actually applied to prediction of lung cancer tumor immune escape and metastasis, and a kit for ATP5B-K259su detection is developed and applied in clinical prognosis diagnosis. Meanwhile, the application is helpful to development of an inhibitor drug taking ATP5B K259 as a target, and provides a new way for lung cancer treatment, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of tumor molecular markers and their applications, specifically relating to the application of succinylated modified ATP5B and its inhibitors and detection reagents, as well as tumor prognostic agents. Background Technology

[0002] Metabolic remodeling is a key characteristic of malignant tumors. It supports tumor growth by maintaining its energy and nutrient requirements, and it regulates immune cell function by secreting specific metabolites into the tumor microenvironment (TME), thus influencing the host's anti-tumor immune response. The core regulatory mechanism of metabolic reprogramming lies in the dynamic changes in metabolic enzyme activity and expression levels. Recent studies have revealed that post-translational modifications (PTMs), as a core mechanism for regulating protein function, dynamically regulate protein activity, localization, and interactions through acetylation, phosphorylation, and ubiquitination, and are widely involved in key biological processes such as signal transduction and metabolic reprogramming. Lysine succinylation is a newly discovered PTM. Compared to lysine methylation and acetylation, succinylation of lysine residues causes greater changes in the charge state of lysine residues, leading to more significant changes in protein chemical properties. In-depth exploration of highly succinylated metabolic enzymes in tumor cells will not only help reveal the crucial role of succinylation modifications in tumor development and progression but may also provide new molecular targets for tumor therapy, promoting the development of novel anti-tumor strategies based on metabolic-immune interactions.

[0003] F0F1 ATP synthase (ATPase) is a protein complex composed of a transmembrane proton channel F0 and an extramembrane catalytic core F1. It catalyzes ATP synthesis via proton transmembrane transport. The β subunit encoded by the ATP5B gene is a key component of the F1 catalytic core, maintaining enzyme activity through its nucleotide binding site. The specific mechanisms by which ATP5B regulates the tumor immune microenvironment and metastasis still require further investigation. Further research into the relationship between ATP5B and tumor immune escape and metastasis will not only help discover new prognostic biomarkers but may also provide a theoretical basis for developing targeted therapy strategies, offering significant guidance for clinical immunotherapy in oncology. Summary of the Invention

[0004] To address the problems existing in the prior art, the purpose of this invention is to address the question of whether the functional level of ATP5B modification can serve as a target for the detection or treatment of tumor immune escape. It has discovered ATP5B K259 succinylation modification (ATP5B-K259su), providing a highly sensitive, highly specific biomarker that can be practically applied to the prediction of tumor immune escape and metastasis. Further development of tumor prognostic agents, as well as the application of inhibitors and detection reagents for this biomarker, are also planned.

[0005] To achieve the above objectives, the present invention is implemented through the following solution:

[0006] The primary objective of this invention is to provide succinylated ATP5B, which is a succinylation modification of lysine residue 259 of ATP5B.

[0007] Specifically, by detecting whether succinylation modification occurs at the ATP5B-K259 site, it can be determined whether tumor immune escape and metastasis are promoted. The presence of succinylation, or a high level of succinylation, indicates the possibility of tumor immune escape and metastasis. This immune escape mainly includes T cell exhaustion-mediated immune escape.

[0008] A second aspect of the present invention aims to provide the use of ATP5B K259 succinylation inhibitors in the preparation of medicaments for treating lung cancer.

[0009] The ATP5B K259 succinylation is the succinylation of lysine at position 259 of ATP5B.

[0010] The ATP5B K259 succinylation inhibitors include:

[0011] .

[0012] The small molecule compound with the above structure was named K259SuIN1 and is used as an intervention for patients with ATP5B K259 hypersuccinylation tumors.

[0013] K259SuIN1 can be purchased from the Enamine website: https: / / enaminestore.com / catalog / Z7158433580?cat=screening-compounds, product number Z7158433580.

[0014] The method for screening small molecule compounds is as follows:

[0015] Step 1: Protein Preparation The 3D structure of ATP5B (human) was downloaded from the RCSB PDB database. The protein was hydrogenated using the Protein Preparationwizard module, missing side chains were added, the B chain was deleted, and energy optimization was performed.

