CN114392344A - Rad51 inhibitor and application thereof - Google Patents

Abstract

The invention discloses a Rad51 inhibitor and application thereof. The Rad51 inhibitor includes Nanog protein. The invention discovers for the first time that the Nanog protein can inhibit Rad51, including strong interaction affinity with Rad51, inhibit Rad51 from being combined with single-stranded DNA (ssDNA), and further inhibit Rad51 from repairing DNA damage, and the Nanog protein can be used as a novel Rad51 inhibitor and has a wide application prospect in the field of medicine preparation.

Description

Rad51 inhibitor and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and relates to a Rad51 inhibitor and application thereof.

Background

Chemotherapy is one of the major therapeutic approaches for malignancies, leading to the death of cancer cells, mainly by producing DNA damage inside the cancer cells. However, many cancer patients have Rad51 recombinase with high expression, and Rad51 is a key factor for mediating Homologous Recombination (HR) to repair DNA damage signal paths without errors, so that the high expression of Rad51 recombinase improves the repair of DNA damage caused by radiotherapy and chemotherapy, thereby inhibiting the death of cancer cells, and the patients have obvious radiosensitivity and poor treatment effect. Therefore, the development of inhibitors against Rad51 is of great interest for improving the effectiveness of cancer chemotherapy treatments.

The inhibitors of Rad51 reported at present are mainly synthetic small molecule compounds, including B02, DIDS, CSB, IBR2, RI-1, Halenaquinone, RS-1 and the like, for example, CN111263756A discloses an inhibitor of RAD51 and is used for treating or preventing diseases related to mitochondrial defects, although various inhibitors of Rad51 exist on the market, the application of the inhibitor in cancer treatment in combination with chemoradiotherapy is not reported, because the synthetic inhibitors generally have the problems of poor stability, strong potential toxicity and the like.

In conclusion, how to provide a Rad51 inhibitor with high stability and low toxicity is one of the problems to be solved in the field of tumor chemotherapy.

Disclosure of Invention

Aiming at the defects and actual requirements of the prior art, the invention provides a Rad51 inhibitor and application thereof, and the invention discovers that the Nanog protein can inhibit Rad51 to repair DNA damage for the first time, and has wide application prospect in the field of tumor chemoradiotherapy.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a Rad51 inhibitor, the Rad51 inhibitor comprising Nanog protein.

In the invention, the Nanog protein is found to be capable of inhibiting Rad51 for the first time, including strong interaction affinity with Rad51, Rad51 and single-stranded DNA (ssDNA) combination are inhibited, and the function of Rad51 in repairing DNA damage is further inhibited, which indicates that the Nanog protein can be used as a novel Rad51 inhibitor, and therefore, the Nanog protein also has a wide application prospect in the field of medicine preparation.

In the invention, the amino acid sequence of the Nanog protein comprises a sequence shown in SEQ ID NO. 1.

SEQ ID NO.1：

MSVGLPGPHSLPSSEEASNSGNASSMPAVFHPENYSCLQGSATEMLCTEAASPRPSSEDLPLQGSPDSSTSPKQKLSSPEADKGPEEEENKVLARKQKMRTVFSQAQLCALKDRFQKQKYLSLQQMQELSSILNLSYKQVKTWFQNQRMKCKRWQKNQWLKTSNGLIQKGSAPVEYPSIHCSYPQGYLVNASGSLSMWGSQTWTNPTWSSQTWTNPTWNNQTWTNPTWSSQAWTAQSWNGQPWNAAPLHNFGEDFLQPYVQLQQNFSASDLEVNLEATRESHAHFSTPQALELFLNYSVTPPGEI。

According to the invention, the Nanog protein inhibits Rad51 binding to ssDNA.

According to the invention, the Nanog protein inhibits Rad51 from repairing double-stranded DNA damage.

In a second aspect, the invention provides the use of Nanog protein for inhibiting Rad51 repair of double-stranded DNA damage for non-disease diagnostic purposes.

The invention discovers that the Nanog protein can inhibit the function of Rad51 in repairing DNA damage for the first time, and shows that the Nanog protein can be widely applied to the process of repairing double-stranded DNA damage by inhibiting Rad51, such as the research on the double-stranded DNA damage repair mechanism and the like.

In a third aspect, the invention provides the use of Nanog in the preparation of Rad51 inhibitors.

In a fourth aspect, the invention provides the use of Nanog in the preparation of a medicament for treating a tumor.

Preferably, the medicament comprises a medicament for radiotherapy and chemotherapy of a tumor.

The invention discovers that the Nanog protein can inhibit the function of Rad51 in repairing DNA damage, and shows that the Nanog protein can be used for developing medicaments for improving the effect of radiotherapy and chemotherapy of malignant tumors.

In a fifth aspect, the present invention provides a pharmaceutical composition comprising Nanog protein.

Preferably, the pharmaceutical composition further comprises an adjuvant.

Preferably, the adjuvant comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrating agent, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, a bacteriostatic agent or a buffer.

