Fox protein as RPA inhibitor in single-stranded DNA repair

Abstract

The present invention relates to a FOX protein as an RPA inhibitor in single-stranded DNA repair. The FOX protein regulates a single-stranded DNA repair pathway through interaction with replication protein A, thereby enabling new identification of an intracellular DNA damage repair mechanism. In addition, the present invention provides the possibility of artificially regulating DNA repair activity by using a specific sequence of a Helix3 region. Therefore, the present invention is expected to provide a novel molecular target for preventing and treating DNA damage-related diseases such as cancer.

Description

FOX protein as an RPA inhibitor in single-stranded DNA repair

[0001] The present invention relates to a FOX protein as an RPA inhibitor in single-stranded DNA repair.

[0002] The present application claims priority based on Korean Patent Applications No. 10-2024-0141299 and No. 10-2025-0149147, filed on October 16, 2024, and October 15, 2025, respectively, and all contents disclosed in the specifications and drawings of said applications are incorporated into the present application.

[0003] Forkhead box (FOX) proteins are a family of proteins containing a conserved DNA-binding motif consisting of approximately 80 to 100 amino acids. To date, more than 50 types of FOX proteins have been identified in humans and are classified into several subgroups based on structural similarities. These proteins are known to be involved in various physiological processes, such as embryonic development, cell differentiation, proliferation, apoptosis, and autophagy.

[0004] The forkhead motif is based on a helix-turn-helix structure consisting of three interconnected helices, with wing-shaped auxiliary structures on both sides. Due to these structural features, FOX proteins are also referred to as "winged helix proteins." While some FOX proteins have been reported to interact with DNA repair-related proteins and participate in the non-homologous end binding (NHEJ) pathway, the specific role of FOX proteins in the DNA repair process has not yet been fully elucidated.

[0005] DNA damage generally occurs as living cells are continuously exposed to endogenous or exogenous factors. Endogenous damage is induced by base hydrolysis, oxidation, and mismatch, while exogenous damage is induced by genotoxic agents such as ultraviolet radiation, radiation, and crosslinking agents. Such damage impairs genomic stability and, if not properly repaired, can lead to mutations, cell cycle arrest, or apoptosis.

[0006] Replication Protein A (RPA) is a major protein that binds to single-stranded DNA in eukaryotic cells and is a heterotrimeric complex composed of three subunits: RPA1, RPA2, and RPA3. RPA plays a pivotal role in DNA replication, recombination, and various repair pathways, stabilizing single-stranded DNA to prevent the formation of secondary structures and degradation. Furthermore, RPA performs an essential role in maintaining genomic stability by acting cooperatively with various repair enzymes during damage recognition, excision, and repair processes. Nevertheless, there are still few reports regarding the molecular mechanisms by which FOX proteins directly regulate DNA damage responses and repair pathways, as well as their pharmacological applications.

[0007] The object of the present invention is a FOX (Forkhead Box) protein-derived peptide characterized by inhibiting single-stranded DNA repair by inhibiting the binding of RPA (Replication Protein A) protein to single-stranded DNA,

[0008] The above FOX protein-derived peptide comprises a peptide represented by the following Formula I:

[0009] GWX3NSX6RHNLX 11 X 12 [Formula I]

[0010] In Formula I,

[0011] X3 is any one selected from the group consisting of Gln, Asn, Glu, His, Lys, and Arg;

[0012] X6 is any one selected from the group consisting of Ile, Leu, and Met;

[0013] X 11 is any one selected from the group consisting of Ser, Thr, Asn, Gly, Ala, and Tyr; and

[0014] X 12 is one selected from the group consisting of Leu, Ile, Val, Met, and Ala.

[0015] Another objective of the present invention is to provide a FOX protein-derived peptide and a gold nanoparticle-DNA aptamer complex formed by specifically binding the FOX protein-derived peptide and the gold nanoparticle-DNA aptamer complex.

[0016] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer, comprising as an active ingredient one or more selected from the group consisting of the FOX (Forkhead Box) protein-derived peptide or a gene encoding the same; and the conjugate.

[0017] Another objective of the present invention is to provide a kit for the prevention or treatment of cancer, comprising the above-mentioned pharmaceutical composition for the prevention or treatment of cancer and instructions.

[0018] Another objective of the present invention is to provide a pharmaceutical composition for enhancing the anticancer effect of an anticancer agent, comprising as an active ingredient one or more selected from the group consisting of the FOX (Forkhead Box) protein-derived peptide or a gene encoding the same; and the conjugate.

[0019]

[0020] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below.

[0021] The present invention relates to a FOX (Forkhead Box) protein-derived peptide characterized by inhibiting single-stranded DNA repair by inhibiting the binding of RPA (Replication Protein A) protein to single-stranded DNA, wherein

[0022] The above FOX protein-derived peptide comprises a peptide represented by the following Formula I:

[0023] GWX3NSX6RHNLX 11 X 12 [Formula I]

[0024] In Formula I,

[0025] X3 is any one selected from the group consisting of Gln, Asn, Glu, His, Lys, and Arg;

[0026] X6 is any one selected from the group consisting of Ile, Leu, and Met;

[0027] X 11 is any one selected from the group consisting of Ser, Thr, Asn, Gly, Ala, and Tyr; and

[0028] X 12 is one selected from the group consisting of Leu, Ile, Val, Met, and Ala.

[0029] In one embodiment of the present invention, X3 is Gln; X6 is Ile; and X 11 is Ser and; and X 12 It may be Leu, but is not limited to this.

[0030] In one embodiment of the present invention, the FOX protein-derived peptide may comprise one or more amino acid sequences selected from the group consisting of SEQ ID NO. 56 and SEQ ID NO. 57, but is not limited thereto.

[0031] In one embodiment of the present invention,

[0032] The above FOX protein-derived peptide may comprise, but is not limited to, an amino acid sequence commonly derived from the Helix3 region of any one or more proteins selected from the group consisting of the following:

[0033] FOXL2 (Forkhead box L2) protein, FOXC1 (Forkhead box C1) protein, FOXI1 (Forkhead box I1) protein, FOXK1 (Forkhead box K1) protein, FOXS1 (Forkhead box S1) protein, FOXF1 (Forkhead box F1) protein, FOXN1 (Forkhead box N1) protein, and FOXR2 (Forkhead box R2) protein.

[0034] In one embodiment of the present invention, the FOX protein-derived peptide may inhibit single-stranded DNA repair by being characterized by one or more selected from the group consisting of the following, but is not limited thereto:

[0035] Inhibiting the DNA binding activity of RPA1 (Replication Protein A1) and RPA3 (Replication Protein A3); and

[0036] Inhibits dimerization between RPA1 (Replication Protein A1) and RPA2 (Replication Protein A2).

[0037] The present invention provides a FOX protein-derived peptide and gold nanoparticle-DNA aptamer conjugate formed by specifically binding the FOX protein-derived peptide and the gold nanoparticle-DNA aptamer complex.

[0038] The present invention provides a pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient one or more selected from the group consisting of the FOX (Forkhead Box) protein-derived peptide or a gene encoding the same; and the conjugate.

[0039] In one embodiment of the present invention, the cancer may be one or more selected from the group consisting of lung cancer, colon cancer, prostate cancer, ovarian cancer, cholangiocarcinoma (CHOL), esophageal cancer (ESCA), cervical cancer, adrenal cancer, head and neck cancer, brain cancer, liver cancer, peritoneal cancer, skin cancer, melanoma of the skin or eye, rectal cancer, anal cancer, perianal cancer, small intestine cancer, endocrine gland cancer, parathyroid cancer, soft tissue sarcoma, urethral cancer, blood cancer, gastric cancer, pancreatic cancer, glioblastoma, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, and thyroid cancer, but is not limited thereto.

[0040] In one embodiment of the present invention,

[0041] The above pharmaceutical composition for cancer prevention or treatment may additionally include a chemo-anticancer agent, but is not limited thereto.

[0042] In one embodiment of the present invention,

[0043] The above chemotherapy agents may be one or more selected from the group consisting of paclitaxel, carboplatin, alimta, oxaliplatin, pemetrexed, cisplatin, gemcitabine, fluorouracil (5-FU), cyclophosphamide, vincristine, etoposide, and doxorubicin, but are not limited thereto.

[0044] In one embodiment of the present invention, the pharmaceutical composition for cancer prevention or treatment is,

[0045] The above-mentioned FOX protein-derived peptide or the gene encoding the same or the above-mentioned conjugate; and the above-mentioned chemical anticancer agent in the form of a mixture; or

[0046] The above-mentioned FOX protein-derived peptide or the gene encoding it or the above-mentioned conjugate; and the above-mentioned chemo-anticancer agent may each be formulated and administered simultaneously, separately, or sequentially, but are not limited thereto.

[0047] The present invention provides a kit for the prevention or treatment of cancer, comprising the above-mentioned pharmaceutical composition for the prevention or treatment of cancer and instructions.

[0048] The present invention provides a pharmaceutical composition for enhancing the anticancer effect of an anticancer agent, comprising as an active ingredient one or more selected from the group consisting of the FOX (Forkhead Box) protein-derived peptide or a gene encoding the same; and the conjugate.

[0049] In one embodiment of the present invention, the pharmaceutical composition for enhancing the anticancer effect of the anticancer agent may be administered simultaneously with, separately from, or sequentially with the anticancer agent, but is not limited thereto.

[0050]

[0051] In addition, the present invention provides a method for preventing or treating cancer, or a method for enhancing the anticancer effect of an anticancer agent, comprising the step of administering to an individual in need of the above a pharmaceutically effective amount a composition comprising: one or more selected from the group consisting of the above FOX (Forkhead Box) protein-derived peptide or a gene encoding the same; and the above conjugate; or the same as an active ingredient.

[0052] In addition, the present invention provides a composition comprising one or more selected from the group consisting of the FOX (Forkhead Box) protein-derived peptide or a gene encoding the same; and the conjugate thereof; or the same as an active ingredient, for use in preventing or treating cancer; or for use in enhancing the anticancer effect of an anticancer agent.

[0053] In addition, the present invention provides a use for preparing a preparation for cancer prevention or treatment, comprising one or more selected from the group consisting of the FOX (Forkhead Box) protein-derived peptide or a gene encoding the same; and the conjugate thereof; or a composition comprising the same as an active ingredient; or a use for preparing a preparation for enhancing the anticancer effect of an anticancer drug.

[0054] According to the FOX protein as an RPA inhibitor in single-stranded DNA repair, the intracellular DNA damage repair mechanism can be newly elucidated by the FOX protein regulating the single-stranded DNA repair pathway through interaction with replication protein A. Furthermore, it suggests the possibility of artificially regulating DNA repair activity using specific sequences in the Helix3 region. Therefore, the present invention is expected to provide a new molecular target for the prevention and treatment of DNA damage-related diseases such as cancer.

