Succinylation modification of RAD51 protein, formulations that promote RAD51 succinylation and their applications

Abstract

This invention relates to the field of biomedical technology, disclosing succinylated modified RAD51 protein, formulations for promoting RAD51 succinylation, and their applications. The method includes: seeding and culturing cells and transfecting them with a tagged RAD51 expression plasmid; adding sodium succinate and an HDAC11 inhibitor to the culture medium for continued culturing to allow intracellular modification accumulation; collecting the supernatant after cell lysis and incubating it with magnetic beads containing anti-tag antibodies under cold conditions; washing and eluting with an elution buffer containing the tagged peptide; performing immunoblotting analysis using antibodies that specifically recognize the modified site to obtain the succinylated modified RAD51 protein. This invention, by adding sodium succinate and an HDAC11 inhibitor, provides a substrate and blocks the demodification pathway. Combined with magnetic bead purification and competitive elution, it avoids disrupting the protein conformation, obtaining a protein that retains its native activity and has a high level of modification, providing a core component for screening targeted drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to succinylation modification of RAD51 protein and preparations and applications that promote RAD51 succinylation. Background Technology

[0002] DNA homologous recombination repair is one of the pathways for maintaining genome stability, and the RAD51 protein acts as a recombinase in this process. In tumor cells, activation of the homologous recombination pathway leads to resistance to chemotherapeutic drugs or PARP inhibitors, limiting clinical efficacy. Therefore, elucidating the functional regulatory mechanism of RAD51 and identifying targets that inhibit RAD51 activity to reverse tumor drug resistance is significant for the development of anti-tumor strategies.

[0003] Post-translational modifications of proteins are mechanisms that regulate the activity of repair proteins. Lysine succinylation is an acylation modification that connects cellular metabolism and signal transduction, and participates in metabolic and stress regulation. However, there is currently a lack of systematic research on whether succinylation modification exists in the RAD51 protein, how this modification affects the homologous recombination activity of RAD51, and how to regulate the sensitivity of tumor cells to DNA-damaging drugs by intervening in this modification.

[0004] Currently, there is no standardized preparation system for succinylated RAD51 protein, making it difficult to obtain proteins that maintain activity and have abundant modification for functional studies. Furthermore, there is a lack of agents capable of specifically upregulating RAD51 succinylation levels intracellularly, thereby inhibiting its repair function and sensitizing chemotherapy. These deficiencies limit the utilization of this modification function and the development of related drugs to reverse drug resistance. Therefore, providing a method for preparing succinylated RAD51 protein and an agent that promotes RAD51 succinylation is a problem that needs to be solved in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides succinylation modification of RAD51 protein, formulations that promote RAD51 succinylation, and their applications. This solves the problem that the lack of preparation systems for succinylation modification of RAD51 protein and modulators for RAD51 succinylation in existing technologies limits the application of this modification function in the development of tumor sensitizing drugs.

[0006] To address the above problems, the present invention provides the following technical solution: This invention provides succinylation modification of RAD51 protein, using the following technical solution: Succinylated RAD51 protein, wherein the succinylated RAD51 protein is prepared by a method comprising the following steps: Cells were seeded in culture dishes and cultured. The cells were then transfected with a tagged RAD51 expression plasmid using a transfection reagent to obtain transfected cells. Sodium succinate and HDAC11 inhibitor were added to the culture medium of the transfected cells, and the cells were cultured for a period of time to accumulate RAD51 succinylation modification in the cells, thus obtaining the modified cells. The modified cells were collected, and a lysis buffer containing protease inhibitors and deacetylase inhibitors was added. The cells were lysed on ice, and the supernatant was collected by centrifugation. Magnetic beads conjugated with anti-tag antibodies were added, and the cells were rotated and incubated under refrigeration to obtain protein-bound magnetic beads. The protein-bound magnetic beads were washed with lysis buffer to remove non-specifically bound proteins, followed by competitive elution with a tagged peptide-containing elution buffer to obtain a purified protein solution. The purified protein solution was subjected to immunoblotting analysis, and the antibody with specific recognition site modification was used to confirm the acquisition of the succinylated modified RAD51 protein.

[0007] By employing the above-described technical solution, the succinylation level of RAD51 protein can be significantly enhanced intracellularly by combining exogenous supplementation of succinate donor (sodium succinate) with a specific blocking inhibitor of the desuccinylation enzyme (HDAC11). HDAC11 has been confirmed as a key desuccinylation enzyme of RAD51, and inhibiting its activity prevents the shedding of modified groups. Sodium succinate, as a metabolic intermediate, increases the intracellular level of succinyl-CoA, providing sufficient substrate for non-enzymatic or enzymatic succinylation reactions. Furthermore, the purification strategy of enriching anti-tag antibody magnetic beads combined with competitive elution of peptides avoids the destruction of protein conformation and stability of modified groups caused by traditional acid-base elution, thereby obtaining full-length RAD51 protein that maintains its native active conformation and has a high degree of modification abundance.

[0008] Preferably, in the preparation method: the confluence of the cells at transfection is 60% to 80%; the time interval between transfection and the addition of sodium succinate is 5 to 8 hours; and the continued culture time is 20 to 28 hours.

[0009] By employing the above-mentioned technical approach, cell confluence is controlled within the range of 60% to 80%, balancing transfection efficiency and cell growth status, and avoiding contact inhibition caused by overly dense cells or transfection toxicity caused by overly sparse cells. The time interval ensures that the exogenous plasmid fully enters the cell and initiates transcription and translation before the introduction of metabolic regulators, allowing the peak of protein synthesis to overlap with the peak of modification accumulation, thereby achieving optimized modified protein yield.

[0010] Preferably, in the preparation method: the final concentration of the added sodium succinate is 30 mM to 50 mM, the final concentration of the HDAC11 inhibitor is 0.5 μM to 1.5 μM, and the deacetylase inhibitor includes NAM and TSA.

[0011] By employing the above-described technical approach, NAM and TSA, as broad-spectrum deacetylase inhibitors, can inhibit the activity of other deacetylases in the Sirtuins and HDACs families, except for HDAC11. This creates a protective environment conducive to high modification levels, preventing non-specific demodification and further stabilizing the succinylation modification site on RAD51. Sodium succinate concentrations of 30 mM to 50 mM are sufficient to drive the modification reaction without causing significant cytotoxicity or osmotic shock.

[0012] This invention also provides a formulation for promoting RAD51 succinylation, employing the following technical solution: An formulation that promotes RAD51 succinylation, said formulation being prepared by a method comprising the following steps: Weigh the lyophilized polypeptide powder and measure the sterile buffer solution to obtain the raw materials for the formulation; Under sterile conditions, the lyophilized polypeptide powder is added to the sterile buffer solution, and stirring is started and continued until no solid particles are visible to the naked eye, thus obtaining a polypeptide solution. The pH value of the polypeptide solution was adjusted by titration with a pH adjuster to obtain a pH-adjusted solution; The pH-adjusted solution was filtered and sterilized using a filter membrane, and the filtrate was collected to obtain a sterile preparation solution. The sterile formulation solution is aseptically dispensed and stored in a frozen environment to obtain the formulation that promotes RAD51 succinylation.