[0016] Step 2: Compound Preparation The 2D format of HY-L0076V (Covalent Screening Library, containing 10,480 commercially available compounds) was processed using the LigRrep Module in Schrödinger software, including hydrogenation and energy optimization, to output 3D structures for virtual screening.

[0017] Step 3: Molecular docking The Covalent docking module was used for virtual screening of covalent docking, with Lys 259 as the center and Lys259 as the reactive amino acid residue. The covalent type was Michael Addition, the docking mode was Virtual screening, and all docking results were output.

[0018] Step 4: Analyze the inhibitory rate of small molecule drugs on tumor cells and the precise blocking of succinylation of ATP5B-K259 site using CCK-8 assay and Western blot assay.

[0019] A third aspect of this invention aims to provide the use of a reagent for detecting the succinylated modified ATP5B in the preparation of tumor prognostic agents, wherein the tumor includes at least one of lung cancer, colon cancer, and head and neck squamous cell carcinoma.

[0020] Furthermore, the reagents include those required for mass spectrometry detection or antibody detection.

[0021] Furthermore, the reagents required for the antibody detection include at least one of the following two peptides that produce antibodies produced by immunizing animals: CESGVINLKDATSK (ATP5B(K259-succ) peptide A) succinylated at position 9 lysine and NLKDATSKVAC (ATP5B(K259-succ) peptide B) succinylated at position 3 lysine.

[0022] The tumor prognostic agents described in this invention specifically refer to agents for predicting tumor immune escape and metastasis.

[0023] This invention provides two succinylated antigens specifically designed for the two ATP5B-K259 sites: CESGVINLKDATSK, lysine succinylated at position 9, and NLKDATSKVAC, lysine succinylated at position 3, for use in animal immunization, purification, and detection.

[0024] A fourth aspect of the present invention aims to provide a tumor prognostic agent, comprising a reagent for detecting the succinylated modified ATP5B, wherein the tumor includes at least one of lung cancer, colon cancer, and head and neck squamous cell carcinoma.

[0025] Furthermore, the reagents include those required for mass spectrometry detection or antibody detection.

[0026] Furthermore, the reagents required for the antibody detection include at least one of the following two peptides that produce antibodies produced by immunizing animals: CESGVINLKDATSK with 9-lysine succinylation and NLKDATSKVAC with 3-lysine succinylation.

[0027] Beneficial effects of the invention

[0028] (1) Through clinical sample analysis, this invention found that the expression of ATP5B-K259su in patients with metastatic lung cancer is significantly higher than that in patients without metastasis. It can be used as a reliable biomarker for predicting tumor immune escape and metastasis, and can be used for the auxiliary diagnosis of tumor immune escape and metastasis; assessing the risk of patients' tumors to develop immune escape and metastasis (risk prediction); and developing and applying a kit based on ATP5B-K259su detection in clinical diagnosis.

[0029] (2) By designing ATP5B-K259su antibodies that specifically target succinylation of ATP5B K259, rapid auxiliary diagnosis of metastatic tumor patients can be achieved.

[0030] (3) It helps in the development of inhibitor drugs targeting ATP5B K259, wherein the inhibitor is a small molecule compound or peptide drug used to block the succinylation of the ATP5B K259 site, suggesting ATP5B K259 succinylation as a molecular target for the treatment of patients with metastatic tumors.

[0031] (4) A drug K259SuIN1 was designed to target and inhibit the succinylation of ATP5B K259. It was found that targeting and inhibiting the succinylation of ATP5B K259 can significantly inhibit the growth and metastasis of lung cancer, and there are no obvious toxic side effects, indicating that it has the potential to be developed into a drug for the treatment of metastatic tumors.