[0055] Figure 1a shows the intracellular interaction between FOX protein and RPA protein. After transforming 293T cells with a FLAG-tagged FOX protein expression plasmid, the intracellular interaction between FOX protein and RPA protein was confirmed through immunoprecipitation (IP) analysis using a FLAG antibody.

[0056] Figure 1b shows the in vitro interaction between FOXL2 and RPA proteins using recombinant proteins. To confirm the in vitro interaction between FOXL2 and RPA proteins, immunoprecipitation (IP) analysis was performed using RPA1 (a), RPA2 (b), RPA3 (c), and FOXL2 recombinant proteins.

[0057] Figure 1c shows the nuclear colocalization of FOX and RPA proteins. After transforming SW480 cells with a designated FLAG-tagged FOX protein expression plasmid, the intracellular and nuclear colocalization of FOX and RPA proteins were confirmed using a confocal microscope. DAPI was used for nucleus staining.

[0058] Figure 2a shows experimental results confirming that FOXL2 reduces the DNA binding levels of RPA1 and RPA3. The DNA binding activity of RPA proteins was measured through chromatin-rich protein fraction analysis in FOXL2 stable-expressing cells. H3 protein and β-Actin were used as indicator proteins for the chromatin fraction and soluble fraction, respectively.

[0059] Figure 2b shows the experimental results confirming that FOXL2 inhibits the binding of RPA to single-stranded DNA in vitro. The in vitro single-stranded DNA binding activity of RPA was confirmed by electrophoretic mobility change analysis (EMSA). 1.25 μM of Cy3-labeled single-stranded DNA was reacted with recombinant proteins of specified concentrations in EMSA buffer, and RPA was used at 25 nM, while FOXL2 was used at concentrations of +: 25 nM, ++: 50 nM, and +++: 75 nM, respectively.

[0060] Figure 2c shows the experimental results confirming that FOXL2 inhibits the formation of a heterotrimer between RPA1 and RPA2. The effect of FOXL2 on the formation of RPA heterotrimers was confirmed through in vitro immunoprecipitation (IP) analysis. Recombinant RPA protein (25 nM) was reacted with FOXL2 protein (0 nM, 25 nM, 50 nM, 75 nM) in NP-40 lysis buffer containing 1% BSA for 1 hour, then immunoprecipitated with an anti-RPA1 antibody and immunoblotted with a designated antibody.

[0061] Figures 3a through 3d show experimental results confirming that the FOX protein inhibits single-stranded DNA repair. To evaluate the effect of the FOX protein on single-stranded DNA repair, native BrdU staining was performed on cells with suppressed FOX gene expression (a) or cells with overexpression (b). Specifically, Figures 3a and 3b represent cells with suppressed FOX gene expression, while Figures 3c and 3d represent cells with overexpression. Cells were treated with 100 μM H₂O₂ for 1 hour, followed by a repair process for an additional 1 hour, and the relative number of BrdU focuses was quantified. A total of 120 cells were analyzed using ImageJ software across three independent experiments, and data were presented as mean ± standard error (SEM) (***p < 0.001).

[0062] Figure 3e shows the results of another experiment confirming that the FOX protein inhibits single-strand break (SSB) repair. To evaluate the effect of the FOX protein on single-strand DNA repair, an alkaline comet assay was performed on cells overexpressing the FOX protein. Cells were treated with 100 μM H₂O₂ for 1 hour, followed by a repair process for 1 hour. In the comet assay, tail moments were measured for a total of 120 cells, and data were presented as the mean ± standard error (SEM) of three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001).

[0063] Figures 3f to 3h show the experimental results confirming that FOX protein inhibits colony formation. To determine the effect of FOX protein expression on colony formation, colony formation analysis was performed using HCT116 or SW480 cells. Cells were cultured after being treated with 100 μM H₂O₂ for 1 hour followed by a 1-hour repair process, and comparisons were made between the treated and untreated groups. The quantified results are shown in Figure 3h, and data are expressed as the mean ± standard error (SEM) of three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001).

[0064] Figure 4a shows the amino acid sequence alignment results and structural description of the forkhead domain of the FOX protein. The forkhead box motifs were compared among all human FOX proteins. Structurally, the forkhead domain has a helix-turn-helix core structure consisting of three helices, with two wings positioned on each side. As a result of motif scanning of the forkhead box domain of the FOX protein, a conserved sequence (12AA-Helix3) consisting of 12 amino acids was identified.

[0065] Figures 4b and 4c show experimental results confirming that the Helix3 region of the FOX protein is the major binding site for the RPA protein. Wild-type (WT) and Helix3-deleted (ΔHelix3) mutants of FOXD3 (Figure 4b) and FOXF1 (Figure 4c) proteins were expressed in 293T cells, respectively, and the differences in interaction affinity with the RPA protein were analyzed.

[0066] Figure 4d shows the experimental results confirming the in vitro interaction between the RPA protein and the Helix3 peptide. Direct interactions with RPA1 (a), RPA2 (b), and RPA3 (c) were confirmed by performing in vitro immunoprecipitation using the His-tagged 12AA-Helix3 peptide and the recombinant protein. Immunoprecipitation was performed using IgG or a designated antibody.

[0067] Figure 4e shows the experimental results confirming the regulatory effect of Helix3 peptide on RPA trimer formation. The regulatory effect of Helix3 peptide on the formation of RPA heterotrimers was analyzed by in vitro immunoprecipitation. Recombinant RPA protein (25 nM) was reacted with FOXL2-derived Helix3 peptides (0 nM, 25 nM, 50 nM, 75 nM) in NP-40 lysis buffer containing 1% BSA for 1 hour, then immunoprecipitated with an anti-RPA1 antibody and immunoblotted with a designated antibody.

[0068] Figure 4f shows the results of another experiment confirming the regulatory effect of the Helix3 peptide on the single-stranded DNA binding activity of RPA. The effect of the Helix3 peptide on the single-stranded DNA binding activity of RPA was evaluated through electrophoretic mobility change analysis (EMSA). Cy3-labeled single-stranded DNA (1.25 μM) was reacted with recombinant proteins (RPA 25 nM, Helix3-His 25, 50, 75 nM; indicated as +, ++, and +++, respectively) in EMSA buffer.

[0069] Figure 5a shows the experimental results confirming the intracellular delivery effect of the 12AA-Helix3 peptide. Effective intracellular delivery of the 12AA-Helix3 peptide was confirmed by confocal microscopy following immunofluorescence staining with anti-His antibody. SW480 cells were cultured with the specified nanoparticle complex for 24 hours. Bright-field images were used to observe cell morphology and visualize gold nanoparticles (AuNP).

[0070] Figure 5b shows the results of the functional analysis experiment of the 12AA-Helix3 peptide. The effect of the 12AA-Helix3 peptide on single-stranded DNA was confirmed through native BrdU focus analysis. DAPI was used for nuclear labeling.

[0071] Figure 6a shows somatic and germline mutations within the forkhead domain. Mutation information for each residue is displayed along with the amino acid sequence alignment of the forkhead domain of the human FOX protein. Residues representing germline mutations are shown in red, residues representing somatic mutations in blue, and residues containing both germline and somatic mutations in green. Germline mutation information was obtained from the NCBI OMIM database (https: / / www.ncbi.nlm.nih.gov / omim), and cancer-related somatic mutation data was extracted from cBioPortal (https: / / www.cbioportal.org / ).

[0072] Figures 6b to 6d show the experimental results confirming the effect of somatic mutations within the Helix3 region of the FOX protein on DNA repair activity.

[0073] Figure 6b was derived by the following method: Somatic mutations within the Helix3 region of the FOX protein were collected from cBioPortal (https: / www.cbioportal.org / ), and the mutation frequency for each residue was calculated. Several FOX protein expression vectors containing mutations specified in the 12AA-Helix3 region were constructed.

[0074] Figure 6c was derived by the following method: native BrdU focus assays were performed on cells transfected with wild-type (WT) or mutant FOX protein under conditions with or without H₂O₂ (100 μM, 1 hour treatment followed by 1 hour repair).

[0075] Figure 6d was derived by the following method: The difference in RPA binding affinity between FOX-WT and FOX-variant was confirmed through immunoprecipitation (IP) analysis using anti-FLAG antibodies.

[0076] Figures 6e to 6h show experimental results confirming that the expression of DNA repair proteins is increased in cancer patients.

[0077] In Figures 6e and 6f, mRNA expression data for DNA repair proteins were obtained from the GENT2 portal (http: / gent2.appex.kr / gent2 / ) based on the U133Plus2 platform as gene expression comparison data between normal tissue and tumor tissue. The sample size for each tissue type is indicated on the right. The relative mRNA expression levels between cancer tissue and normal tissue were compared and displayed as a heatmap, where red shading indicates high expression and green shading indicates low expression.

[0078] In Figures 6g and 6h, RNA-seq data were obtained using the multi-gene analysis function provided by GEPIA2 (http: / gepia2.cancer-pku.cn / #index). Differences in RNA expression levels between cancer tissue and normal tissue were shown as heatmaps, and the size of each sample is indicated at the bottom.

[0079] Figure 7a shows the results of cell viability analysis of various 12AA-Helix3-derived peptides in SW480 cancer cells. Cell viability analysis was performed on SW480 cells after treatment with each peptide for 24 hours. Data are expressed as the mean ± standard error (SEM) of three independent experiments. Statistical significance is indicated by the letters a, b, and c (p < 0.05), and the amino acid sequences of each peptide are as follows: Peptide_1 (derived from FOXL2 / C1 / I1 / K1 / S1), Peptide_2 (derived from FOXF1 / N1), and Peptide_3 (derived from FOXR2).

[0080] Figures 7b to 7e show the sensitization effects of 12AA-Helix3-derived peptide monotherapy and chemotherapy on various cancer cells. Cell viability was normalized relative to the control group and expressed as the mean ± standard error (SEM) of three independent experiments. Different letters indicate significant differences between groups (p < 0.05).

[0081] Figure 7b shows the results of measuring the cell viability of A549 lung cancer cells treated with 12AA-Helix3-derived peptide and Paclitaxel (10 μM, A) or Carboplatin (200 μM, B).

[0082] Figure 7c shows the results of measuring the cell viability of SW480 colon cancer cells treated with 12AA-Helix3-derived peptide and Paclitaxel (40 μM, C) or Carboplatin (200 μM, D).

[0083] Figure 7d shows the results of measuring the cell viability of PC-3 prostate cancer cells treated with 12AA-Helix3-derived peptide and Paclitaxel (20 nM, E) or Carboplatin (200 μM, F).

[0084] Figure 7e shows the results of measuring the cell viability of OV90 ovarian cancer cells treated with 12AA-Helix3-derived peptide and Paclitaxel (1 μM, G) or Carboplatin (200 μM, H).