[0013] By employing the above-described technical solution, and through strictly controlled dissolution, pH adjustment, and sterile filtration processes, the homogeneity, sterility, and bioactivity stability of the peptide formulation are ensured. This preparation process solves the problem of peptide aggregation or degradation in solution, providing a standardized dosage form for subsequent cell or animal experiments.

[0014] Preferably, the amino acid sequence of the polypeptide in the lyophilized polypeptide powder is formed by fusing the sequence of the cell-penetrating peptide with the sequence shown in SEQ ID NO:7, wherein the sequence of the cell-penetrating peptide is shown in SEQ ID NO:9.

[0015] By employing the above-described technical solution, the cell-penetrating peptide shown in SEQ ID NO:9 can effectively carry functional polypeptides across the cell membrane into the cytoplasm or nucleus, overcoming the barrier that hinders polypeptide drug entry into cells. The sequence shown in SEQ ID NO:7 is a specific sequence designed based on the RAD51 interaction interface. After entering the cell, this sequence can mimic or induce conformational changes in endogenous RAD51, or competitively bind to demodifying enzymes, thereby specifically upregulating the succinylation level of RAD51.

[0016] Preferably, the stirring is carried out at an environment of 4℃-30℃, the stirring speed is 200rpm-600rpm, and the stirring time is 10 minutes-30 minutes; the sterile buffer is PBS buffer; and the filter membrane is a polyethersulfone filter membrane.

[0017] By employing the above technical solution, gentle stirring speed and temperature control prevented the peptides from denaturing and precipitating due to excessive shear force or thermal effects. Polyethersulfone (PES) membranes were chosen because they have low protein adsorption properties, which significantly reduces the loss of active ingredients during filtration and ensures the accuracy of formulation concentration.

[0018] Preferably, the mass of the lyophilized polypeptide powder weighed is 10mg-100mg; the volume of the sterile buffer measured is 10mL-20mL; and the final concentration of the polypeptide in the formulation promoting RAD51 succinylation is 1mg / mL-10mg / mL.

[0019] Preferably, the pH adjuster is hydrochloric acid or sodium hydroxide solution, which adjusts the pH of the polypeptide solution to 6.5-7.8.

[0020] By adopting the above technical solution, the pH value is controlled within the physiological range of 6.5 to 7.8, which not only ensures the solubility of the peptides, but also avoids local acid-base stimulation or inflammatory reactions caused by the injection of the preparation into the organism.

[0021] Preferably, in the preparation method, the lyophilized polypeptide powder in step one is prepared by solid-phase synthesis, which includes: using Rink Amide MBHA resin with a degree of substitution of 0.3 mmol / g to 0.8 mmol / g, mixing Fmoc-protected amino acids, condensing agent HBTU and organic base DIEA in a molar ratio of 3 to 8:3 to 8:6 to 16 to carry out a condensation reaction; after the synthesis is completed, pyrolysis is carried out using a pyrolysis buffer composed of trifluoroacetic acid, water and triisopropylsilane.

[0022] By employing the above-mentioned technical solution and using Rink Amide MBHA resin for solid-phase synthesis, the final peptide obtained has an amidated C-terminus. C-terminal amidation removes the negative charge of the natural carboxyl group, making the peptide terminus closer to the peptide bond state in natural proteins. This increases the peptide's resistance to carboxypeptidase in vivo, thereby significantly prolonging the formulation's half-life and bioavailability in vivo.

[0023] This invention also provides the application of succinylation modification of RAD51 protein and formulations that promote RAD51 succinylation, using the following technical solution: The application of the succinylation modification of RAD51 protein in the preparation of a kit for screening drugs targeting RAD51 post-translational modifications; Alternatively, its application in the preparation of tumor chemotherapy sensitizers, radiotherapy sensitizers, or PARP inhibitor sensitizers; The drug enhances the sensitivity of tumor cells to chemotherapy, radiotherapy, or PARP inhibitors by upregulating RAD51 succinylation levels and inhibiting RAD51-mediated homologous recombination repair.

[0024] By employing the above-described technical solution, the modified protein prepared according to this invention can be used as a standard or screening substrate, facilitating the construction of a drug screening platform for discovering small molecule compounds that regulate RAD51 modification levels. Furthermore, RAD51 is a protein in the DNA homologous recombination repair pathway, and its succinylation modification can inhibit the interaction between RAD51 and BRCA2, thereby suppressing homologous recombination repair efficiency. The formulation provided by this invention, by increasing the succinylation level of RAD51, can inhibit the ability of tumor cells to repair DNA damage, hindering tumor cells from repairing DNA damage caused by chemotherapy drugs or radiotherapy, thereby significantly enhancing the sensitivity of tumor cells to chemotherapy drugs, radiotherapy, or PARP inhibitors, and has application value in the preparation of drugs to overcome tumor drug resistance.

[0025] This invention provides succinylation modification of RAD51 protein, formulations that promote RAD51 succinylation, and their applications. It has the following beneficial effects: 1. This invention involves adding sodium succinate and an HDAC11 inhibitor to the cell culture medium after transfection with a tagged RAD51 expression plasmid. Sodium succinate provides the substrate for the modification reaction, while the HDAC11 inhibitor blocks the demodification pathway, thereby effectively accumulating succinylated RAD51 in cells. Simultaneously, purification and competitive elution are performed using magnetic beads coupled with anti-tag antibodies, avoiding the destruction of protein conformation by strong acids and bases. This yields succinylated RAD51 protein that retains its native activity and has a high level of modification, providing a stable core component for screening kits targeting RAD51 post-translational modifications.

[0026] 2. This invention prepares a formulation comprising a polypeptide formed by fusing the sequence of a cell-penetrating peptide with the sequence shown in SEQ ID NO: 7. The cell-penetrating peptide carries the functional sequence into the cell to specifically upregulate the succinylation level of RAD51. The preparation process strictly controls the polypeptide dissolution, pH adjustment, and sterile filtration, effectively solving the problems of aggregation or degradation that easily occur during the preparation and storage of polypeptides. This ensures the uniformity and bioactivity stability of the formulation that promotes RAD51 succinylation, thus providing a standardized formulation for subsequent applications.