[0032] (5) The present invention also found that the combination of the ATP5B K259 succinylation inhibitor K259SuIN1 and the CTLA-4 antibody can effectively inhibit the growth and metastasis of lung cancer, which is beneficial to the development of drug compositions for treating metastatic tumors and methods for treating metastatic tumors. Attached Figure Description

[0033] Figure 1 : Identification results of ATP5B K259 hypersuccinylation modification in metastatic lung adenocarcinoma;

[0034] in Figure 1 A represents the volcano plot analysis of differentially succinylated modified proteins; Figure 1 B is the secondary mass spectrum of ATP5B K259 site succinylation. Figure 1 C is a schematic diagram of the mutation of the lysine (K) codon AAA at position 259 of ATP5B to the arginine (R) codon CGA; Figure 1 D represents the use of recombinant protein GST-ATP5B. WT / GST-ATP5B K259RThe succinylation level of ATP5B was detected by in vitro succinylation assay; Succinyl-CoA indicates the addition of succinyl-CoA, Succ indicates pansuccinylation modified antibody, and GST-ATP5B indicates recombinant ATP5B protein.

[0035] Figure 2 Preparation, purification, specificity and functional verification results of specific ATP5B-K259su antibody;

[0036] in Figure 2 A represents the ability of ELISA to recognize two modified antigen peptides, ATP5B (K259-succ) peptide A, ATP5B (K259-succ) peptide B, and the unmodified ATP5B peptide. Figure 2 B represents the ability of Dot blot to detect the recognition of ATP5B-K259su by two modified antigen peptides, ATP5B (K259-succ) peptide A, ATP5B (K259-succ) peptide B, and the unmodified ATP5B peptide. Figure 2 C represents the expression level of ATP5B-K259su in clinical samples detected by Western blot, N1-5 represent 5 non-metastatic lung cancer samples, and M1-5 represent 5 metastatic lung cancer samples.

[0037] Figure 3 Results of ATP5B-K259su antibody labeling of tumor metastasis and immune escape; among which Figure 3 Image A shows representative images of the protein expression levels of ATP5B-K259su, CD8, IFN-γ, and CTLA-4 in lung cancer, colorectal cancer, and head and neck squamous cell carcinoma in the metastatic and non-metastatic groups, analyzed by immunohistochemistry. Figure 3 B represents the immunohistochemical score and CD8 expression level of ATP5B-K259su protein in lung cancer, colorectal cancer, and head and neck squamous cell carcinoma in the metastatic and non-metastatic groups. + T cells, IFN-γ + T cells and CTLA-4 + Statistical analysis of T cell infiltration; Figure 3 C represents the diagnostic efficacy of ATP5B-K259su antibody for patients with metastatic tumors, analyzed using ROC curves. Figure 3 D represents the CD8 group in the ATP5B-K259su high and low expression groups. + T cells, IFN-γ + T cells and CTLA-4 + Statistical analysis of T cell infiltration.

[0038] Figure 4K259SuIN1 targets ATP5B K259 succinylation, resulting in reduced tumor cell viability;

[0039] in Figure 4 A is the two-dimensional chemical formula and structural formula of K259SuIN1; Figure 4 B represents the half-maximal inhibitory concentration (IC50) of K259SuIN1 against tumor cells, as determined by the CCK8 assay. Figure 4 C represents the protein levels of ATP5B and ATP5B-K259su detected by Western blot after treating tumor cells with K259SuIN1 for 48 hours. DMSO represents the solvent control group.

[0040] Figure 5 Results of the immunotherapeutic effect of K259SuIN1-sensitized CTLA-4 antibody in mice;

[0041] in Figure 5 A is to use in vivo imaging technology to detect the distribution of tumors in mice; Figure 5 B is a representative image of a mouse lung tumor; Figure 5 C represents the statistical analysis of the total intensity of the mouse in vivo imaging signal; Figure 5 D represents the statistical analysis of the distal transfer rate in mice; Figure 5 E represents mouse survival curve analysis; Figure 5 F represents the mouse body weight change curve;

[0042] DMSO+IgG was the isotype antibody solvent control group (n=8); K259SulN1+IgG was the inhibitor effect control group in the context of isotype antibody (n=8); DMSO+Anti-CTLA-4 was the antibody monotherapy control group (n=8); and K259SulN1+Anti-CTLA-4 was the antibody-inhibitor combination therapy group (n=8). Detailed Implementation

[0043] The following examples are intended to further illustrate the present invention, but not to limit it.