[0085] DNA damage commonly occurs in organisms and cells due to continuous stress from endogenous and exogenous factors. To maintain genomic stability, appropriate DNA repair pathways are activated depending on the type of damage to remove damaged DNA. Forkhead box (FOX) proteins are a family of proteins containing highly conserved DNA-binding motifs consisting of 80 to 100 amino acids, and they have been reported to be involved in various processes such as embryonic development, differentiation, proliferation, apoptosis, and autophagy. However, the effects of FOX proteins on regulating DNA repair pathways have not been clearly elucidated.

[0086] In the present invention, the Replication Protein A (RPA) heterotrimeric protein, a major single-stranded DNA binding protein in eukaryotic cells, was identified as a novel universal interacting protein with FOX protein. FOX protein reduced the dimerization of RPA1 and RPA2 subunits and inhibited RPA's binding to single-stranded DNA; consequently, the accumulation of single-stranded DNA (ssDNA), reduced repair of single-stranded break (SSB) DNA, and reduced colony formation were observed. These phenomena were measured by native BrdU staining, alkaline comet analysis, and colony formation analysis, respectively.

[0087] Analysis of the forkhead box domain motif revealed that a 12-amino acid sequence (12AA-helix3) within the helix 3 region is a key site mediating the binding of FOX protein and RPA. When the 12AA-helix3 peptide was delivered into the nucleus using a gold nanoparticle-based peptide delivery system, SSB repair activity was inhibited. Additionally, FOX proteins with somatic mutations within the helix 3 region found in cancer patients were shown to have weakened inhibitory activity against the SSB DNA repair pathway.

[0088] Additionally, in silico analysis of mRNA expression confirmed that both PARP1 and RPA are upregulated in tumor tissues. Taken together, these results demonstrate that the FOX protein plays a novel functional role in the SSB DNA repair pathway and suggest a new molecular mechanism by which the FOX protein acts by regulating RPA.

[0089] At this time, the present invention confirmed that residues of the Helix3 region within the forkhead domain in most FOX proteins are highly conserved, and that the Helix3 region is a key site for interaction with DNA repair proteins, and confirmed that mutants in which Helix3 is deleted in FOXD3 and FOXF1 proteins cannot bind to RPA proteins. Based on these experimental results, it was identified that the Helix3 region of FOX proteins is a key residue mediating binding with RPA proteins.

[0090] Subsequently, through precise analysis of the motif of the forkhead box domain, a conserved sequence consisting of 12 amino acids commonly present in FOX proteins was identified. It was confirmed that the 12AA-Helix3 peptide represented by this sequence inhibited ssDNA repair when administered alone or as a conjugate using a gold nanoparticle-DNA aptamer as a carrier, and excellent anticancer effects against various types of cancer were confirmed.

[0091] Accordingly, the present invention relates to a FOX (Forkhead Box) protein-derived peptide characterized by inhibiting single-stranded DNA repair by inhibiting the binding of RPA (Replication Protein A) protein to single-stranded DNA, wherein

[0092] The above FOX protein-derived peptide comprises a peptide represented by the following Formula I:

[0093] GWX3NSX6RHNLX 11 X 12 [Formula I]

[0094] In Formula I,

[0095] X3 is any one selected from the group consisting of Gln, Asn, Glu, His, Lys, and Arg;

[0096] X6 is any one selected from the group consisting of Ile, Leu, and Met;

[0097] X 11 is any one selected from the group consisting of Ser, Thr, Asn, Gly, Ala, and Tyr; and

[0098] X 12 is one selected from the group consisting of Leu, Ile, Val, Met, and Ala.

[0099] In the present invention, “FOX protein-derived peptide” may refer to a peptide containing an amino acid sequence derived from the helix 3 region of the FOX protein. Accordingly, “FOX protein-derived peptide” can be used interchangeably with “peptide derived from the helix 3 region of the FOX protein.” “Peptide derived from the helix 3 region of the FOX protein” may refer to a peptide derived from an amino acid sequence region that binds to the RPA protein among the structurally corresponding sites of the helix 3 of the FOX protein, and which exhibits inhibitory activity on the binding of RPA to DNA. In one embodiment of the present invention, 12AA-Helix3 peptide 1 (or FH-helix peptide 1), which consists of 12 conserved amino acids in the helix 3 region of the FOX protein, was derived, and variants 12AA-Helix peptide 2 and 12AA-Helix3 peptide 3 were derived therefrom. Among these, 12AA-Helix peptide 1 was identified as exhibiting ssDNA repair pathway inhibitory activity and the resulting anticancer effect. Therefore, “FOX protein-derived peptide” can be understood interchangeably with “peptide derived from the helix 3 region of FOX protein,” “12AA-Helix3 peptide 1,” “peptide 1,” or “12AA-Helix3 peptide.”

[0100] In the present invention, it was confirmed that the FOX protein, the helix 3 region of the FOX protein, or a peptide conservedly derived from the helix 3 region of the FOX protein regulates the ssDNA repair pathway by inhibiting the binding of RPA to DNA. Therefore, in the present invention, the FOX protein, the helix 3 region of the FOX protein, or a peptide derived from the helix 3 region of the FOX protein can function as an inhibitor of RPA, but is not limited thereto.

[0101] Meanwhile, in Formula I of the present invention, according to one embodiment of the present invention, when a 7th arginine (R) mutation, a 5th serine (S>A) mutation, and a 10th leucine (L>I) mutation are applied based on the N-terminus of Sequence No. 56, which consists of 12 amino acids, the ssDNA repair activity is absent or significantly reduced, so the sequence having the sequence homology may be characterized in that no mutation occurs at the corresponding position.

[0102] In addition, in Formula I of the present invention,

[0103] X3 can be any one selected from the group consisting of Gln, Asn, Glu, and His. Since Gln, Asn, Glu, and His all possess polar side chains and contribute to hydrogen bonding networks and solubility regulation at protein surfaces or binding sites, they can maintain the same function even when mutually substituted at sites where charge specificity is not critical. In particular, Gln and Asn are amide derivatives that are charge-free and capable of both donating and accepting hydrogen bonds, making them the most conservative substitution group. Additionally, Gln-Glu is a substitution that changes only the charge to negative while preserving binding geometry, as their chain lengths and volumes are similar, thus having a high probability of maintaining function. Since His has a pKa near physiological pH, it allows for reversible switching between positive and neutral states, which means function can also be preserved through substitution between Gln / Asn / His.

[0104] Alternatively, X3 may be any one selected from the group consisting of Gln, Asn, Glu, His, Lys, and Arg. Lys and Arg possess a positively charged functional group at the end of a long hydrophobic alkyl chain that is basic and positively charged, and perform a common role of providing electrostatic bonding, salt bridge formation, and hydrogen bonding with a negatively charged surface at physiological pH; therefore, most of the same function can be preserved through K↔R complement substitution at positions where charge retention is critical.

[0105] X6 may be any one selected from the group consisting of Ile, Leu, and Met. Ile, Leu, and Met are all alkyl / thioether amino acids with large hydrophobic side chains that are involved in protein core packing, membrane-transmembrane helix stabilization, and hydrophobic interactions; therefore, their structure and stability are easily maintained even when they are mutually substituted. Ile and Leu are virtually isomers with nearly identical volume and hydrophobicity, making them the top priority substitution group. Ile / Leu-Met is highly suitable for substitution as it has similar volume and hydrophobicity, and the -S- group of Met is a flexible straight chain that does not significantly disrupt packing. Met may be preferred when weak sulfur interactions and flexibility are required, and for this hydrophobic role, the three amino acids can be substituted to perform the same functionality.

[0106] X 11 ... may be any one selected from the group consisting of Ser, Thr, Asn, Gly, Ala, and Tyr. Ser, Thr, Asn, Gly, Ala, and Tyr are residues used for fine-tuning of structure and function, such as controlling surface polarity, hydrogen bonding, tuning size and flexibility, and, in some cases, accommodating modifications like phosphorylation; they can be mutually substituted while maintaining the same function. Ser-Thr is the most conservative polar / uncharged pair possessing a hydroxyl group and can be mutually substituted at phosphorylation sites. Ser / Thr-Asn shares a hydrogen bonding pattern, making it easy to maintain bonding surface polarity, and can even achieve strength-tuning effects due to differences in functional groups (-OH vs. amide). Gly-Ala is a compactness and structural flexibility tuning pair that acts as an amino acid frequently substituted for controlling loop, turn, and helix stabilization. Ser / Thr-Tyr has functional equivalence due to the retention of -OH groups, and since the aromatic ring of Tyr increases volume and reduces water retention, substitutions based on identical functionality can be established on surfaces and in wide pockets.

[0107] X 12is any one selected from the group consisting of Leu, Ile, Val, Met, and Ala. Since Leu, Ile, Val, Met, and Ala are representative hydrophobic residues involved in protein core formation, hydrophobic interactions, and the stabilization of membrane protein helices, they can perform the same structural functions even when mutually substituted. Ile-Leu-Val is the most conservative due to its similar hydrophobic and packing properties as a β-branched alkyl group, and among them, Ile-Leu may be the highest equivalence. Ile / Leu-Met has similar volume and hydrophobicity, and the flexible straight chain of Met does not significantly impair packing, making it a high potential for substitution. Ala is hydrophobic and very small, so it can be equivalently substituted at sites where volume reduction is permitted.

[0108] In one embodiment of the present invention, X3 is Gln; X6 is Ile; and X 11 is Ser and; and X 12 It may be Leu, but is not limited to this.

[0109] In one embodiment of the present invention, the FOX protein-derived peptide may comprise one or more amino acid sequences selected from the group consisting of SEQ ID NO. 56 and SEQ ID NO. 57, but is not limited thereto.

[0110] Meanwhile, in one embodiment of the present invention, 12AA-Helix3 peptides 2 and 3 each have Lys located at the X3 position; Val or Thr located at the X6 position; and X 11 Cys is located at the position, and X 12 Phe is located at the position. Given that each peptide did not exhibit significant inhibition of the ssDNA repair pathway or anticancer activity, X3 may not contain Lys; X6 may not contain Val or Thr, and X 11 may not include Cys, and X 12It may not include Phe, but is not limited to this.

[0111] In one embodiment of the present invention, the FOX protein-derived peptide may bind to a his tag, but is not limited thereto. In this case, "His tag" refers to a polyhistidine sequence added to the N-terminus or C-terminus of a protein to facilitate the expression, purification, and detection of the protein. The his tag generally consists of six histidine residues and nickel (Ni²⁺) + ) or cobalt (Co² + It can bind to a metal-affinity resin through selective binding with ions. By utilizing these binding characteristics, proteins to which His tags are attached can be selectively separated or detected. In the present invention, the His tag is not necessarily limited to six histidine residues, and the number and arrangement of histidine residues can be adjusted according to the protein expression efficiency, solubility, or purification efficiency.

[0112] In the present invention, the his tag may include 4 to 10 histidine residues. For example, it may be 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 4, 5, 6, 7, 8, 9, or 10.