[0027] 3. This invention upregulates the succinylation level of RAD51 by using the aforementioned RAD51-promoting succinylation agent, inhibits RAD51-mediated homologous recombination repair, reduces the cell's ability to repair DNA damage, and thereby enhances the sensitivity of tumor cells to chemotherapy, radiotherapy or PARP inhibitors, thus having applications in the preparation of tumor chemotherapy sensitizers, radiotherapy sensitizers or PARP inhibitor sensitizers. Attached Figure Description

[0028] Figure 1 This is a diagram illustrating the working mode of the DR-GFP reporting system used in this invention. Figure 2 This is a graph showing the relevant data on succinyl-CoA inhibition of homologous recombination repair in this invention; Figure 3 This is a graph showing the data related to how succinylation of RAD51 affects the formation of RAD51 foci and inhibits homologous recombination repair in this invention. Figure 4 This is a schematic diagram (A) of the main structural domains of the human RAD51 protein of the present invention, and a graph showing the succinylation-related data of RAD51. Figure 5 This is a graph showing the relevant data of HDAC11 being phosphorylated by ATM and then desuccinylated RAD51 in this invention; Figure 6 This is a graph showing the relevant data on how RAD51 succinylation promotes tumor chemosensitivity in this invention. Figure 7 This is a graph showing the data related to the increase in tumor killing effect of chemotherapeutic drugs by the peptide of this invention promoting RAD51 succinylation. Figure 8 This is a graph showing the tumor-killing data of the present invention using peptides in combination with olaparib in animals. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, preparation examples, embodiments, comparative examples, and test examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Preparation Examples 1-3: Preparation Example 1: This preparation example provides a polypeptide that promotes RAD51 succinylation, and its preparation method includes the following steps: Rink Amide MBHA resin with a degree of substitution of 0.3 mmol / g was placed in a solid-phase reactor, and dichloromethane (DCM) was added to swell the resin at 20 °C for 15 minutes. The solvent was removed by filtration. A DMF solution containing 20% ​​(v / v) piperidine was added to the resin, and the mixture was shaken at 20 °C for 5 minutes to remove the Fmoc protecting group. The reaction solution was removed by filtration, and this deprotection operation was repeated once. The resin was then washed 5 times with DMF. The condensation reaction was carried out one by one from the C-terminus to the N-terminus according to the polypeptide sequence. The Fmoc-protected amino acid, condensing agent HBTU and organic base DIEA were dissolved in DMF at a molar ratio of 3:3:6 (relative to the resin loading) and activated for 3 minutes before being added to the resin. The reaction was carried out at 20°C with constant temperature shaking for 30 minutes. After each condensation reaction, the resin was washed three times with DMF, and the completeness of the reaction was confirmed using the ninhydrin assay. The above deprotection and condensation steps were repeated until the assembly of the full-length sequence HLYVSPWGGLLRLADEF was completed. Finally, the N-terminal Fmoc protecting group was removed, the resin was condensed with methanol, and dried under nitrogen. A lysis buffer consisting of trifluoroacetic acid, water, and triisopropylsilane in a volume ratio of 95:2.5:2.5 (10 mL / g resin) was added, and the reaction was carried out at 20°C in the dark for 1.5 hours. The filtrate was collected by filtration. Ten volumes of ice-cold diethyl ether were added to the filtrate to precipitate the crude peptide. The precipitate was collected by centrifugation at 4000 rpm for 5 minutes. The precipitate was purified by preparative HPLC using a C18 reversed-phase column. Mobile phase A was an aqueous solution containing 0.1% (v / v) trifluoroacetic acid, and mobile phase B was an acetonitrile solution containing 0.1% (v / v) trifluoroacetic acid. The flow rate was 10 mL / min, and elution was performed at a detection wavelength of 220 nm using a gradient elution program with the concentration of mobile phase B increasing linearly from 10% to 60%, for a total elution time of 40 minutes. The main peak fraction was collected and freeze-dried to obtain a white powdery peptide with a purity of 95.2%.

[0031] Preparation Example 2: This preparation example provides a polypeptide that promotes RAD51 succinylation, and its preparation method includes the following steps: Rink Amide MBHA resin with a degree of substitution of 0.5 mmol / g was placed in a solid-phase reactor, and dichloromethane (DCM) was added to swell the resin at 25 °C for 30 minutes. The solvent was removed by filtration. A DMF solution containing 20% ​​(v / v) piperidine was added to the resin, and the mixture was shaken at 25 °C for 10 minutes to remove the Fmoc protecting group. The reaction solution was removed by filtration. This deprotection operation was repeated once, followed by washing the resin five times with DMF. The condensation reaction was carried out one by one from the C-terminus to the N-terminus of the polypeptide sequence. The Fmoc-protected amino acid, condensing agent HBTU and organic base DIEA were dissolved in DMF at a molar ratio of 5:5:10 (relative to the resin loading) and activated for 3 minutes before being added to the resin. The reaction was carried out at 25°C with constant temperature shaking for 60 minutes. After each condensation reaction, the resin was washed four times with DMF, and the completeness of the reaction was confirmed using the ninhydrin assay. The above deprotection and condensation steps were repeated until the assembly of the full-length sequence HLYVSPWGGLLRLADEF was completed. Finally, the N-terminal Fmoc protecting group was removed, the resin was condensed with methanol, and dried under nitrogen. A lysis buffer consisting of trifluoroacetic acid, water, and triisopropylsilane in a volume ratio of 95:2.5:2.5 (liquid-to-solid ratio 12 mL / g resin) was added, and the reaction was carried out at 25°C in the dark for 2.5 hours. The filtrate was collected by filtration. Twelve volumes of ice-cold diethyl ether were added to the filtrate to precipitate the crude peptide. The precipitate was collected by centrifugation at 4500 rpm for 8 minutes. The precipitate was purified by preparative HPLC using a C18 reversed-phase column. Mobile phase A was an aqueous solution containing 0.1% (v / v) trifluoroacetic acid, and mobile phase B was an acetonitrile solution containing 0.1% (v / v) trifluoroacetic acid. The flow rate was 12 mL / min, and elution was performed at a detection wavelength of 220 nm using a gradient elution program where the concentration of mobile phase B increased linearly from 20% to 70%, for a total elution time of 50 minutes. The main peak fraction was collected and freeze-dried to obtain a white powdery peptide with a purity of 98.5%.