Preferably, the dosage form of the pharmaceutical composition comprises any one of suspension, granules, capsules, powder, tablets, emulsions, solutions, dripping pills or injections.

Compared with the prior art, the invention has the following beneficial effects:

the invention discovers for the first time that the Nanog protein can inhibit Rad51, including strong interaction affinity with Rad51, inhibit Rad51 from being combined with single-stranded DNA (ssDNA), and further inhibit Rad51 from repairing DNA damage, and the Nanog protein can be used as a novel Rad51 inhibitor and has a wide application prospect in the field of medicine preparation.

Drawings

FIG. 1 is a graph showing the results of the purification of Nanog and Rad51 proteins;

FIG. 2 is a graph showing the results of GST pull down experiments on Nanog and Rad51 proteins;

FIG. 3 is a graph showing the results of EMSA experiments;

FIG. 4 is a drawing showing the result of Western blotting experiment;

FIG. 5 is a diagram showing the result of the quantitative statistical analysis of the Western blotting experiment.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.

Example 1

This example demonstrates that Nanog is able to interact directly with Rad 51.

Respectively constructing clones of Nanog (GST tag) and Rad51(His tag) on prokaryotic expression vectors, respectively constructing Rad51 (fused with GST tag) and Nanog (fused with His tag) into PET30a and pGEX6p-1 expression vectors, transferring constructed plasmids into BL21 protein expression escherichia coli, inducing protein expression and purifying, wherein the result is shown in figure 1 (protein bands with corresponding sizes are marked with "+"), performing GST fusion protein sedimentation (GST pull down) experiment by using the purified protein, respectively mixing 10 mu g of the Nanog protein and GST protein (contrast) fused with GST tag with 10 mu g of Rad5 protein fused with His tag, supplementing the reaction system to 500 mu L, taking 1% of the mixed solution of the reaction system as Input, reserving the sample for detection, and shaking the table (120r/min) at 16 ℃ for 18 hours.

The results of the GST pull down experiment are shown in fig. 2, where ". star" is labeled as Nanog, a GST-tagged target protein, which co-precipitates Rad51, indicating that Nanog can interact directly with Rad51 protein-protein.

Example 2

This example demonstrates that Nanog can inhibit the binding of Rad51 to ssDNA.

Rad51 is a DNA binding protein that, after recruitment to the site of damage, replaces RPA to bind to ssDNA to form nucleoprotein filaments for homologous recombination-mediated DNA damage repair, to elucidate that Nanog inhibits Rad51 binding to ssDNA, gel migration Experiments (EMSA) were performed using the in vitro purified protein of example 1, purified Nanog and Rad51 were desalted and then each reactant was added as in Table 1 and reacted for 20 min at 25 ℃.

TABLE 1

The results of EMSA experiments are shown in FIG. 3, wherein "-" indicates that no corresponding component is added, "+" indicates that the corresponding component is added, a 90nt ssDNA is labeled with biotin (biotin), Rad51 can be combined with the ssDNA, so that when non-denatured polypropylene gel electrophoresis is performed, the migration rate of a protein-DNA complex is slower than that of a non-combined probe, an electrophoresis blocking band is formed, purified Nanog protein is added on the basis of a Rad51 and ssDNA system, and the quantitative finding of the blocking band shows that the electrophoresis blocking effect is obviously weakened after the Nanog is added, which indicates that the Nanog is combined with Rad51, so that the amount of Rad51 combined with the ssDNA is reduced, and the Rad51 is interfered with the ssDNA.

Example 3

This example demonstrates that Nanog can inhibit the function of Rad51 to repair double-stranded lesions.

In order to explore that Nanog can inhibit repair of double-stranded damage by Rad51 in combination with CPT (DNA double-stranded damage inducer) treatment causing DNA double-stranded damage, the influence of different Nanog expression levels on Rad51 DNA double-stranded damage repairing activity in 293T cells was examined by western blotting experiments, Rad51 and different concentrations of Nanog (+: 0.1. mu.g, + +: 0.5. mu.g, + ++: 1. mu.g) were overexpressed in 293T cells, after 44 hours of expression, 2. mu.M CPT was used for 4 hours, the cells were collected, histone and holoprotein were extracted to examine the level of gamma H2AX (DNA double-stranded damage marker) and cell expression, respectively.

The results of the Western blotting experiments are shown in FIG. 4, FIG. 5 is the result of the quantitative statistical analysis of the results of FIG. 4, Image J is used to quantify the gray values of the Anti-gamma H2AX band and the Anti-H3 band, the gray value of the Anti-gamma H2AX band is divided by the gray value of the Anti-H3 band to obtain the relative gamma H2AX expression amount, the experiments are not less than 3 times (ttest,. about.P <0.001), it can be seen from FIGS. 4 and 5 that no-load of Nanog and no-load of Rad51 are expressed in two groups of 293T cells, one of which is treated with 2. mu.M CPT (DNA double-strand damage inducer) for 4 hours, the other is not treated, the CPT treatment significantly increases the level of gamma H2AX (DNA double-strand damage marker), which indicates that CPT causes double-strand DNA damage, after over-expression of 51, the level of gamma.H 2AX decreases, which indicates that Rad51 repairs double-strand damage, and when the level of Nanog increases in the cells, leads to the increase of the level of gamma H2AX, and shows that the over-expressed Nanog successfully inhibits the repair of Rad51 to double-stranded DNA damage; furthermore, this inhibitory activity increased with increasing levels of Nanog expression, demonstrating the specificity and effectiveness of Nanog in inhibiting Rad51 activity.