[0113] In the present invention, the FOX protein-derived peptide may include a sequence having sequence homology with one or more amino acid sequences selected from the group consisting of SEQ ID NO. 56 and SEQ ID NO. 57 at a level capable of performing the same function.

[0114] The concept of a nucleic acid molecule of the polynucleotide sequence of the present invention (used interchangeably with the base sequence) includes functional equivalents of the nucleic acid molecule constituting it, for example, variants in which some base sequences of the nucleic acid molecule have been modified by deletion, substitution, or insertion, but which can perform the same function as the nucleic acid molecule. That is, it may include base sequences having sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% with the base sequence of the present invention. For example, it includes polynucleotides having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology.

[0115] The “% of sequence homology” for a polynucleotide is determined by comparing two optimally arranged sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for the optimal arrangement of the two sequences.

[0116] In addition, the same concept as the polynucleotide sequence can be applied to the amino acid sequence of the present invention. That is, the amino acid sequence is a concept that includes variants capable of performing the same functional action as the one described above, and that is, it may include nucleotide sequences having sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% with respect to the amino acid sequence indicated by the sequence number described above in the present invention. For example, it includes an amino acid sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology.

[0117] In one embodiment of the present invention,

[0118] The above FOX protein-derived peptide may comprise, but is not limited to, an amino acid sequence commonly derived from the Helix3 region of any one or more proteins selected from the group consisting of the following:

[0119] FOXL2 (Forkhead box L2) protein, FOXC1 (Forkhead box C1) protein, FOXI1 (Forkhead box I1) protein, FOXK1 (Forkhead box K1) protein, FOXS1 (Forkhead box S1) protein, FOXF1 (Forkhead box F1) protein, FOXN1 (Forkhead box N1) protein, and FOXR2 (Forkhead box R2) protein.

[0120] In one embodiment of the present invention, the FOX protein-derived peptide may include amino acids commonly derived from one or more helix 3 regions selected from the group consisting of FOXL2 protein, FOXC1 protein, FOXI1 protein, FOXK1 protein, and FOXS1 protein, but is not limited thereto.

[0121] In an embodiment of the present invention, amino acid sequences commonly derived from the helix 3 regions of FOXL2 protein, FOXC1 protein, FOXI1 protein, FOXK1 protein, and FOXS1 protein among the FOX protein family were derived, and it was confirmed that the peptide represented by the said amino acid sequence is a region that interacts with RPA protein. In addition, it was confirmed that when treated with this, ssDNA repair inhibitory activity is exhibited and therapeutic effects are shown against various types of cancer. Accordingly, in one embodiment of the present invention, the FOX protein-derived peptide may include amino acids commonly derived from the helix 3 regions of FOXL2 protein, FOXC1 protein, FOXI1 protein, FOXK1 protein, and FOXS1 protein, but is not limited thereto.

[0122] Meanwhile, in the present invention, when the FOX protein-derived peptide has an amino acid sequence commonly derived from the helix 3 region of the FOXF1 (Forkhead box F1) protein and the FOXN1 (Forkhead box N1) protein; and when the amino acid sequence is derived from the helix 3 region of the FOXR2 (Forkhead box R2) protein, it was found that the cancer cell death effect is poor, and the ssDNA repair inhibitory activity is also found to be poor. Therefore, the FOX protein-derived peptide can be characterized by being derived from the helix 3 region of a specific family protein of the FOX protein.

[0123] In one embodiment of the present invention, the FOX protein-derived peptide may inhibit single-stranded DNA repair by being characterized by one or more selected from the group consisting of the following, but is not limited thereto:

[0124] Inhibiting the DNA binding activity of RPA1 (Replication Protein A1) and RPA3 (Replication Protein A3); and

[0125] Inhibits dimerization between RPA1 (Replication Protein A1) and RPA2 (Replication Protein A2).

[0126] In the present invention, the FOX protein-derived peptide can inhibit single-stranded DNA repair by inhibiting the binding of RPA protein to single-stranded DNA as described above, but is not limited thereto.

[0127] The present invention provides a FOX protein-derived peptide and gold nanoparticle-DNA aptamer conjugate formed by specifically binding the FOX protein-derived peptide and the gold nanoparticle-DNA aptamer complex.

[0128] At this time, in the present invention, the anti-DNA aptamer of the DNA aptamer may include the base sequence of SEQ ID NO. 62, but is not limited thereto.

[0129] In the present invention, “gold nanoparticle-DNA aptamer” is a nanoparticle-based carrier used to efficiently deliver peptide or nucleic acid materials into cells. The carrier ensures stability by covalently bonding a DNA aptamer to the surface of gold nanoparticles using thiol groups (-SH), and improves cell membrane recognition and binding efficiency. In addition, aptamer-bound gold nanoparticles enable selective delivery to target cells while minimizing cytotoxicity, thereby increasing the intracellular delivery efficiency of therapeutic peptides.

[0130] In the present invention, it was confirmed that the inhibitory activity of the ssDNA repair pathway is excellent when the 12AA-Helix3 peptide is delivered in combination with a gold nanoparticle-DNA aptamer. However, the delivery of the 12AA-Helix3 peptide alone may be limited to delivery via AuNPs. Therefore, the 12AA-Helix3 peptide of the present invention may be in a form combined with a histag, and the histag may be, for example, 6XHis, but is not limited thereto.

[0131] Meanwhile, the method of binding the gold nanoparticle-DNA aptamer and the FOX protein-derived peptide of the present invention is not limited. For example, they may be bound through a linker or directly bound, and in this case, binding refers to all types of binding, such as covalent bonds and non-covalent bonds, and may be directly connected by a covalent bond due to known gene recombination technology, etc., and may be bound through any method generally performed in the art.

[0132] In the present invention, “protein,” “polypeptide,” or “peptide” are used interchangeably and refer to a polymer of amino acid residues, for example, as generally found in proteins in their natural state.

[0133] The present invention provides a pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient one or more selected from the group consisting of the FOX (Forkhead Box) protein-derived peptide or a gene encoding the same; and the conjugate.

[0134] In one embodiment of the present invention, the cancer may be one or more selected from the group consisting of lung cancer, colon cancer, prostate cancer, ovarian cancer, cholangiocarcinoma (CHOL), esophageal cancer (ESCA), cervical cancer, adrenal cancer, head and neck cancer, brain cancer, liver cancer, peritoneal cancer, skin cancer, melanoma of the skin or eye, rectal cancer, anal cancer, perianal cancer, small intestine cancer, endocrine gland cancer, parathyroid cancer, soft tissue sarcoma, urethral cancer, blood cancer, gastric cancer, pancreatic cancer, glioblastoma, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, and thyroid cancer, but is not limited thereto.

[0135] In the present invention, colon cancer may include colon adenocarcinoma (COAD), cervical cancer may include cervical squamous cell carcinoma (CESC), adrenal cancer may include pheochromocytoma and paraganglioma (PCPG), head and neck cancer may include head and neck squamous cell carcinoma (HNSC), and liver cancer may include hepatocellular carcinoma (LIHC).

[0136] In one embodiment of the present invention, it was confirmed that RPA mRNA is significantly overexpressed in brain cancer, cervical cancer, liver cancer, cervical squamous cell carcinoma (CESC), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), esophageal cancer (ESCA), head and neck squamous cell carcinoma (HNSC), hepatocellular carcinoma (LIHC), and pheochromocytoma and paraganglioma (PCPG). The FOX protein-derived peptide of the present invention inhibits single-stranded DNA damage by inhibiting RRA protein activity, and thus can exhibit a therapeutic effect against the above cancers. For example, after confirming that RPA mRNA is overexpressed in colon adenocarcinoma, which is a type of colon cancer, it was confirmed that the FOX protein-derived peptide of the present invention exhibits an excellent anticancer effect when colon cancer is treated. Therefore, it can be understood that therapeutic activity will also be exhibited against colon adenocarcinoma.

[0137] In the present invention, the composition may be administered in combination with a chemotherapy agent, as it exhibited a synergistic effect against various types of cancer when treated together with a chemotherapy agent. Accordingly, in one embodiment of the present invention, the pharmaceutical composition for cancer prevention or treatment may additionally include a chemotherapy agent, but is not limited thereto.

[0138] In one embodiment of the present invention,

[0139] The above chemotherapy agents may be one or more selected from the group consisting of paclitaxel, carboplatin, alimta, oxaliplatin, pemetrexed, cisplatin, gemcitabine, fluorouracil (5-FU), cyclophosphamide, vincristine, etoposide, and doxorubicin, but are not limited thereto.

[0140] In the present invention, “combined administration” may be achieved by administering individual components of a therapeutic regimen simultaneously, sequentially, or individually. A combined therapeutic effect is obtained by administering two or more drugs simultaneously or sequentially, or by administering them alternately at regular or indeterminate intervals. The combined therapeutic regimen is not limited thereto, but may be defined as one that provides a synergistic effect while being therapeutically superior to the efficacy obtained by administering one or the rest of the components of the combined therapeutic regimen at a normal dose, provided, for example, through the degree of response, response rate, time to disease progression, or survival period.

[0141] In the present invention, “chemical anticancer agent” may refer to a first-generation anticancer agent also referred to as a “cytotoxic anticancer agent” or “chemical drug anticancer agent.”

[0142] In one embodiment of the present invention, the pharmaceutical composition for cancer prevention or treatment is,

[0143] The above-mentioned FOX protein-derived peptide or the gene encoding the same or the above-mentioned conjugate; and the above-mentioned chemical anticancer agent in the form of a mixture; or

[0144] The above-mentioned FOX protein-derived peptide or the gene encoding it or the above-mentioned conjugate; and the above-mentioned chemo-anticancer agent may each be formulated and administered simultaneously, separately, or sequentially, but are not limited thereto.

[0145] In the case where the composition of the present invention is in the form of a mixed agent, it may be a form for simultaneous administration of the FOX protein-derived peptide or the gene encoding therein or the conjugate thereof; and the anticancer agent.

[0146] In the case where the composition of the present invention is in a form in which the above substances are each formulated and administered simultaneously, separately, or sequentially, the composition may be a pharmaceutical composition for concomitant administration for simultaneous or sequential administration, comprising: a first pharmaceutical composition comprising a pharmaceutically effective amount of the FOX protein-derived peptide, a gene encoding the same, or the conjugate thereof as an active ingredient; and a second pharmaceutical composition comprising a pharmaceutically effective amount of the chemo-anticancer agent as an active ingredient. In this case, in the case of sequential administration, the order of administration is not limited, and the administration regimen may be appropriately adjusted according to the patient's condition, etc.

[0147] That is, if the above pharmaceutical composition is a pharmaceutical composition for concomitant administration for sequential administration, the composition may be such that the FOX protein-derived peptide or the gene encoding it, or the conjugate (“first component”) is administered first, followed by the administration of the chemotherapy agent (“second component”), and the reverse order is also possible.