[0032] Preparation Example 3: This preparation example provides a polypeptide that promotes RAD51 succinylation, and its preparation method includes the following steps: Rink Amide MBHA resin with a degree of substitution of 0.8 mmol / g was placed in a solid-phase reactor, and dichloromethane (DCM) was added to swell the resin at 30 °C for 45 minutes. The solvent was removed by filtration. A DMF solution containing 20% ​​(v / v) piperidine was added to the resin, and the mixture was shaken at 30 °C for 15 minutes to remove the Fmoc protecting group. The reaction solution was removed by filtration, and this deprotection operation was repeated once. The resin was then washed five times with DMF. The condensation reaction was carried out one by one from the C-terminus to the N-terminus of the polypeptide sequence. The Fmoc-protected amino acid, condensing agent HBTU and organic base DIEA were dissolved in DMF at a molar ratio of 8:8:16 (relative to the resin loading) and activated for 5 minutes before being added to the resin. The reaction was carried out at 30°C with constant temperature shaking for 90 minutes. After each condensation reaction, the resin was washed five times with DMF, and the completeness of the reaction was confirmed using the ninhydrin assay. The above deprotection and condensation steps were repeated until the assembly of the full-length sequence HLYVSPWGGLLRLADEF was completed. Finally, the N-terminal Fmoc protecting group was removed, the resin was condensed with methanol, and dried under nitrogen. A lysis buffer consisting of trifluoroacetic acid, water, and triisopropylsilane in a volume ratio of 95:2.5:2.5 (liquid-to-solid ratio 15 mL / g resin) was added, and the reaction was carried out at 30°C in the dark for 4.0 hours. The filtrate was collected by filtration. 15 times the volume of ice-cold diethyl ether was added to the filtrate to precipitate the crude peptide. The precipitate was collected by centrifugation at 5000 rpm for 10 minutes. The precipitate was purified by preparative HPLC using a C18 reversed-phase column. Mobile phase A was an aqueous solution containing 0.1% (v / v) trifluoroacetic acid, and mobile phase B was an acetonitrile solution containing 0.1% (v / v) trifluoroacetic acid. The flow rate was 15 mL / min, and elution was performed at a detection wavelength of 220 nm using a gradient elution program where the concentration of mobile phase B linearly increased from 30% to 80%, for a total elution time of 60 minutes. The main peak fraction was collected and freeze-dried to obtain a white powdery peptide with a purity of 96.8%.

[0033] Examples 1-6: Example 1

[0034] This embodiment provides a succinylated modified RAD51 protein, the preparation method of which includes the following steps: S01. HEK293T cells were seeded in culture dishes with a diameter of 10 cm. When the cell confluence reached 60%, wild-type RAD51 expression plasmid with FLAG tag was transfected into the cells using liposome transfection reagent. S02. Five hours after transfection, sodium succinate and HDAC11 inhibitor at a final concentration of 30 mM and 0.5 μM were added to the culture medium and cultured for another 20 hours to accumulate RAD51 succinylation modification in the cells. S03. Collect cells, add lysis buffer containing 1% (volume fraction) protease inhibitor and 1 μM deacetylase inhibitors NAM and TSA, lyse on ice for 20 minutes, centrifuge to collect supernatant, add magnetic beads conjugated with anti-FLAG antibody, and incubate at 4°C for 3 hours. S04. Wash the magnetic beads twice with lysis buffer to remove non-specifically bound proteins, and then use elution buffer containing FLAG peptide for competitive elution to obtain a purified protein solution. S05. Take a small amount of the elution product for immunoblotting analysis. Use an antibody that specifically recognizes the succinylation of lysine at position 285 of RAD51 to confirm that high-purity succinylated modified RAD51 protein has been obtained. Example 2

[0035] This embodiment provides a succinylated modified RAD51 protein, the preparation method of which includes the following steps: S01. HEK293T cells were seeded in culture dishes with a diameter of 10 cm. When the cell confluence reached 70%, wild-type RAD51 expression plasmid with FLAG tag was transfected into the cells using liposome transfection reagent. S02. Six hours after transfection, sodium succinate and HDAC11 inhibitor at a final concentration of 40 mM and 1.0 μM were added to the culture medium and cultured for another 24 hours to accumulate RAD51 succinylation modification in the cells. S03. Collect cells, add lysis buffer containing 1% (volume fraction) protease inhibitor and 2 μM deacetylase inhibitors NAM and TSA, lyse on ice for 30 minutes, centrifuge to collect supernatant, add magnetic beads conjugated with anti-FLAG antibody, and incubate at 4°C for 4 hours. S04. Wash the magnetic beads three times with lysis buffer to remove non-specifically bound proteins, and then use elution buffer containing FLAG peptide for competitive elution to obtain a purified protein solution. S05. Take a small amount of the elution product for immunoblotting analysis. Use an antibody that specifically recognizes the succinylation of lysine at position 285 of RAD51 to confirm that high-purity succinylated modified RAD51 protein has been obtained. Example 3

[0036] This embodiment provides a succinylated modified RAD51 protein, the preparation method of which includes the following steps: S01. HEK293T cells were seeded in culture dishes with a diameter of 10 cm. When the cell confluence reached 80%, wild-type RAD51 expression plasmid with FLAG tag was transfected into the cells using liposome transfection reagent. S02. Eight hours after transfection, sodium succinate and HDAC11 inhibitor at a final concentration of 50 mM and 1.5 μM were added to the culture medium and cultured for another 28 hours to accumulate RAD51 succinylation modification in the cells. S03. Collect cells, add lysis buffer containing 1% (volume fraction) protease inhibitor and 3 μM deacetylase inhibitors NAM and TSA, lyse on ice for 40 minutes, centrifuge to collect supernatant, add magnetic beads conjugated with anti-FLAG antibody, and incubate at 4°C for 5 hours. S04. Wash the magnetic beads four times with lysis buffer to remove non-specifically bound proteins, and then use elution buffer containing FLAG peptide for competitive elution to obtain a purified protein solution. S05. Take a small amount of the elution product for immunoblotting analysis. Use an antibody that specifically recognizes the succinylation of lysine at position 285 of RAD51 to confirm that high-purity succinylated modified RAD51 protein has been obtained. Example 4

[0037] This embodiment provides a formulation that promotes the succinylation of RAD51, and its preparation method includes the following steps: S01. Weigh 10 mg of the lyophilized polypeptide powder obtained in Preparation Example 1 and measure 10 mL of sterile PBS buffer. S02. In a sterile laminar flow hood, add the peptide powder to PBS buffer, turn on magnetic stirring at 4°C, stir at 200 rpm for 10 minutes until no solid particles are visible to the naked eye. S03. Titrate the solution to adjust the pH to 6.5 using a 0.10 mol / L hydrochloric acid or sodium hydroxide solution; S04. The solution is filtered and sterilized using a polyethersulfone filter membrane with a pore size of 0.20 μm. The filtrate is collected to obtain a preparation that promotes the succinylation of RAD51 with a final concentration of 1 mg / mL. S05. Aseptically dispense the obtained preparation into cryovials and store them in a -60°C freezer. Example 5