[0044] Example 1: Identification of ATP5B K259 hypersuccinylation modification in metastatic lung adenocarcinoma.

[0045] Five cases of primary lung adenocarcinoma without metastasis and five cases of primary lung adenocarcinoma with metastasis were sent to Hangzhou Jingjie Biotechnology Co., Ltd. for 4D label-free quantitative proteomics and lysine succinylation modification proteomics (Ksucc) combined analysis. After grinding the tissue samples in liquid nitrogen, total protein was extracted using lysis buffer. Protein concentration was determined using the BCA method. Equal volumes of protein were reduced with dithiothreitol, alkylated with iodoacetamide, and then digested overnight with trypsin at 37 °C. The resulting peptides were desalted and dried using a C18 column and then dissolved in 0.1% formic acid solution. The peptide solution was separated using a NanoElute ultra-high performance liquid chromatography system (Bruker), followed by mass spectrometry data acquisition. Raw mass spectrometry data were retrieved and analyzed using MaxQuant. Volcano plot analysis of differentially succinylated proteins showed that ATP5B succinylation was most significantly upregulated (…). Figure 1 A). Further analysis of the succinylation modification data revealed that ATP5B K259 underwent succinylation ( ). Figure 1 B). Simultaneously, an ATP5B K259 desuccinylation mutant plasmid was constructed: the lysine (K) codon AAA at this site was mutated to the arginine (R) codon CGA, thereby simulating the desuccinylation state at this site. Figure 1 C), and in vitro succinylation experiments using the purified recombinant protein GST-ATP5B-WT / K259R (P06576, https: / / www.uniprot.org / ) revealed that desuccinylation of ATP5B K259 downregulated the succinylation level of ATP5B. Figure 1 D).

[0046] Example 2: Preparation, purification, specificity and functional verification of specific ATP5B-K259su antibody

[0047] Preparation of the ATP5B-K259su antibody

[0048] Step 1: Immunogen Design

[0049] Based on the human ATP5B protein sequence and modification type, two modified antigen peptides were designed and synthesized for animal immunization, purification, and detection; simultaneously, one unmodified control peptide was designed and synthesized for purification and detection. Peptide information is as follows:

[0050] ATP5B (K259-succ) peptide A: CESGVINLKDATSK, succinylated at position 9 (lysine);

[0051] ATP5B (K259-succ) peptide B: NLKDATSKVAC succinylated at position 3 (lysine);

[0052] ATP5B unmodified peptide: CESGVINLKDATSK;

[0053] The full-length polypeptide sequence of ATP5B is as follows: MLGFVGRVAAAPASGALRRLTPSASLPPAQLLLRAAPTAVHPVRDYAAQTSPSPKAGAATGRIVAVIGAVVDVQFDEGLPPILNALEVQGRETRLVLEVAQHLGESTVRTIAMDGTEGLVRGQKVLDSG APIKIPVGPETLGRIMNVIGEPIDERGPIKTKQFAPIHAEAPEFMEMSVEQEILVTGIKVVDLLAPYAKGGKIGLFGGAGVGKTVLIMELINNVAKAHGGYSVFAGVGERTREGNDLYHEMIESGVINL K DATSKVALVYGQMNEPPGARARVALTGLTVAEYFRDQEGQDVLLFIDNIFRFTQAGSEVSALLGRIPSAVGYQPTLATDMGTMQERITTTKKGSITSVQAIYVPADDLTDPAPATTFAHLDATVLSRAIAELGI YPAVDPLDTSRIMDPNIVGSEHYDVARGVQKILQDYKSLQDIIAILGMDELSEEDKLTVSRARKIQRFLSQPFQVAEVFTGHMGKLVPLKETIKGFQQILAGEYDHLPEQAFYMVGPIEEAVAKADKLAEEHSS;

[0054] Step 2: Immunogen Mass Spectrometry Detection

[0055] Mass spectrometry was used to determine the accuracy of the sequences of the two peptides.

[0056] Step 3: Serum Screening

[0057] Four SPF experimental-grade New Zealand white rabbits (purchased from Shanghai Yishang Biotechnology Co., Ltd.) were immunized multiple times with ATP5B (K259-succ) peptide A. Small amounts of serum were collected and subjected to ELISA to evaluate the titer and specificity of the antiserum.