[0148] The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric granules, liquids, eye drops, oxylic agents, emulsions, suspensions, ethanol tablets, troches, fragrances, limonene adzes, tablets, sustained-release tablets, enteric tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, warning agents, lotions, pastes, sprays, inhalants, patches, sterile injectable solutions, or aerosols, according to conventional methods, and the external preparation may have a formulation such as a cream, gel, patch, spray, ointment, warning agent, lotion, liniment, paste, or cataplasm.

[0149] Carriers, excipients, and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0150] When formulating, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0151] Excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium monohydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, refined lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethylcellulose (HPMC), HPMC 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, primogel, etc., as additives to tablets, powders, granules, capsules, pills, and lozenges according to the present invention; Gelatin, gum arabic, ethanol, agar powder, cellulose phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium casein, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch paste, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc. may be used as binders, and hydroxypropylmethylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate, anhydrous silica, 1-hydroxypropylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, D-sorbitol solution, hard anhydrous silica, etc. disintegrants;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium pods, kaolin, petroleum jelly, sodium stearate, cocoa paste, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silica, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and hard anhydrous silica may be used.

[0152] As additives to the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearic acid sucroses, polyoxyethylene sorbitol fatty acid esters (tween esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, water ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. may be used.

[0153] In the syrup preparation according to the present invention, a solution of white sugar, other sugars or sweeteners, etc. may be used, and if necessary, flavorings, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers, viscosity enhancers, etc. may be used.

[0154] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0155] In the suspension agent according to the present invention, suspending agents such as acacia, tragacanthus, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, and HPMC 2910 may be used, and surfactants, preservatives, stabilizers, coloring agents, and fragrances may be used as needed.

[0156] The injectable preparation according to the present invention comprises solvents such as distilled water for injection, 0.9% sodium chloride injection solution, Ringer's injection solution, dextrose injection solution, dextrose + sodium chloride injection solution, PEG, lactated Ringer's injection solution, ethanol, propylene glycol, non-volatile oils—sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; solubilizing agents such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nijungtinamide, hexamine, and dimethylacetamide; and buffers such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums. It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; sulfating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and sodium bisulfite acetone; non-inflammatory agents such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium CMC, sodium alginate, Tween 80, and aluminum monostearate.

[0157] The suppository according to the present invention contains cocoa dough, lanolin, Witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, lanette wax, glycerol monostearate, Tween or Spandex, Imhausen, monollene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydrocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydrocote Bases such as (Hydrokote) 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Masa-MF, Masupol, Masupol-15, Neosupostal-N, Paramount-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), suppository base type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wekovi (W, R, S, M, Fs), and Tegestor triglyceride base (TG-95, MA, 57) may be used.

[0158] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the extract. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used.

[0159] Liquid preparations for oral administration include suspensions, oral liquids, emulsions, and syrups; in addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0160] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, a “pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient’s disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0161] The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by considering all the above-mentioned factors, and this can be easily determined by a person skilled in the art to which the present invention belongs.

[0162] The pharmaceutical composition of the present invention may be administered to an individual by various routes. All modes of administration are expected, for example, oral administration, subcutaneous injection, intraperitoneal administration, intramuscular injection, intrathecal (intradural) injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, ear administration, nasal administration, inhalation, spray through the mouth or nose, skin administration, transdermal administration, etc.

[0163] The pharmaceutical composition of the present invention is determined by the type of active ingredient drug, along with various relevant factors such as the disease to be treated, the route of administration, the patient's age, gender, weight, and the severity of the disease.

[0164] In the present invention, the term “individual” refers to a subject requiring disease risk prediction, diagnosis, prognosis prediction, or treatment, and more specifically, may refer to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cattle, but is not limited thereto.

[0165] In the present invention, “administration” means providing a predetermined composition of the present invention to an individual by any appropriate method.

[0166] The present invention provides a kit for the prevention or treatment of cancer, comprising the above-mentioned pharmaceutical composition for the prevention or treatment of cancer and instructions.

[0167] In the present invention, “kit” refers to a tool that additionally includes a formulation or a substance for the function, storage, etc., of the kit to enable the use of the kit claimed in the present invention. In addition to the above substances, the kit of the present invention may include other components, compositions, solutions, devices, etc., that are typically required for the storage and processing methods thereof. At this time, each component may be applied one or more times without limitation on the number of times, there is no restriction on the order in which each substance is applied, and the application of each substance may proceed simultaneously or sequentially.

[0168] In the present invention, the kit may include a container; instructions; etc. The container may serve to package the material and may also serve to store and secure it. The material of the container may take the form, for example, a bottle, a tub, a sachet, an envelope, a tube, an ampoule, etc., and may be formed partially or wholly from plastic, glass, paper, foil, wax, etc. The container may be equipped with a cap that is initially part of the container or can be attached to the container by mechanical, adhesive, or other means and is fully or partially detachable, and may also be equipped with a stopper that allows access to the contents by a syringe needle. The kit may include an outer package, and the outer package may include instructions regarding the use of the components.

[0169] The present invention provides a pharmaceutical composition for enhancing the anticancer effect of an anticancer agent, comprising as an active ingredient one or more selected from the group consisting of the FOX (Forkhead Box) protein-derived peptide or a gene encoding the same; and the conjugate.

[0170] Meanwhile, the composition (or active ingredient thereof) according to the present invention can enhance the anticancer effect of an anticancer drug and reduce side effects, and the dosage of an anticancer drug with side effects can be minimized through appropriate combination therapy. In the present invention, "enhancing the anticancer effect" may include all effects that can consequently strengthen the function of the anticancer drug. For example, this concept encompasses not only enhancing the anticancer effect of the anticancer drug, such as inhibiting tumor growth, inhibiting tumor metastasis, and inhibiting tumor recurrence, but also enhancing the anticancer effect by inhibiting the formation of resistance or tolerance in cancer cells to the anticancer drug. That is, the active ingredient of the present invention can be used as a compound for combination administration with known anticancer drugs for the purpose of enhancing the anticancer effect. In other words, the active ingredient of the present invention can be used for combination administration with an anticancer drug to enhance the anticancer effect of said anticancer drug.

[0171] In one embodiment of the present invention, the pharmaceutical composition for enhancing the anticancer effect of the anticancer agent may be administered simultaneously with, separately from, or sequentially with the anticancer agent, but is not limited thereto.

[0172] In the present invention, the pharmaceutical composition for enhancing the anticancer effect of the anticancer agent may be in the form of a mixture comprising the FOX protein-derived peptide or the gene encoding the same, or the conjugate thereof; and the chemical anticancer agent, but is not limited thereto.

[0173] The content of the active ingredient or anticancer agent of the present invention can be appropriately adjusted according to the symptoms of the disease, the degree of progression of symptoms, the condition of the patient, etc. For example, it may be 0.0001 to 99.9% by weight or 0.001 to 50% by weight based on the total weight of the composition, but is not limited thereto. The above content ratio is a value based on the dry weight after removing the solvent.

[0174] In the present invention, when the term “comprising” is used, it means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0175] Throughout the specification of this invention, terms such as “approximately,” “substantially,” etc., are used to mean at or near the stated value when inherent manufacturing and material tolerances are presented in the said sense, and are used to prevent unscrupulous infringers from unfairly exploiting the disclosed content in which precise or absolute values ​​are mentioned to aid in understanding the invention.

[0176] Throughout the specification of the present invention, the term “combination thereof” included in a Markush-style expression means one or more mixtures or combinations selected from a group consisting of components described in the Markush-style expression, and means including one or more selected from said group consisting of said components.

[0177] The terms used in this invention have been selected based on currently widely used general terms, taking into account their functions within the invention; however, these terms may vary depending on the intent of those skilled in the art, case law, the emergence of new technologies, etc. Additionally, in specific cases, terms have been arbitrarily selected by the applicant, and in such cases, their meanings will be described in detail in the relevant description of the invention. Therefore, the terms used in this invention must be defined not merely by their names, but based on their meanings and the overall content of the invention.

[0178] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention, and the scope of the invention is not limited by the following embodiments.

[0179]

[0180] [Example]

[0181]

[0182] Plasmid

[0183] pcMV-Myc-FOXL2 wild-type and ΔAla, ΔFH, and FH mutant expression vectors were constructed according to known methods (Jin et al., 2020, Foxl2 directs DNA double-strand break repair pathways by differentially interacting with ku. Nature Communications. 11 (1):2010.). Plasmids for FOX protein expression were generated by PCR using specified templates and primers. The PCR products were cleaved with specified restriction enzymes (Takara Bio, Shiga, Japan) and ligated to p3XFLAG-CMV-10 (Sigma-Aldrich, St. Louis, MO, USA). The restriction enzymes, primers, and templates used are shown in Table 1.

[0184]

[0185]

[0186]

[0187]

[0188] pCMV-Myc-FOXL2 ΔFH1, ΔFH2, ΔFH3, ΔHelix3, p3XFLAG-CMV10-FOXD3-ΔHelix3, and p3XFLAG-CMV10-FOXF1-ΔHelix3 mutants were constructed by recombinant PCR using the following primer pairs: FOXL2-ΔFH1-F / FOXL2-ΔFH1-R, FOXL2-ΔFH2-F / FOXL2-ΔFH2-R, FOXL2-ΔFH3-F / FOXL2-ΔFH3-R, FOXL2-ΔHelix3-F / FOXL2-ΔHelix3-R, FOXD3-ΔHelix3-F / FOXD3-ΔHelix3-R, and FOXF1-ΔHelix3-F / FOXF1-ΔHelix3-R. The oligonucleotide sequences of the recombinant PCR primers are shown in Table 2.

[0189]

[0190]

[0191]

[0192] The pcDNA3-HA-RPA1 construct was amplified by PCR using RPA1-F (5'-CTC-GGA-TCC-ATG-TAC-CCA-TAC-GAT-GTT-CCA-GAT-TAC-GCT-ATG-GTC-GGC-CAA-CTG-AGC-GA-3') and RPA1-R (5'-CGC-GGT-ACC-TCA-CAT-CAA-TGC-ACT-TCT-CC-3') primers and then ligated to pcDNA3 using BamHI and KpnI restriction enzyme cleavage sites. Myc-RPA2 was amplified using RPA2-F (5'-CCC-AAG-CTT-ATG-GCA-TCA-ATG-CAG-AAG-CTG-ATC-TCA-GAG-GAG-GAC-CTG-ATG-TGG-AAC-AGT-GGA-TTC-GA-3') and RPA2-R (5'-CGC-GGT-ACC-TTA-TTC-TGC-ATC-TGT-GGA-TT-3') primers and cloned into the pcDNA3 vector using HindIII and KpnI restriction enzyme cleavage sites. The primers used for mutagenesis of pcDNA3-HA-RPA1 and pcDNA3-Myc-RPA2 are presented in Table 3.