[0038] This embodiment provides a formulation that promotes the succinylation of RAD51, and its preparation method includes the following steps: S01. Weigh 50 mg of the lyophilized polypeptide powder obtained in Preparation Example 2 and measure 15 mL of sterile PBS buffer. S02. In a sterile laminar flow hood, add the peptide powder to PBS buffer, turn on magnetic stirring at 25°C, stir at 400 rpm for 20 minutes until no solid particles are visible to the naked eye. S03. Titrate the solution with a 0.15 mol / L hydrochloric acid or sodium hydroxide solution to adjust the pH to 7.2; S04. The solution was filtered and sterilized using a polyethersulfone filter membrane with a pore size of 0.22 μm. The filtrate was collected to obtain a formulation that promotes the succinylation of RAD51 with a final concentration of 5 mg / mL. S05. Aseptically dispense the obtained preparation into cryovials and store them in a -70°C freezer. Example 6

[0039] This embodiment provides a formulation that promotes the succinylation of RAD51, and its preparation method includes the following steps: S01. Weigh 100 mg of the lyophilized polypeptide powder obtained in Preparation Example 3 and measure 20 mL of sterile PBS buffer. S02. In a sterile laminar flow hood, add the peptide powder to PBS buffer, turn on magnetic stirring at 30°C, stir at 600 rpm for 30 minutes until no solid particles are visible to the naked eye. S03. Titrate the solution with a 0.20 mol / L hydrochloric acid or sodium hydroxide solution to adjust the pH to 7.8; S04. The solution was filtered and sterilized using a polyethersulfone filter membrane with a pore size of 0.24 μm. The filtrate was collected to obtain a formulation that promotes the succinylation of RAD51 with a final concentration of 10 mg / mL. S05. Aseptically dispense the obtained preparation into cryovials and store them in a -80°C freezer.

[0040] Comparative Examples 1-4: Comparative Example 1: This comparative example provides an unmodified wild-type RAD51 protein. The difference from Example 2 is that in step S02, sodium succinate and HDAC11 inhibitor are not added to the culture medium, and only conventional complete culture medium is used for culture. The remaining cell transfection, lysis, purification and identification steps are the same.

[0041] Comparative Example 2: Compared with Example 5, the difference is that the amino acid sequence of the synthesized polypeptide in the preparation step is HLYVSPWGGLGLRWELPLDAF, while the other synthesis parameters and drug preparation steps are the same.

[0042] Comparative Example 3: Compared with Example 5, the difference is that the amino acid sequence of the synthesized polypeptide in the preparation step is LLRLADEF (containing only the functional sequence), while the other synthesis parameters and drug preparation steps are the same.

[0043] Comparative Example 4: Compared with Example 5, the difference is that the amino acid sequence of the synthesized polypeptide in the preparation step is HLYVSPWGG (containing only cell-penetrating peptides), while the other synthesis parameters and drug preparation steps are the same.

[0044] Test Examples 1-7: Test Example 1: Detection of the effect of intracellular succinyl-CoA levels on homologous recombination repair efficiency Experimental description: This experiment aims to investigate the regulatory effect of changes in succinyl-CoA concentration in the cellular metabolic microenvironment on the efficiency of DNA homologous recombination repair.

[0045] Experimental steps: HEK293T cells were selected as experimental subjects and randomly divided into a gene interference group and an exogenous treatment group. For the gene interference group, liposome transfection reagents were used to transfect the cells with interfering plasmids targeting succinyl-CoA synthase OGDH or DLD, and interfering plasmids targeting the depleting enzymes SUCLG2 or SUCLA2. Cells transfected with empty vectors served as a control. For the exogenous treatment group, sodium succinate at concentrations of 0 mmol / L, 20 mmol / L, and 40 mmol / L were added to the cell culture medium for incubation.

[0046] Twenty-four hours after treatment, a subset of cells were collected to determine the relative content of succinyl-CoA within the cells. Simultaneously, each group of cells was co-transfected with the DR-GFP reporter plasmid and the I-SceI endonuclease plasmid, using the I-SceI enzyme to cleave DNA intracellularly, generating double-strand breaks. Forty-eight hours after transfection, cells were collected, and the proportion of cells expressing green fluorescent protein was detected by flow cytometry; this proportion directly reflects the homologous recombination repair efficiency. Furthermore, cellular proteins were extracted for immunoprecipitation and Western blotting analysis to detect the modification status of the homologous recombination core protein RAD51 and to screen related regulatory enzymes.

[0047] Experimental data:

[0048] Experimental conclusion: Combining the experimental data in Table 1 and the appendix Figure 2 Analysis of the experimental results showed that the intracellular succinyl-CoA level was significantly negatively correlated with the homologous recombination repair efficiency.

[0049] As shown in Table 1, when the synthases OGDH or DLD were inhibited by gene interference, the intracellular content of succinyl-CoA decreased, and the corresponding homologous recombination repair efficiency increased to more than 1.5 times that of the control group. Conversely, when the depleting enzymes SUCLG2 or SUCLA2 were inhibited, or when exogenous sodium succinate was added, leading to the accumulation of succinyl-CoA, the homologous recombination repair efficiency was significantly inhibited.

[0050] Combined with appendix Figure 2 F to Appendix Figure 2 The mechanistic study of I revealed that succinyl-CoA, as an acyl donor, mediates succinylation modification of RAD51, a core protein in the homologous recombination pathway. High levels of succinyl-CoA promote the accumulation of modified RAD51, thereby inhibiting its repair function.

[0051] Test Example 2: Detection of the Regulation of RAD51 Succinylation Modification on RAD51 Spot Formation and Homologous Recombination Repair Function Experimental description: This experiment aims to verify the specific mechanism by which RAD51 succinylation modification affects the homologous recombination repair pathway mediated by the cell function.

[0052] Experimental steps: The experiment was divided into two parts: homologous recombination efficiency detection and RAD51 spot formation detection.

[0053] In the homologous recombination efficiency detection section, a stable integration DR-GFP reporter system was selected (see Appendix). Figure 1 HEK293T cells were transfected with shRNA plasmids targeting OXCT1, HDAC11, or RAD51 for single-gene or double-gene knockdown. Twenty-four hours after transfection, I-SceI plasmid was co-transfected to induce DNA damage. The proportion of GFP-positive cells was detected by flow cytometry after 48 hours.

[0054] In the RAD51 spot formation detection section, U2OS cells were selected and pretreated for 24 hours with either the OGDH inhibitor CPI-613, sodium succinate, or the HDAC11 inhibitor. Subsequently, they were treated with 6 Gy of ionizing radiation to induce DNA double-strand breaks. After irradiation, the cells were restored to culture, fixed, and immunofluorescence stained. Using RAD51-specific antibodies, the number of RAD51 spots in each cell nucleus was counted under a confocal microscope. The control group values ​​were normalized to 1.00, and the relative changes between the treatment groups were calculated.

[0055] Experimental data:

[0056] Experimental conclusion: Based on the experimental data in Table 2 and the appendix Figure 3 A to Appendix Figure 3 The results analysis of D showed that the succinylation modification state of RAD51 had a negative regulatory effect on its function.