[0058] Step 4: Antibody Quality Control

[0059] Sufficient rabbit serum was collected and purified using an immunogen polypeptide column affinity assay. The purified antibody was then analyzed by ELISA, DotBlot, and Western blot.

[0060] The binding activity of ATP5B-K259su antibody was assessed by ELISA and Dot blot experiments. The results showed that ATP5B-K259su antibody exhibited significant specific recognition ability for the succinylated peptide of ATP5B K259. Figure 2 AB). Meanwhile, Western blot analysis showed that the ATP5B-K259su antibody effectively identified ATP5B K259 succinylation in succinylated modified omics samples. Figure 2 C).

[0061] Example 3: ATP5B-K259su antibody labeling of tumor metastasis and immune escape.

[0062] Immunohistochemical analysis was performed on clinical tissue samples from both metastatic and non-metastatic sites.

[0063] The specific steps are as follows:

[0064] (1) Pretreatment of glass slides

[0065] (a) Baked slices: Bake at 65°C for 3 hours to enhance tissue adhesion.

[0066] (b) Dewaxing and rehydration: Environmentally friendly dewaxing solution (15 minutes x 2 times).

[0067] (c) Gradient ethanol: 100% → 90% → 80% → 70% (5 minutes / gradient × 2 times).

[0068] (d) PBS rinse: Rinse with 1×PBS for 5 minutes.

[0069] (2) Antigen retrieval

[0070] Antigen retrieval was performed using citrate retrieval solution (pH 6.0) as follows: After microwaving on high to a boil, place a glass slide in the microwave and microwave on medium for 8 minutes. Let the slide stand at room temperature for 8 minutes, then repeat the heating for 8 minutes. Allow the slide to cool naturally for 30 minutes. Finally, wash with 1×PBS for 5 minutes each time for 3 times.

[0071] (3) Transparent

[0072] Prepare a solution containing 0.5% Triton X-100 using 1×PBS, immerse the slide in the solution, and incubate at room temperature for 1 hour to enhance cell membrane permeability. After incubation, wash with 1×PBS for 5 minutes.

[0073] (4) Blocking endogenous peroxidase

[0074] Remove any remaining water from the slide. Use an immunohistochemistry hydrophobic pen to draw a circle around the tissue to form a hydrophobic barrier. Add the peroxidase inhibitor, ensuring complete coverage of the tissue. Let it stand at room temperature for 20 minutes to block the action of endogenous peroxidase. After removing the reagent, wash with 1×PBS for 5 minutes each time, 3 times.

[0075] (5) Closed and primary antibody incubation

[0076] Prepare 10% goat serum using 1×PBS, add it dropwise to the tissue, and incubate at room temperature for 1 hour. Discard the blocking solution and place the slide horizontally in a humidified chamber. Prepare the corresponding working dilutions according to the instructions for each antibody. The primary antibodies used include: ATP5B-K259 succinylated modified specific antibody (prepared in Example 2), CD8A antibody (catalog number A11856SP, Wuhan Aibotek Biotechnology Co., Ltd.), IFN-γ monoclonal antibody (catalog number A27845, Wuhan Aibotek Biotechnology Co., Ltd.), and CTLA-4 antibody (catalog number sc-376016, Santa Cruz Biotechnology (Shanghai) Co., Ltd.). Gently drop the prepared working solutions of each primary antibody onto the surface of the tissue section, ensuring that the antibody working solutions completely wet and cover the entire tissue area; cover with a humidified incubation chamber and incubate overnight at 4 ℃.

[0077] (6) Signal amplification and color development

[0078] After discarding the primary antibody, wash the tissue three times (5 minutes each) with 1×PBS on a shaker. Then place the slide in a humidified chamber, add reaction enhancement solution to cover the tissue, and incubate at room temperature for 20 minutes. Afterward, wash with 1×PBS on a shaker to completely remove the primary antibody. Next, add enzyme-labeled polymer enhancement solution, incubate at 37°C for 1 hour, and then wash with 1×PBS on a shaker to completely remove the primary antibody. Prepare a fresh chromogenic solution (DAB) at a 1:20 ratio under dark conditions, gently add it to the tissue surface, and observe the colorimetric reaction under an optical microscope. When the color development is complete, immerse the slide in distilled water to terminate the reaction.