[0193]

[0194]

[0195]

[0196] Human embryonic kidney cell line 293T (ATCC, Manassas, VA, USA) and human colon cancer cell lines SW480 and HCT116 were cultured in Dulbecco modified Eagle medium (DMEM; Caisson, North Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Caisson) and 1% penicillin-streptomycin (P / S; Caisson) at 37°C and 5% CO₂. Human adult germ cell (GCT)-derived KGN cells (Riken, Tsukuba, Japan) were cultured in DMEM / F12 medium containing 10% FBS and 1% P / S. FOXL2-deficient (KO) cells (Jin et al. 2020) and FOXL2-stable expressing cells (Choi et al. 2022, Foxl2 and foxa1 cooperatively assemble on the tp53 promoter in alternative dimer configurations. Nucleic acids research. 50 (15):8929-8946.) were prepared according to known methods and cultured under the same conditions as KGN cells. Transformation of 293T cells was performed using polyethyleneimide (PEI; Polysciences Inc., Warrington, PA, USA), and transformation of SW480, HCT116, and KGN cells was performed using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

[0197]

[0198] Reagents and Antibodies

[0199] H₂O₂ (216763) was purchased from Sigma-Aldrich. Methylmethanesulfonic acid (MMS; M320170) was purchased from Toronto Research Chemicals (Toronto, ON, Canada). Anti-β-actin (sc-47778), anti-RPA1 (sc-48425), and anti-Tubulin (SC-9104) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-H3 (05-928) was purchased from Upstate. Anti-FLAG (#14793), anti-Myc (#2276S), and anti-RPA2 (#52448S) were purchased from Cell Signaling Technology (Danvers, MA, USA). Alexa Fluor 546 stained goat anti-mouse IgG and Alexa Fluor 488 stained goat anti-rabbit IgG were purchased from Invitrogen (Waltham, Massachusetts, USA).

[0200]

[0201] Immunoprecipitation and immunoblot analysis

[0202] Cells were transformed with a specified plasmid or treated with chemical reagents for a specified time. Immunoprecipitation and immunoblot analysis were performed according to known methods (Jin et al. 2020). Specifically, after treatment, cell lysates were incubated overnight at 4°C with a specified antibody using Dynabeads Protein G (Thermo Scientific, Rockford, IL). Subsequently, the immunoprecipitated proteins were eluted, heat-denatured at 100°C, separated by SDS-PAGE, and transferred to a PVDF membrane (Millipore, Bedford, MA, USA). After antibody reaction, protein bands were visualized using an Amersham Imager 600 (GE Healthcare Life Sciences, Amersham, Buckinghamshire, UK), and band intensity was quantified using OptiQuant software (Bio-Rad Laboratories, Hercules, CA, USA).

[0203]

[0204] Confocal microscopy and immunofluorescence analysis

[0205] Cells (4 × 10^4) were seeded onto circular coverslips in 24-well plates. After overnight incubation, the cells were transformed with the specified plasmid and fixed with 4% paraformaldehyde for 15 minutes. Subsequently, they were permeated with 0.2% Triton X-100 for 15 minutes and blocked for 1 hour in a blocking buffer consisting of PBS containing 1% BSA at room temperature. The cells were incubated with the specified antibodies in a 1% BSA solution at 4°C overnight and washed three times with PBS-T (0.1% Tween-20 in PBS). They were then stained with Alexa Fluor 546 goat anti-mouse IgG (1:1000; Invitrogen) and Alexa Fluor 488 goat anti-rabbit IgG (1:1000; Invitrogen) at room temperature for 1 hour. After washing three times with PBS-T, the slides were mounted using Fluoroshield™ with DAPI mounting solution (ImmunoBioScience Crop, Mukilteo, WA, USA). Fluorescence signals were observed using a Zeiss LSM 800 confocal laser scanning microscope (Carl Zeiss, Gottingen, Germany).

[0206]

[0207] Chromatin-rich protein fraction

[0208] Chromatin-enriched protein fractionation was performed as follows. Specifically, cells were cultured overnight in 60 mm culture dishes followed by plasmid transformation or reagent treatment. Subsequently, soluble proteins and chromatin-enriched proteins were separated according to known methods (Boetefuer et al. 2018, Poly(adp-ribose) polymerase 1 (parp1) promotes oxidative stress-induced association of cockayne syndrome group b protein with chromatin. The Journal of biological chemistry. 293(46):17863-17874; Lake et al. 2015, The csb chromatin remodeler and ctcf architectural protein cooperate in response to oxidative stress. Nucleic Acids Research. 44(5):2125-2135.). Briefly, cells were washed with PBS and dissolved in 200 μl of Buffer B (150 mM NaCl, 0.5 mM MgCl₂, 20 mM HEPES pH 8.0, 10% Glycerol, 0.5% Triton X-100, 1 mM DTT). After centrifugation at 15,000 rpm at 4°C for 20 minutes, 6× SDS loading buffer was added to the supernatant as the soluble fraction. The precipitate was resuspended in 60 μl of 1× SDS loading buffer and sonicated at 25% amplitude for 10 seconds to be used as the chromatin-rich fraction. All samples were denatured in a 100°C heat block for 10 minutes before use in immunoblot analysis.

[0209]

[0210] Colony Formation Assay

[0211] 500 cells transformed with plasmids were seeded into 6-well plates. After cell attachment, the medium was replaced with fresh medium after treatment with H₂O₂ for 1 hour or without treatment. Cells were cultured under standard conditions (37°C, 5% CO₂) for 14 days, with the medium replaced every 2 days. After culture, the cells were fixed in 4% paraformaldehyde at room temperature for 15 minutes, followed by staining with 0.5% (w / v) crystal violet solution (containing 20% ​​methanol) for 30 minutes. Excess stain was removed by washing with running water and air-dried. Colony images were captured using a flatbed scanner, and counted using ImageJ software.

[0212]

[0213] Comet Assay

[0214] Cells were cultured overnight in 6-well plates before undergoing transformation or reagent treatment. The alkaline comet assay was performed according to known methods to detect single-strand breaks (Olive and Banath 2006, The comet assay: A method to measure DNA damage in individual cells. Nature protocols. 1(1):23-29.). Briefly, cells were suspended in ice-cold PBS and mixed with 1% low-melting point agarose. The mixture was applied to an agarose-coated slide with a cover glass and coagulated at 4°C. The coagulated cells were lysed overnight at 4°C in A1 alkaline lysis buffer (1.2 M NaCl, 100 mM Na₂EDTA, 0.1% sodium lauryl sarcosinate, 0.26 M NaOH, pH > 13). Subsequently, the samples were washed with an A2 alkaline rinse and electrophoresis buffer (0.03 M NaOH, 2 mM Na₂EDTA, pH 12.3). Electrophoresis was performed at 20 V for 20 minutes. DNA was stained with 10 μg / mL of propidium iodide (Sigma-Aldrich) for 20 minutes, fluorescence images were captured using a confocal microscope, and tail moments were measured using the OpenComet tool in ImageJ software (NIH, Bethesda, MD, USA).

[0215]

[0216] BrdU / ssDNA analysis

[0217] The original form BrdU assay was performed according to a known method for ssDNA detection (Caron et al. 2019, Poly(adp-ribose) polymerase-1 antagonizes DNA resection at double-strand breaks. Nat Commun. 10(1):2954.). In summary, cells were transformed with a specified plasmid or siRNA for 24 hours, pretreated with 10 μM BrdU (B23151, ThermoFisher) for at least 16 hours, and then treated with a specified DNA damage-inducing reagent. The in situ fraction was treated with pre-extraction buffer 1 (10 mM PIPES pH 7.0, 100 mM NaCl, 300 mM Sucrose, 3 mM MgCl₂, 0.5% Triton X-100) and buffer 2 (10 mM Tris pH 7.5, 3 mM MgCl₂, 10 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate) on ice for 10 minutes. After washing three times with PBS, cells were fixed with 4% PFA and permeated with 0.5% Triton X-100. Subsequently, the cells were incubated overnight at 4°C with anti-BrdU antibody (#PA5-32256, Invitrogen), washed with PBS-T, and stained with Alexa Fluor 488 goat anti-rabbit IgG for 1 hour. Nuclei were stained with DAPI mounting solution and mounted on slides. The BrdU focus was observed with a Zeiss LSM 800 confocal microscope, and the images were analyzed using ImageJ software.

[0218]

[0219] Analysis of Changes in Electric Mobility (EMSA)

[0220] In vitro protein-ssDNA interactions were analyzed by EMSA using a modified version of the known method (Lei et al. 2021, Crosstalk between cst and rpa regulates rad51 activity during replication stress. Nature Communications. 12(1):6412.). Cy3-labeled ssDNA (1.25 μM; Cy3-5'-TTA-GAG-CTT-AAT-TGC-TGA-ATC-TGG-TGC-TGT-GGG-TGA-ACC-TGC-AGG-TGG-GCA-AAG-A-3') was reacted with recombinant protein in 10 μl of EMSA buffer (35 mM Tris-HCl pH 7.5, 50 mM KCl, 2.5 mM MgCl₂, 1 mM DTT, 100 ng / μL BSA) at 37°C for 20 minutes. Subsequently, the samples were electrophoresed on 5% SDS-PAGE and separated in 0.5× TBE buffer (89 mM Tris, 89 mM Borate, 2 mM EDTA, pH 8) at 100 V for 1 hour (Hellman and Fried 2007, Electrophoretic mobility shift assay (emsa) for detecting protein-nucleic acid interactions. Nature protocols. 2(8):1849-1861.). The gel was imaged using the FOBI Fluorescence In Vivo Imaging System (CELLGENTEK, Daejeon, Korea) and analyzed with ImageJ software.

[0221]

[0222]

[0223]

[0224] Preparation of gold nanoparticle-DNA aptamer-peptide complex

[0225] His-tagged FH-Helix 3 peptides (peptide_1: GWQNSIRHNLSLHHHHHH-NH2; peptide_2: GWKNSVRHNLSLHHHHHH-NH2; peptide_3: GWKSTIHYNLCFHHHHHH-NH2) were purchased from Cosmo GENETECH (Seoul, Korea). Peptide conjugation was performed according to a known method (Yeom et al. 2016, Gold nanoparticle-DNA aptamer conjugate-assisted delivery of antimicrobial peptide effectively eliminates intracellular salmonella enterica serovar typhimurium. Biomaterials. 104:43-51.). First, the Anti-His aptamer (5'-GCT-ATG-GGT-GGT-CTG-GTT-GGG-ATT-GGC-CCC-GGG-AGC-TGG-C-A10-Thiol-3') was conjugated according to known techniques (Ryou et al. 2014, Gold nanoparticle-DNA aptamer composites as a universal carrier for in vivo delivery of biologically functional proteins. Journal of controlled release: official journal of the Controlled Release Society. 196:287-294.). Subsequently, the aptamer-conjugated AuNPs were reacted at 80°C for 5 minutes, after which 1 nM AuNP-AptHis and 10 μM His-tagged peptide were reacted in 1× AMP buffer (200 mM Tris-HCl pH 8.8, 1 mM MgCl₂, 200 mM NaOH) for 10 minutes. Then, 0.5 μg of Lipofectamine 3000 was added and equilibrated in 1× PBS for 10 minutes, then treated with cells and cultured at 37℃ for 24 hours.