[0057] Regarding the efficiency of homologous recombination repair, see attached... Figure 3 A and Appendix Figure 3 As shown in B, when knocked down (in the attached) Figure 2 When the enzyme OXCT1, which was identified as promoting modification, was used, the level of intracellular succinylation decreased and the homologous recombination repair efficiency was significantly increased to 1.68 times that of the control group.

[0058] Regarding RAD51 spot formation, see attached... Figure 3 C to Appendix Figure 3 As shown in D, CPI-613 treatment reduced the supply of succinyl-CoA, thereby reducing the modification level and leading to an increase in the number of RAD51 spots induced by ionizing radiation to 1.58 times that of the control group, indicating that the low modification state is conducive to the recruitment of RAD51 at the damage site.

[0059] Test Example 3: Identification and functional verification of lysine residue 285 of RAD51 protein as a major succinylation modification site. Experimental description: This experiment aims to identify the specific amino acid sites at which RAD51 protein undergoes succinylation modification, verify the regulatory role of the modification state at these sites on homologous recombination repair function, and simultaneously verify the quality and specificity of the succinylated modified RAD51 protein prepared in Examples 1 to 3 of this invention.

[0060] Experimental steps: The experiment was divided into three parts: mutant function screening, site specificity verification, and protein preparation verification.

[0061] Expression plasmids for RAD51 wild-type and point mutants K64R, K73R, and K285R were constructed. These plasmids were transfected into HEK293T cells integrating the DR-GFP system, and simultaneously transfected with the I-SceI plasmid to induce DNA damage. After 48 hours of culture, the proportion of green fluorescent protein-positive cells was detected by flow cytometry to assess the impact of mutations at different sites on homologous recombination repair efficiency.

[0062] HEK293T cells were transfected with either wild-type RAD51 or K285R plasmids, and simultaneously transfected with shRNAs targeting OXCT1 or HDAC11 for gene interference. Cellular proteins were extracted, and Western blotting was performed using an antibody that specifically recognizes succinylation of lysine at position 285 of RAD51 to observe changes in the modification level at this site after the deletion of the modifying enzyme or the demodifying enzyme.

[0063] Succinylated RAD51 protein prepared in Examples 1-3 and unmodified wild-type RAD51 protein prepared in Comparative Example 1 were taken respectively. The concentrations of the above proteins were adjusted to the same level of 0.5 mg / mL, and 2 μL of each was spotted onto nitrocellulose membranes for dot blot hybridization experiments. Incubation and color development were performed using an antibody that specifically recognizes the succinylation of lysine at position 285 of RAD51. The relative intensity of modification levels of each protein group was analyzed by grayscale scanning.

[0064] Experimental data:

[0065] Experimental conclusion: Based on the experimental data in Table 3 and the appendix Figure 4 Analysis of the relevant results determined that lysine K285 at position 285 is a key site for succinylation modification of RAD51 and its regulatory function.

[0066] Regarding the efficiency of homologous recombination repair, see attached... Figure 4As shown in Table C and Table 3, compared with the control group expressing wild-type RAD51, the repair efficiency of cells expressing K64R or K73R mutants showed no significant change or only slight fluctuations. However, the homologous recombination repair efficiency of cells expressing K285R mutants was significantly increased, reaching 1.78 times that of the control group.

[0067] Regarding the verification of site specificity and regulatory mechanisms, see attached... Figure 4 D and appendix Figure 4 As shown in E, detection using specific antibodies revealed that knockdown of the pro-modification enzyme OXCT1 resulted in a decrease in succinylation signal at the K285 site of wild-type RAD51, while knockdown of the demodification enzyme HDAC11 resulted in an increase in the signal at this site.

[0068] Test Example 4: Detection of the regulation of HDAC11 phosphorylation and its interaction with RAD51 by the DNA damage response pathway Experimental description: This experiment aims to investigate how intracellular signaling pathways regulate the functional state of desuccinylase HDAC11 after DNA damage occurs in cells.

[0069] Experimental steps: HEK293T cells were selected and transfected with the HDAC11 plasmid expressing the FLAG tag. The cells were randomly divided into three groups: blank control group (solvent treatment, no radiation), radiation model group (solvent treatment, 6 Gy ionizing radiation), and inhibitor group (pre-treated with 10 μmol / L of ATM inhibitor KU55933 for 24 hours, followed by 6 Gy ionizing radiation).

[0070] After irradiation treatment and recovery culture for 1 hour, cells were lysed and total protein was extracted. Immunoprecipitation was performed using magnetic beads conjugated with anti-FLAG antibody to enrich the intracellular HDAC11 protein complex. Subsequent protein electrophoresis and Western blotting analysis were performed. The phosphorylation level of HDAC11 in the precipitate was detected using anti-p-SQ / TQ antibody, and the amount of endogenous RAD51 protein bound to HDAC11 was detected using anti-RAD51 antibody. The total amount of HDAC11 detected using anti-FLAG antibody was used as an internal control. The relative phosphorylation level and interaction strength of each group were calculated using grayscale analysis.

[0071] Experimental data:

[0072] Experimental conclusion: Based on the experimental data in Table 4 and the appendix Figure 5 A to Appendix Figure 5 The analysis of results B confirmed the existence of the signal axis from DNA damage to ATM to HDAC11 to RAD51.

[0073] Regarding HDAC11 phosphorylation modification, see attached... Figure 5 As shown in Figure A, the phosphorylation level of HDAC11 in the unirradiated control group remained at a low basal level. (Combined with...) Figure 5 Domain analysis of D revealed the presence of a potential ATM phosphorylation site, TQ-motif, in the HDAC11 protein sequence.

[0074] Regarding protein-protein interactions, as shown in the appendix Figure 5 As shown in B, ionizing radiation treatment increased the interaction strength between HDAC11 and RAD51 to 2.88 times that of the control group, indicating that DNA damage signaling promotes the recruitment of demodification enzymes to the substrate.

[0075] Test Example 5: RAD51 succinylation modification enhances tumor chemosensitivity and its clinical relevance. Experimental description: This experiment aims to explore the intrinsic link between RAD51 succinylation modification and tumor genomic instability and chemotherapeutic drug sensitivity, and to verify the feasibility of overcoming tumor drug resistance by intervening in this modification state.

[0076] Experimental steps: In the bioinformatics analysis section, transcriptome data and clinical information of breast cancer patients were downloaded from the Cancer Genome Atlas database. The samples were divided into high expression group and low expression group according to the expression level of BRCA1 gene, and the correlation between HDAC11 expression level and genomic scar score, i.e. homologous recombination defect score, was analyzed within each group.