In conclusion, the invention discovers for the first time that the Nanog protein can inhibit Rad51, including the strong interaction affinity with Rad51, inhibit the combination of Rad51 and single-stranded DNA (ssDNA), and further inhibit the function of Rad51 in repairing DNA damage, and shows that the Nanog protein can be used as a novel Rad51 inhibitor and has a wide application prospect in the field of medicine preparation.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Sequence listing

<110> university of capital education

<120> Rad51 inhibitor and application thereof

<130> 2022-01-17

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<170> PatentIn version 3.3

<210> 1

<211> 305

<212> PRT

<213> Artificial sequence

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Met Ser Val Gly Leu Pro Gly Pro His Ser Leu Pro Ser Ser Glu Glu

1 5 10 15

Ala Ser Asn Ser Gly Asn Ala Ser Ser Met Pro Ala Val Phe His Pro

20 25 30

Glu Asn Tyr Ser Cys Leu Gln Gly Ser Ala Thr Glu Met Leu Cys Thr

35 40 45

Glu Ala Ala Ser Pro Arg Pro Ser Ser Glu Asp Leu Pro Leu Gln Gly

50 55 60

Ser Pro Asp Ser Ser Thr Ser Pro Lys Gln Lys Leu Ser Ser Pro Glu

65 70 75 80

Ala Asp Lys Gly Pro Glu Glu Glu Glu Asn Lys Val Leu Ala Arg Lys

85 90 95

Gln Lys Met Arg Thr Val Phe Ser Gln Ala Gln Leu Cys Ala Leu Lys

100 105 110

Asp Arg Phe Gln Lys Gln Lys Tyr Leu Ser Leu Gln Gln Met Gln Glu

115 120 125

Leu Ser Ser Ile Leu Asn Leu Ser Tyr Lys Gln Val Lys Thr Trp Phe

130 135 140

Gln Asn Gln Arg Met Lys Cys Lys Arg Trp Gln Lys Asn Gln Trp Leu

145 150 155 160

Lys Thr Ser Asn Gly Leu Ile Gln Lys Gly Ser Ala Pro Val Glu Tyr

165 170 175

Pro Ser Ile His Cys Ser Tyr Pro Gln Gly Tyr Leu Val Asn Ala Ser

180 185 190

Gly Ser Leu Ser Met Trp Gly Ser Gln Thr Trp Thr Asn Pro Thr Trp

195 200 205

Ser Ser Gln Thr Trp Thr Asn Pro Thr Trp Asn Asn Gln Thr Trp Thr

210 215 220

Asn Pro Thr Trp Ser Ser Gln Ala Trp Thr Ala Gln Ser Trp Asn Gly

225 230 235 240

Gln Pro Trp Asn Ala Ala Pro Leu His Asn Phe Gly Glu Asp Phe Leu

245 250 255

Gln Pro Tyr Val Gln Leu Gln Gln Asn Phe Ser Ala Ser Asp Leu Glu

260 265 270

Val Asn Leu Glu Ala Thr Arg Glu Ser His Ala His Phe Ser Thr Pro

275 280 285

Gln Ala Leu Glu Leu Phe Leu Asn Tyr Ser Val Thr Pro Pro Gly Glu

290 295 300

Ile

305

Claims (10)

1. A Rad51 inhibitor, wherein the Rad51 inhibitor includes Nanog protein.

2. The Rad51 inhibitor according to claim 1, wherein the amino acid sequence of the Nanog protein includes the sequence shown in SEQ ID No. 1.

3. The Rad51 inhibitor according to claim 1 or 2, wherein the Nanog protein inhibits Rad51 binding to ssDNA.

4. The Rad51 inhibitor according to any one of claims 1-3, wherein the Nanog protein inhibits Rad51 repair of double stranded DNA damage.

Use of Nanog protein for inhibiting Rad51 repair of double-stranded DNA damage for non-disease diagnostic purposes.

Use of Nanog protein in the preparation of Rad51 inhibitors.

Application of Nanog protein in preparing medicine for treating tumor;

preferably, the medicament comprises a medicament for radiotherapy and chemotherapy of a tumor.

8. A pharmaceutical composition comprising Nanog.

9. The pharmaceutical composition of claim 8, further comprising an adjuvant;

preferably, the adjuvant comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrating agent, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, a bacteriostatic agent or a buffer.

10. The pharmaceutical composition according to claim 8 or 9, wherein the dosage form of the pharmaceutical composition comprises any one of suspension, granules, capsules, powders, tablets, emulsions, solutions, drop pills or injections.

Images