[0226]

[0227]

[0228]

[0229] Cell viability analysis

[0230] Cell viability after treatment with peptides and chemical reagents was measured using the cell counting method. Cells were harvested using trypsin-EDTA solution and resuspended in fresh medium. 10 μL of the cell suspension was mixed with 10 μL of trypan blue solution, and 10 μL of the mixture was added dropwise to a hemocytometer. Cells within the four large grids of the hemocytometer were counted under an optical microscope. Cells that did not absorb trypan blue and appeared clear were classified as viable cells, while cells that were stained and appeared blue were classified as non-viable cells. Cell viability was calculated as the ratio of viable cells to the total number of cells.

[0231]

[0232] Statistical analysis

[0233] A non-coordinated two-sided Student's t-test was performed using GraphPad PRISM (San Diego, CA, USA) for comparison with the control group. A p < 0.05 value was considered statistically significant.

[0234]

[0235] Example 1. Identification of RPA, a novel interacting protein of FOX protein

[0236] To identify novel interacting proteins of the FOX protein, an immunoprecipitation analysis was performed using a FLAG-tagged FOX protein expression plasmid.

[0237]

[0238] As a result, it was confirmed that most FOX proteins, excluding FOXJ1, FOXM1, FOXN3, FOXP3, and FOXR2, interact with RPA proteins within cells, and the binding affinity of the interaction varied depending on the type of protein (Fig. 1a). In addition, these interactions were also confirmed in an in vitro immunoprecipitation analysis using FOXL2 and RPA recombinant proteins (Fig. 1b).

[0239]

[0240] Additionally, the intracellular localization of FOX proteins and RPA was confirmed using immunofluorescence staining with a confocal microscope. RPA proteins were mainly distributed within the nucleus, and most FOX proteins, with the exception of FOXM1 and FOXR2, were observed to colocalize with RPA within the nucleus (Fig. 1c).

[0241]

[0242] Taken together, these results suggest that RPA protein acts as a general substrate for FOX protein.

[0243]

[0244] Example 2. Confirmation of the inhibitory activity of FOX protein on the interaction between RPA protein and ssDNA

[0245] Example 2-1. Confirmation of reduction in binding levels between RPA protein and ssDNA by FOX protein

[0246] RPA protein is a major single-stranded DNA binding protein in eukaryotic cells that performs the function of coating ssDNA to prevent the formation of secondary structures and protecting it from degradation by nucleases. Therefore, it is possible that FOX protein regulates the ssDNA binding activity of RPA by interacting with RPA protein. To verify this possibility, in this example, the DNA binding activity of RPA in FOXL2 stable-expressing cells and control cells was analyzed using chromatin-rich protein fractionation.

[0247]

[0248] As a result, when DNA damage was induced, the DNA binding activity of RPA1 and RPA3 increased in control cells, but this increase was not observed in FOXL2 stable-expressing cells (Fig. 2a). This means that FOXL2 inhibits the DNA binding of RPA1 and RPA3.

[0249]

[0250] In addition, to confirm the direct interaction between ssDNA and RPA, electrophoretic mobility change analysis (EMSA) was performed using Cy3-labeled ssDNA and recombinant protein. As a result, FOXL2 inhibited the DNA binding activity of the RPA complex in a concentration-dependent manner (Fig. 2b).

[0251]

[0252] Example 2-2. Confirmation of the inhibitory activity of FOX protein dimerization between RPA1 and RPA2

[0253] The RPA complex is a heterotrimeric protein composed of three subunits of 70 kDa, 32 kDa, and 14 kDa, which forms a stable and highly soluble complex through the binding of the three subunits. In contrast, the monomer or dimer forms exhibit low stability and solubility. Given that the FOX protein can interact with RPA1, RPA2, and RPA3 respectively, it is possible that these interactions may affect the dimerization or trimerization of the RPA proteins.

[0254] To verify this, in vitro analysis using recombinant protein was performed in this example.

[0255]

[0256] As a result, FOXL2 inhibited the binding of RPA1 and RPA2, but no significant change was observed in the binding between RPA1 and RPA3 (Fig. 2c).

[0257]

[0258] In summary, these results suggest that FOXL2 inhibits the DNA binding activity of RPA1 and RPA3, while simultaneously interfering with the binding between RPA1 and RPA2.

[0259]

[0260] Example 3. Confirmation of the inhibitory activity of FOX protein on ssDNA repair action

[0261] Since the interaction between the FOX protein and the RPA protein was shown to inhibit the binding of RPA to ssDNA and interfere with the formation of heterotrimers, it was hypothesized that if DNA damage occurs in cells overexpressing the FOX protein, ssDNA would not be protected by RPA, and the unprotected ssDNA would be degraded, thereby inhibiting single-stranded DNA repair. To verify this, in this example, intracellular ssDNA levels were measured using a native bromodeoxyuridine (BrdU)-based assay.

[0262] Analysis results showed that when the expression of the FOX gene was suppressed, ssDNA accumulation decreased (Figs. 3a and 3b), while conversely, when the FOX gene was overexpressed, the amount of nuclear ssDNA increased (Figs. 3c and 3d).

[0263]

[0264] Additionally, the effect of FOX protein on single-stranded DNA repair was evaluated using alkaline comet analysis and rapid microanalysis to measure DNA unwinding rates.

[0265] As a result, in cells expressing most of the FOX proteins, the tail moment increased and the DNA unwinding rate accelerated, which means that the FOX proteins inhibit single-strand break (SSB) repair (Fig. 3e).

[0266]

[0267] Finally, colony formation analysis was performed to evaluate long-term survival and proliferation ability after DNA damage.

[0268] As a result, FOXD3 and FOXF1 proteins were found to inhibit colony formation in colon cancer cells regardless of the presence or absence of DNA damage (Figs. 3f to 3h).

[0269]

[0270] Synthesizing all these experimental results, it has been proven that the FOX protein plays a role in inhibiting the single-stranded DNA repair process within cells.

[0271]

[0272] Example 4. Identification of the key interaction site mediating binding with RPA protein: Helix3 region of FOX protein

[0273] Example 4-1. Identification of key amino acid residues involved in binding with RPA protein

[0274] To identify the precise binding site of the FOX protein, amino acid sequence alignment of the forkhead box domain was performed.

[0275] As a result, it was confirmed that residues of the Helix3 region within the forkhead domain are highly conserved in most FOX proteins (Fig. 4a), which suggests that the Helix3 region may be a key site for interaction with DNA repair proteins.

[0276]

[0277] Accordingly, mutant expression plasmids in which Helix3 is deleted in FOXD3 and FOXF1 proteins were constructed, and the interaction with RPA proteins was confirmed by immunoprecipitation (IP) analysis.

[0278] As a result, FOX proteins containing the Helix3 deletion mutation were found to be unable to bind to the RPA protein (Figs. 4b and 4c).

[0279]

[0280] These results suggest that the Helix3 region of the FOX protein is a key residue mediating binding with the RPA protein.

[0281]

[0282] In addition, a precise analysis of the motif of the forkhead box domain mentioned above confirmed a conserved sequence consisting of 12 amino acids that is commonly present in the FOX protein (Fig. 4a).

[0283]

[0284] In vitro immunoprecipitation was performed using the synthesized His (6x)-tagged 12-amino acid 12AA-Helix3 peptide 1) and recombinant RPA protein, and it was confirmed that the 12AA-Helix3 peptide directly binds to the RPA protein (Fig. 4c).

[0285]

[0286] In summary, it can be seen that the conserved sequence region consisting of 12 amino acids within the Helix3 region, particularly the forkhead domain, is a key interaction site that mediates binding with the RPA protein.

[0287]

[0288] Example 4-2. Confirmation of Functional Regulation of RPA Protein by FH-Helix3

[0289] To more specifically confirm the effect of the Helix3 region on the function of the RPA protein, experiments were performed using 12AA-Helix3 peptide 1.

[0290] As a result, 12AA-Helix3 peptide 1 inhibited the interaction between RPA1 and RPA2 (Fig. 4d), which was consistent with the results using FOXL2 recombinant protein.

[0291]

[0292] In addition, the single-stranded DNA binding activity of RPA was inhibited by 12AA-Helix3 peptide 1, which showed the same pattern as the action of the FOXL2 full-length protein (Fig. 4e).

[0293]

[0294] From the above results, it was confirmed that 12AA-Helix3 peptide 1 performs the function of regulating the trimer formation and single-stranded DNA binding of RPA protein.

[0295]

[0296] Example 5. Confirmation of the inhibitory effect of a gold nanoparticle-DNA aptamer complex loaded with 12AA-Helix3 peptide on SSB repair.

[0297] After confirming the function of the 12AA-Helix3 region in vitro, a gold nanoparticle-based delivery system was used to deliver Helix3 peptide 1 into cells. The intracellular delivery efficiency of Helix3 peptide 1 was confirmed by immunofluorescence analysis using His antibodies and observed through confocal microscopy after staining with Alexa 488.

[0298] The dark dots in the bright-field image represent gold nanoparticles delivered into the cell. As a result, the peptide was effectively introduced into the cell using the gold nanoparticle delivery system, and no morphological changes were observed (Fig. 5a).

[0299]

[0300] Subsequently, a focus assay of native BrdU was performed to evaluate whether single-strand break (SSB) repair was inhibited. As a result, BrdU-labeled ssDNA signals increased in cells treated with Helix3 peptide 1, indicating that Helix3 peptide 1 inhibits the SSB repair pathway (Fig. 5b).

[0301]

[0302] Example 6. Confirmation of the dependence of DNA repair inhibition by FOX protein on somatic mutations and RPA expression levels

[0303] Example 6-1. Confirmation of the effect of somatic mutations within the Helix3 region on DNA repair activity

[0304] As 12AA-Helix3 peptide 1, a Helix3 region of the forkhead domain (FH), was identified as the major binding site of the RPA protein, it was hypothesized that somatic mutations present in that region could affect DNA repair pathways. To verify this, in this example, information on cancer-related mutations occurring in the 12-amino acid (12AA-Helix3 peptide 1) motif within the Helix3 region was obtained from the cBioportal database, and the mutation frequency was calculated (Fig. 6a).

[0305] As a result, the arginine (R) residue at the 7th position was found to be the most frequently mutated (Fig. 6b).

[0306]

[0307] In addition, several mutant FOX proteins were produced, and their DNA repair activity was analyzed by comparing them to the wild-type (WT) FOX protein.