[0077] In the clinical sample testing section, tumor tissues from breast cancer patients treated with chemotherapy were collected to prepare tissue microarrays. Immunohistochemical staining was performed using an antibody that specifically recognizes succinylated lysine at position 285 of RAD51. The modification level was assessed based on the staining intensity, and survival curves were plotted using patient survival data to analyze the relationship between the modification level and clinical prognosis.

[0078] For the cellular drug sensitivity assay, triple-negative breast cancer cell lines Hs578T, MDA-MB-231, and HCC1806 were selected. The basal level of succinylation at lysine 285 of RAD51 in each cell line was detected using co-immunoprecipitation and Western blotting. Cells were then seeded in 96-well plates and treated with gradient concentrations of olaparib. For the drug-resistant MDA-MB-231 line, a combination therapy group was established. After a certain culture time, cell viability was measured using CCK8 reagent, and the half-maximal inhibitory concentration (IC50) was calculated to evaluate drug sensitivity.

[0079] Experimental data:

[0080] Experimental conclusion: Based on the experimental data in Table 5 and the appendix Figure 6 The analysis results confirmed that RAD51 succinylation modification is a key factor determining the genomic stability and chemosensitivity of tumor cells.

[0081] In database analysis, the expression level of HDAC11 was negatively correlated with the homologous recombination defect score in BRCA1 high-expression samples. This means that as a demodification enzyme, the high expression of HDAC11 reduces the succinylation level of RAD51, thereby maintaining the homologous recombination repair function and reducing genomic instability.

[0082] Clinical sample analysis showed that patients with high levels of RAD51 lysine succinylation at position 285 exhibited a better prognostic trend after chemotherapy, which was correlated with tumor malignancy due to high homologous recombination deficiency scores, but also suggested that these patients had homologous recombination repair defects. Hs578T cells, naturally possessing high levels of RAD51 succinylation modification, exhibited significant homologous recombination repair deficiency characteristics, and were therefore highly sensitive to the PARP inhibitor olaparib, which utilizes a synthetic lethal mechanism. Conversely, MDA-MB-231 cells with low modification levels showed resistance.

[0083] Test Example 6: Detection of the pharmacodynamic and structure-activity relationship of peptides blocking the interaction between HDAC11 and RAD51 and sensitizing chemotherapeutic drugs. Experimental description: This experiment, based on the interaction domain between the C-terminus of the RAD51 protein and the desuccinylase HDAC11, aims to screen and verify peptide drugs that can competitively block their binding.

[0084] Experimental steps: Based on the appendix Figure 7 The immunoprecipitation results shown in Figure A confirm that the C-terminus of RAD51 is the key region for physical interaction with HDAC11. The α-helix structure of this region was analyzed using AlphaFold, and eight candidate peptides P1 to P8 (sequences SEQ ID NO:1-SEQ ID NO:8, respectively) and a disordered peptide Pscr were designed, as shown in Table 1. These peptides were added to HEK293T cell lysates, and their ability to block the interaction between HDAC11 and RAD51 was detected by immunoprecipitation. Simultaneously, the effect of each peptide treatment on homologous recombination repair efficiency was detected using the DR-GFP system.

[0085] The optimal active sequence P7 was selected to construct the drug molecule. To verify the effect of different drug formulation processes on efficacy, the formulations prepared in Examples 4-6 were selected. These three formulations were diluted to the same final concentration with culture medium to treat U2OS cells, and their effect on enhancing the succinylation level of RAD51 K285 was detected.

[0086] Subsequently, Example 5 was set up, namely, P7 sequence fused with membrane-penetrating peptide; Comparative Example 2 was set up, namely, Pscr disordered sequence fused with membrane-penetrating peptide; Comparative Example 3 was set up, namely, containing only P7 core sequence but no membrane-penetrating peptide; and Comparative Example 4 was set up, namely, containing only membrane-penetrating peptide.

[0087] The number of RAD51 spots formed in the nucleus of U2OS cells treated with different peptides under ionizing radiation-induced precipitation was detected using immunofluorescence. The sensitivity of triple-negative breast cancer cell lines MDA-MB-231 and HCC1806 to the chemotherapeutic drug olaparib under different peptide pretreatments was detected using the CCK8 assay, and cell survival curves were plotted.

[0088] Experimental data:

[0089] Experimental conclusion: Combined with Table 6 and Appendix Figure 7 Data analysis confirmed that the formulations for promoting RAD51 succinylation provided in Examples 4, 5 and 6 all have significant biological activity, and the active ingredients are rationally and effectively designed.

[0090] Appendix Figure 7 A to Appendix Figure 7 The results showed that the C-terminus of RAD51 is a key region for physical interaction with HDAC11. Based on this, the P7 peptide designed can specifically inhibit the interaction between HDAC11 and RAD51 and reduce the efficiency of homologous recombination repair in cells.

[0091] Table 6 shows that the drug preparation process provided by this invention has good stability and can obtain highly efficient drug formulations within the set parameter range. The indicators in Example 5 are slightly better, demonstrating the effect of process parameter optimization.

[0092] The survival curve results are attached. Figure 7 F and appendix Figure 7 As shown in G, the treatment in Example 5 suppressed the survival curve of triple-negative breast cancer cells under olaparib treatment, achieving drug resistance reversal. However, Comparative Example 2 and other comparative examples did not show any sensitizing effect.

[0093] Test Example 7: In vivo pharmacodynamic evaluation of peptide drug P7 in combination with olaparib in a breast cancer PDX model Experimental description: This experiment aims to verify the antitumor efficacy of the present invention's peptide P7 combined with the poly-ADP ribose polymerase inhibitor olaparib at the in vivo animal level using a more clinically relevant human breast cancer xenograft model, namely the PDX model.

[0094] Experimental steps: Four PDX tumor tissues from breast cancer, preserved in liquid nitrogen, were resuscitated. Proteins were extracted, and the succinylation level of lysine at position 285 of RAD51 was detected using immunoprecipitation and Western blotting. Sample number 3, with the lowest succinylation level, was selected for model construction. This tumor tissue was cut into approximately 1 mm pieces. 3 Small pieces were inoculated subcutaneously into athymic nude mice under gas anesthesia. The tumors were allowed to grow to approximately 600 mm. 3 We conducted passages to construct a cohort of tumor-bearing mice for drug efficacy evaluation.

[0095] Once the tumors in the mice reached a measurable size, they were randomly divided into 6 groups. The dosing regimen was as follows: olaparib was administered intraperitoneally every other day at a dose of 10 mg / kg body weight; peptide P7 or disordered peptide Pscr was administered intraperitoneally daily at a dose of 10 mg / kg body weight. During the dosing period, the major and minor axes of the tumors were measured periodically using calipers, and the tumor volume was calculated and growth curves plotted. At the end of the experiment, the nude mice were sacrificed, and the tumor tissue was completely dissected and weighed. A portion of the tumor tissue was lysed to extract proteins, and immunoprecipitation analysis was performed to detect the succinylation level of lysine at position 285 of RAD51 in the tumor tissue of each treatment group.