[0079] (7) Counterstaining and mounting

[0080] Shake off the water from the slide, add hematoxylin for staining for 30 seconds and then quickly discard it. Immerse the slide in distilled water for 20 minutes to re-blue. Then dehydrate the slide in a gradient of 75% ethanol for 5 minutes, 85% ethanol for 5 minutes, 95% ethanol for 5 minutes, and anhydrous ethanol for 5 minutes. After the slide has dried, seal it with neutral resin, allow it to air dry at room temperature, and then place it under a microscope to acquire images.

[0081] Immunohistochemical analysis was performed on the expression of ATP5B-K259su, CD8, IFN-γ, and CTLA-4 in metastatic and non-metastatic tumor tissues and the tumor microenvironment. The expression of ATP5B-K259su antibody and CD8 were also statistically analyzed. + T cells, IFN-γ + T cells and CTLA-4 + The extent of T cell infiltration in tumor tissue, and clinical sample information are shown in Tables 1-3.

[0082] Table 1. Clinical information of 30 lung cancer patients

[0083] Table 2. Clinical information of 30 patients with colon cancer

[0084] Table 3. Clinical information of 30 patients with head and neck squamous cell carcinoma.

[0085] The expression of ATP5B-K259su, CD8, IFN-γ, and CTLA-4 in metastatic and non-metastatic tumor tissues and the tumor microenvironment was further analyzed using clinical lung cancer, colon cancer, and head and neck squamous cell carcinoma tissues. Figure 3 (AB), indicating that ATP5B-K259su is highly expressed in metastatic tumor tissues, CD8 + T cells, IFN-γ + T cell infiltration is reduced in metastatic tumor tissue, CTLA-4 + T cell infiltration is increased in metastatic tumor tissues; ATP5B-K259su is detected in tumor cells, so immunohistochemical scoring is used, while CD8... + IFN-γ + and CTLA-4 + This test measures the degree of T-cell infiltration, so the number of positive cells in the field of view is used to express the result. Analysis based on ROC curves showed that the ATP5B-K259su antibody exhibited significant diagnostic value for patients with metastatic tumors. The area under the curve (AUC) of this antibody in different cancer types was higher than the high diagnostic efficacy threshold of 0.90 (Lung cancer AUC: 0.9043, Colon cancer AUC: 0.9118; Head and neck squamous cell carcinoma AUC: 0.9219). Figure 3 C). Further, based on ATP5B-K259su expression, the groups were divided into a low-expression group and a high-expression group. Statistical analysis was performed on the CD8 expression levels in the two groups. + T cells, IFN-γ + T cells and CTLA-4+ T cell infiltration status ( Figure 3 D), the results showed that CD8 in the ATP5B-K259su high expression group + T cells, IFN-γ + The infiltration of T cells was significantly lower in the ATP5B-K259su low expression group than in the CTLA-4 group. + T cell infiltration was significantly higher in the group with low ATP5B-K259su expression than in the group with low ATP5B-K259su expression. These results indicate that ATP5B-K259su antibody is positively correlated with tumor metastasis and the immunosuppressive microenvironment.

[0086] Example 4: K259SuIN1 targets ATP5B K259 succinylation, reducing tumor cell viability.

[0087] Analysis showed that K259SuIN1 irreversibly blocked the succinylation of ATP5B K259 by forming a covalent bond with Lys259 of ATP5B, a hydrogen bond with Asn257 (bond distance 1.9 Å), and a hydrogen bond and a salt bridge with Glu252 of the target protein. Figure 4 A). The CCK-8 assay showed that K259SuIN1 had a significant inhibitory effect on the growth of human tumor cells 95D (lung cancer cells, purchased from Suzhou Xinsaimei Biotechnology Co., Ltd.). Figure 4 B). Western blot analysis confirmed that ATP5B K259 succinylation was efficiently inhibited in cells treated with K259SuIN1 without affecting the total protein expression of ATP5B. Figure 4 (C) indicates that K259SuIN1 can precisely block the succinylation modification of ATP5B K259 without interfering with the stability and abundance of the target protein. K259SuIN1 is available from the enamine website https: / / enaminestore.com / catalog / Z7158433580?cat=screening-compounds, catalog number Z7158433580.