[0308] As a result (Fig. 6c), the FOX protein containing a mutation at the 7th arginine residue showed significantly weakened single-strand break (SSB) repair inhibitory activity (Fig. 6c). In addition, the FOX protein with mutations at the 5th serine (S>A) and 10th leucine (L>I) residues also failed to inhibit SSB repair.

[0309]

[0310] The interaction between these variant FOX proteins and RPA proteins was further confirmed by immunoprecipitation (IP) analysis.

[0311] As a result (Fig. 6d), the FOX protein containing the 7th arginine variant failed to bind to RPA1 and RPA3, but its binding affinity to RPA2 was weakened and not completely lost. Additionally, the 5th serine (S>A) and 10th leucine (L>I) variant FOX proteins also failed to bind to RPA1 and RPA3. These results suggest that a single variant alone is not sufficient to completely eliminate the strong binding between the FOX protein and RPA2.

[0312]

[0313] Example 6-2. Increase in DNA repair protein expression in cancer tissue

[0314] In addition to mutations in the FOX protein, the clinical significance was evaluated by analyzing how the expression level of the RPA protein changes in cancer tissues. To this end, Affymetrix U133Plus2 microarray data was analyzed using the GENT2 portal.

[0315] As a result (Figs. 6e and 6f), RPA1 and RPA2 mRNA expression was significantly increased in brain, cervical, colon, and liver cancer tissues compared to normal tissue. In addition, RPA3 mRNA was increased in blood, brain, breast, cervical, colon, and liver cancer tissues compared to normal tissue.

[0316]

[0317] Additionally, analysis of RNA-seq data extracted from GEPIA2 revealed that mRNA expression of RPA protein was significantly increased in various cancers, including cervical squamous cell carcinoma (CESC), cholangiocarcinoma (CHOL), colorectal adenocarcinoma (COAD), esophageal carcinoma (ESCA), head and neck squamous cell carcinoma (HNSC), hepatocellular carcinoma (LIHC), and pheochromocytoma and paraganglioma (PCPG) (Figs. 6g and 6h).

[0318]

[0319] Overexpression of the DNA repair protein RPA is highly likely to be associated with the development and progression of cancer, and inhibiting RPA in cancer cells can inhibit cancer growth. Therefore, the 12AA-Helix3 peptide has been developed as an efficient RPA inhibitor and has been confirmed to have potential for application in cancer treatment.

[0320]

[0321] Example 7. Confirmation of broad antitumor effects resulting from inhibition of RPA and ssDNA repair by 12AA-Helix3-derived peptides

[0322] Example 7-1. Selection of 12AA-Helix3-Derived Peptides

[0323] To elucidate the mechanism related to DNA (ssDNA) repair disturbance, screening experiments were performed on three types of peptides 1 to 3 in Table 5 using SW480 colon cancer cells.

[0324] As a result (Fig. 7a), peptide_1 (GWQNSIRHNLSL) derived from FOXL2 / C1 / I1 / K1 / S1 exhibited the strongest inhibitory activity, reducing cell viability to approximately 0.79 compared to the control group. Peptide_2 derived from FOXF1 / N1 showed a viability of 0.85, while peptide_3 derived from FOXR2 showed a viability of approximately 0.96, indicating almost no toxicity.

[0325] Therefore, peptide_1 was used in subsequent experiments, and the inhibitory effect of ssDNA repair mediated by RPA inhibition was evaluated under monotherapy and chemotherapy combination therapy conditions.

[0326]

[0327] Example 7-2. Confirmation of excellent anticancer activity by 12AA-Helix3-derived peptide

[0328] In this example, the anticancer effect of the 12AA-Helix3-derived peptide selected in Example 7-1 was analyzed when applied to various types of cancer. According to the experimental results, consistent with the mechanism of action targeting the ssDNA repair mechanism, peptide_1 exhibited significant anticancer activity in various cancer cell lines and showed a synergistic effect when combined with chemotherapy agents.

[0329]

[0330] The results were confirmed as shown in Figures 7b to 7e.

[0331] First, in A549 lung cancer cells, survival was reduced with peptide_1 alone, and further reduced when combined with 10 μM paclitaxel or 200 μM carboplatin (Fig. 7b).

[0332] Moderate cell death was induced in SW480 colon cancer cells with peptide_1 alone, and survival was significantly reduced when combined with 40 μM paclitaxel or 200 μM carboplatin, confirming that the peptide alleviates chemotherapy resistance in colon cancer cells (Fig. 7c).

[0333] In addition, peptide_1 alone reduced survival in PC-3 prostate cancer cells, and when combined with 20 nM paclitaxel or 200 μM carboplatin, the cell death effect increased compared to chemotherapy alone (Fig. 7d).

[0334] Finally, in OV90 ovarian cancer cells, survival was inhibited by peptide_1 monotherapy, and apoptosis induction was significantly increased when combined with 1 μM paclitaxel or 200 μM carboplatin. The combination group showed a statistically significant difference compared to the monotherapy group and the control group (Fig. 7e).

[0335]

[0336] From the above results, it was confirmed that peptide_1 exhibits broad anticancer activity as a monotherapy by inhibiting RPA and disrupting ssDNA repair, and can enhance therapeutic effects in various cancer cell lines when combined with paclitaxel and carboplatin.

[0337]

[0338] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0339] According to the FOX protein as an RPA inhibitor in single-stranded DNA repair, the intracellular DNA damage repair mechanism can be newly elucidated by the FOX protein regulating the single-stranded DNA repair pathway through interaction with replication protein A. Furthermore, it suggests the possibility of artificially regulating DNA repair activity using specific sequences in the Helix3 region. Therefore, the present invention is expected to provide a new molecular target for the prevention and treatment of DNA damage-related diseases such as cancer, and thus its industrial applicability is recognized.

Claims

1. A FOX (Forkhead Box) protein-derived peptide characterized by inhibiting single-stranded DNA repair by inhibiting the binding of RPA (Replication Protein A) protein to single-stranded DNA, The above FOX protein-derived peptide comprises a peptide represented by the following Formula I: GWX3NSX6RHNLX 11 X 12 [Formula I] In Formula I, X3 is any one selected from the group consisting of Gln, Asn, Glu, His, Lys, and Arg; X6 is any one selected from the group consisting of Ile, Leu, and Met; X 11 is any one selected from the group consisting of Ser, Thr, Asn, Gly, Ala, and Tyr; and X 12 is one selected from the group consisting of Leu, Ile, Val, Met, and Ala.

2. In Paragraph 1, The above X3 is Gln; the above X6 is Ile, and X 11 is Ser and; and X 12 is a FOX protein-derived peptide that is Leu.

3. In Paragraph 1, The above FOX protein-derived peptide comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO. 56 and SEQ ID NO.

57.

4. In Paragraph 1, The above FOX protein-derived peptide comprises an amino acid sequence commonly derived from the Helix 3 region of one or more proteins selected from the group consisting of: FOXL2 (Forkhead box L2) protein, FOXC1 (Forkhead box C1) protein, FOXI1 (Forkhead box I1) protein, FOXK1 (Forkhead box K1) protein, FOXS1 (Forkhead box S1) protein, FOXF1 (Forkhead box F1) protein, FOXN1 (Forkhead box N1) protein, and FOXR2 (Forkhead box R2) protein.

5. In Paragraph 1, The above FOX protein-derived peptide inhibits single-stranded DNA repair by being characterized by one or more selected from the group consisting of the following: Inhibiting the DNA binding activity of RPA1 (Replication Protein A1) and RPA3 (Replication Protein A3); and Inhibits dimerization between RPA1 (Replication Protein A1) and RPA2 (Replication Protein A2).

6. A FOX protein-derived peptide and gold nanoparticle-DNA aptamer conjugate formed by specifically binding the FOX protein-derived peptide of claim 1 and the gold nanoparticle-DNA aptamer complex.

7. A pharmaceutical composition for the prevention or treatment of cancer, comprising as an active ingredient any one or more selected from the group consisting of a FOX (Forkhead Box) protein-derived peptide or a gene encoding the same according to any one of claims 1 to 5; and a conjugate according to claim 6.

8. In Paragraph 7, A pharmaceutical composition for the prevention or treatment of cancer, characterized in that the above cancer is one or more selected from the group consisting of lung cancer, colon cancer, prostate cancer, ovarian cancer, cholangiocarcinoma (CHOL), esophageal cancer (ESCA), cervical cancer, adrenal cancer, head and neck cancer, brain cancer, liver cancer, peritoneal cancer, skin cancer, melanoma of the skin or eye, rectal cancer, anal cancer, perianal cancer, small intestine cancer, endocrine gland cancer, parathyroid cancer, soft tissue sarcoma, urethral cancer, blood cancer, gastric cancer, pancreatic cancer, glioblastoma, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, and thyroid cancer.

9. In Paragraph 7, The above pharmaceutical composition for cancer prevention or treatment additionally comprises a chemo-anticancer agent, and A pharmaceutical composition for the prevention or treatment of cancer, characterized in that the above-mentioned chemo-anticancer agent is one or more selected from the group consisting of paclitaxel, carboplatin, alimta, oxaliplatin, pemetrexed, cisplatin, gemcitabine, fluorouracil (5-FU), cyclophosphamide, vincristine, etoposide, and doxorubicin.

10. In Paragraph 7, The above pharmaceutical composition for cancer prevention or treatment is, The above-mentioned FOX protein-derived peptide or the gene encoding the same or the above-mentioned conjugate; and the above-mentioned chemical anticancer agent in the form of a mixture; or A pharmaceutical composition for the prevention or treatment of cancer, characterized in that the above-mentioned FOX protein-derived peptide or a gene encoding the same or the above-mentioned conjugate; and the above-mentioned chemo-anticancer agent are each formulated and administered simultaneously, separately, or sequentially.

11. A kit for the prevention or treatment of cancer, comprising the pharmaceutical composition for the prevention or treatment of cancer according to claim 7 and instructions.

12. A pharmaceutical composition for enhancing the anticancer effect of an anticancer agent, comprising as an active ingredient any one or more selected from the group consisting of a FOX (Forkhead Box) protein-derived peptide or a gene encoding the same according to any one of claims 1 to 5; and a conjugate according to claim 6.

13. A method for preventing or treating cancer, comprising the step of administering to an individual in need of the same a pharmaceutically effective amount a composition selected from the group consisting of a peptide derived from a FOX (Forkhead Box) protein of any one of claims 1 to 5 or a gene encoding the same; and a conjugate of claim 6 as an active ingredient.

14. Use for cancer prevention or treatment of a composition comprising, as an active ingredient, any one or more selected from the group consisting of a FOX (Forkhead Box) protein-derived peptide or a gene encoding the same according to any one of claims 1 to 5; and a conjugate according to claim 6.

15. Use for manufacturing a preparation for the prevention or treatment of cancer comprising, as an active ingredient, one or more selected from the group consisting of a peptide derived from a FOX (Forkhead Box) protein of any one of claims 1 to 5 or a gene encoding the same; and a conjugate of claim 6.

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