[0096] Experimental data:

[0097] Experimental conclusion: Combining the experimental data in Table 7 and the appendix Figure 8 Comprehensive analysis confirmed that peptide P7 can effectively reverse the resistance of breast cancer to olaparib in vivo, and has good safety.

[0098] During the sample screening stage, as shown in the attached document... Figure 8 As shown in Figure A, PDX sample 3 exhibits the lowest level of RAD51 succinylation and a higher level of HDAC11.

[0099] Regarding tumor growth inhibition, such as... Figure 8 C and appendix Figure 8 As shown in Figure D, treatment with either peptide P7 or the disordered peptide Pscr alone did not result in significant differences in tumor volume and weight compared to the control group. This indicates that blocking the interaction between HDAC11 and RAD51 alone is insufficient to induce tumor cell death in the absence of DNA damage inducers, which also reflects the low single toxicity of this peptide.

[0100] Regarding the verification of molecular mechanisms, see attached... Figure 8 As shown in E, biochemical analysis of tumor tissues revealed that high levels of RAD51 lysine succinylation modification at position 285 were detected only in tumor samples from the olaparib combined with P7 treatment group.

[0101] Appendix: Amino acid sequences of the eight polypeptides (P1-P8): SEQ ID NO:1: PERLLAVAER.

[0102] SEQ ID NO:2: TDHQTQLLY.

[0103] SEQ ID NO:3: YQASAMMVE.

[0104] SEQ ID NO:4: RGELSARQ.

[0105] SEQ ID NO:5: ELSARQMH.

[0106] SEQ ID NO:6: LARFLRML.

[0107] SEQ ID NO:7: LLRLADEF.

[0108] SEQ ID NO:8: QVDGAAMF.

[0109] Cell membrane penetration peptide amino acid sequence: SEQ ID NO:9:HLYVSPWGG.

Claims

1. Succinylated modification of RAD51 protein, characterized in that, It is prepared through the following steps: Cells were seeded in culture dishes and cultured. The cells were then transfected with a tagged RAD51 expression plasmid using a transfection reagent to obtain transfected cells. Sodium succinate and HDAC11 inhibitor were added to the culture medium of the transfected cells, and the cells were cultured for a period of time to accumulate RAD51 succinylation modification in the cells, thus obtaining the modified cells. The modified cells were collected, and a lysis buffer containing protease inhibitors and deacetylase inhibitors was added. The cells were lysed on ice, and the supernatant was collected by centrifugation. Magnetic beads conjugated with anti-tag antibodies were added, and the cells were rotated and incubated under refrigeration to obtain protein-bound magnetic beads. The protein-bound magnetic beads were washed with lysis buffer to remove non-specifically bound proteins, followed by competitive elution with a tagged peptide-containing elution buffer to obtain a purified protein solution. The purified protein solution was subjected to immunoblotting analysis, and the antibody with specific recognition site modification was used to confirm the acquisition of the succinylated modified RAD51 protein.

2. The succinylated modified RAD51 protein according to claim 1, characterized in that, The confluence of cells at the time of transfection was 60%-80%; the time interval between transfection and the addition of sodium succinate was 5-8 hours; and the culture time was 20-28 hours.

3. The succinylated modified RAD51 protein according to claim 1, characterized in that, The final concentration of the added sodium succinate is 30mM-50mM, and the final concentration of the HDAC11 inhibitor is 0.5μM-1.5μM; the deacetylase inhibitor includes NAM and TSA.

4. An formulation that promotes RAD51 succinylation, characterized in that, It is prepared through the following steps: Weigh the lyophilized polypeptide powder and measure the sterile buffer solution to obtain the raw materials for the formulation; Under sterile conditions, the lyophilized polypeptide powder is added to the sterile buffer solution, and stirring is started and continued until no solid particles are visible to the naked eye, thus obtaining a polypeptide solution. The pH value of the polypeptide solution was adjusted by titration with a pH adjuster to obtain a pH-adjusted solution; The pH-adjusted solution was filtered and sterilized using a filter membrane, and the filtrate was collected to obtain a sterile preparation solution. The sterile formulation solution is aseptically dispensed and stored in a frozen environment to obtain the formulation that promotes RAD51 succinylation.

5. The formulation for promoting RAD51 succinylation according to claim 4, characterized in that, The amino acid sequence of the polypeptide in the lyophilized polypeptide powder is formed by fusing the sequence of the cell-penetrating peptide with the sequence shown in SEQ ID NO:7, wherein the sequence of the cell-penetrating peptide is shown in SEQ ID NO:

9.

6. The formulation for promoting RAD51 succinylation according to claim 4, characterized in that, The stirring is carried out at 4℃-30℃, with a stirring speed of 200rpm-600rpm and a stirring time of 10-30 minutes; the sterile buffer is PBS buffer; and the filter membrane is a polyethersulfone filter membrane.

7. The formulation for promoting RAD51 succinylation according to claim 4, characterized in that, The mass of the lyophilized polypeptide powder weighed is 10mg-100mg; the volume of the sterile buffer measured is 10mL-20mL; and the final concentration of the polypeptide in the formulation that promotes RAD51 succinylation is 1mg / mL-10mg / mL.

8. The formulation for promoting RAD51 succinylation according to claim 4, characterized in that, The pH adjuster is hydrochloric acid or sodium hydroxide solution, used to adjust the pH of the polypeptide solution to 6.5-7.

8.

9. The formulation for promoting RAD51 succinylation according to claim 4, characterized in that, The lyophilized polypeptide powder is prepared by a solid-phase synthesis method, which includes: The Fmoc-protected amino acids, condensing agent HBTU, and organic base DIEA were mixed in a molar ratio of (3-8):(3-8):(6-16) using Rink Amide MBHA resin with a degree of substitution of 0.3 mmol / g-0.8 mmol / g to carry out a condensation reaction. After the synthesis was completed, the mixture was cleaved using a cleavage solution composed of trifluoroacetic acid, water, and triisopropylsilane.

10. The application of succinylation modification of RAD51 protein and formulations that promote RAD51 succinylation, characterized in that, The use of the succinylated modified RAD51 protein according to any one of claims 1-3 in the preparation of a kit for screening drugs targeting RAD51 post-translational modifications; Alternatively, the use of the formulation for promoting RAD51 succinylation according to any one of claims 4-9 in the preparation of tumor chemotherapy sensitizers, radiotherapy sensitizers or PARP inhibitor sensitizers; The drug enhances the sensitivity of tumor cells to chemotherapy, radiotherapy, or PARP inhibitors by upregulating RAD51 succinylation levels and inhibiting RAD51-mediated homologous recombination repair.

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