[0088] Example 5: Immunotherapy of K259SuIN1 sensitizing CTLA-4.

[0089] After subcutaneous injection of LLC-luc cells (mouse lung cancer cells, purchased from Jiangsu Kaiji Biotechnology Co., Ltd.) into C57BL / 6 mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) on day 0, the mice were divided into four groups: DMSO+IgG as the isotype antibody solvent control group (n=8); K259SulN1+IgG as the inhibitor effect control group in the background of isotype antibody (n=8); DMSO+Anti-CTLA-4 as the antibody monotherapy control group (n=8); and K259SulN1+Anti-CTLA-4 as the antibody-inhibitor combination therapy group (n=8). In this embodiment, IgG2b was used. For 7 consecutive days, mice were intraperitoneally injected with either IgG2b (purchased from Leinco Technologies, clone MCP-11, I-119, distributed by Shanghai Qifa Experimental Reagent Co., Ltd.) (200 μg / mouse), Anti-CTLA-4 (purchased from Leinco Technologies, clone 9D9, C2855, distributed by Shanghai Qifa Experimental Reagent Co., Ltd.) (200 μg / mouse), K259SuIN1 (100 mg / kg), or DMSO (100 μL / mouse). In vivo imaging technology was used to detect tumor distribution in the mice. Figure 5 A) Dissect the mouse and record the size of the lung tumor ( Figure 5 B) Statistical analysis of the total signal intensity of whole-body in vivo imaging of mice ( Figure 5 C) and mouse distal transfer rate ( Figure 5 D) Survival curve analysis Figure 5 E) and weight change analysis ( Figure 5 Experimental methods such as F) were used to investigate the enhancing effect of K259SuIN1 on CTLA-4 immunotherapy. The results showed that the combination therapy group of K259SuIN1 and anti-CTLA-4 antibody exhibited a more significant anti-tumor growth effect and a more significant reduction in distant metastasis rate compared to the single-agent therapy group (K259SuIN1 or anti-CTLA-4 antibody), and the combination treatment group had a better prognosis. Furthermore, the combined use of K259SuIN1 and anti-CTLA-4 antibody did not induce systemic toxicity within the effective dose range.

Claims

1. Succinylated modified ATP5B, characterized in that, It is a succinylation modification of lysine at position 259 of ATP5B.

2. The application of ATP5B K259 succinylation inhibitors in the preparation of lung cancer therapeutic drugs, characterized in that, The ATP5B K259 succinylation is the succinylation of lysine at position 259 of ATP5B; The ATP5B K259 succinylation inhibitors include: 。 3. The use of the reagent for detecting the succinylated modified ATP5B as described in claim 1 in the preparation of tumor prognostic agents, wherein the tumor includes at least one of lung cancer, colon cancer, and head and neck squamous cell carcinoma.

4. The application according to claim 3, characterized in that, The reagents mentioned include those required for mass spectrometry or antibody detection.

5. The application according to claim 4, characterized in that, The reagents required for the antibody detection include at least one of the following two peptides that produce antibodies produced by immunizing animals: CESGVINLKDATSK with succinylated lysine at position 9 and NLKDATSKVAC with succinylated lysine at position 3.

6. A tumor prognostic agent, characterized in that, The reagent includes a reagent for detecting succinylated modified ATP5B as described in claim 1, wherein the tumor includes at least one of lung cancer, colon cancer, and head and neck squamous cell carcinoma.

7. The formulation according to claim 6, characterized in that, The reagents mentioned include those required for mass spectrometry or antibody detection.

8. The formulation according to claim 7, characterized in that, The reagents required for the antibody detection include at least one of the following two peptides that produce antibodies produced by immunizing animals: CESGVINLKDATSK with succinylated lysine at position 9 and NLKDATSKVAC with succinylated lysine at position 3.

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