Fox protein serving as PARP1 inhibitor in DNA repair

Abstract

The present invention relates to a FOX protein serving as a PARP1 inhibitor in DNA repair. A novel interaction mechanism between the FOX protein and PARP1 was identified, and the FOX protein was found to act as a direct regulator in a DNA damage repair process. A 12AA-helix3 peptide consisting of 12 amino acids according to the present invention can improve DNA repair efficiency by promoting a homologous recombination repair pathway. Therefore, the present invention can be effectively applied to the development of: a novel DNA repair promoter based on the FOX protein-PARP1 interaction; and a composition for treating cancer.

Description

FOX protein as a PARP1 inhibitor in DNA repair

[0001] The present invention relates to a FOX protein as a PARP1 inhibitor in DNA repair.

[0002] The present application claims priority based on Korean Patent Applications No. 10-2024-0141310 and No. 10-2025-0149155, 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] As organisms and cells are continuously exposed to stress from endogenous and exogenous factors, DNA damage commonly occurs. To maintain genomic stability, appropriate DNA repair pathways are activated depending on the type of damage to remove damaged DNA.

[0004] There are major repair pathways within cells, including non-homologous end joining (NHEJ), homologous recombination (HR), base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR).

[0005] Poly[ADP-ribose] polymerases (PARPs) are a group of enzymes that form poly[ADP-ribose] polymerases by transferring ADP-ribose residues from NAD+ to a recipient protein; this process is called PARylation. PARP1 is a key factor that recognizes damaged DNA breaks (double-strand breaks and single-strand breaks). Upon activation, it recruits repair proteins and mediates repair responses through PARylation. PARP1 plays a crucial role in chromatin regulation, transcription, replication, recombination, apoptosis, and DNA repair, and is known as an important target protein in therapeutic strategies targeting the DNA damage response (DDR) in cancer cells.

[0006] The forkhead box (FOX) protein family consists of transcription factor proteins that possess a highly conserved forkhead DNA binding motif (forkhead box domain, FH domain). FOX proteins regulate the expression of various genes related to embryonic development, differentiation, cell cycle regulation, apoptosis, autophagy, immune response regulation, and cancer development. However, the mechanism by which FOX proteins regulate DNA repair pathways has not yet been clearly elucidated, and research on this is currently very limited.

[0007] The object of the present invention is a FOX (Forkhead Box) protein-derived peptide characterized by promoting the homologous recombination (HR) repair pathway of DNA by inhibiting the binding of PARP1 (Poly (ADP-ribose) polymerase 1) protein to DNA,

[0008] The above FOX protein-derived peptide provides a FOX protein-derived peptide comprising 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 promoting the homologous recombination (HR) repair pathway of DNA by inhibiting the binding of PARP1 (Poly(ADP-ribose) polymerase 1) protein to 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. 51 and SEQ ID NO. 52, 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 promote a homologous recombination repair pathway of DNA by being characterized by one or more selected from the group consisting of the following, but is not limited thereto:

[0035] Interacting with the PARP1 protein to regulate PARP1's DNA recognition ability by inhibiting PARP1's binding to DNA damage sites; and

[0036] Induces poly(ADP-ribosyl)ization (PARylation) of PARP1.

[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 colorectal cancer, breast cancer, 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, 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 FOX protein as a PARP1 inhibitor in DNA repair, a novel interaction mechanism between FOX protein and PARP1 has been identified, revealing that FOX protein acts as a direct regulator in the DNA damage repair process. The 12AA-helix3 peptide of the present invention, composed of 12 amino acids, can improve DNA repair efficiency by promoting the homologous recombination (HR) repair pathway. Therefore, the present invention can be usefully applied to the development of novel DNA repair promoters and cancer treatment compositions based on the FOX protein-PARP1 interaction.

[0055] Figure 1a shows the results of identifying PARP1, a new interacting protein of the FOX protein.

[0056] Figures 1b and 1c show experimental results confirming that the binding domain of PARP1 is the forkhead domain. To identify the PARP1 binding domain of FOXL2, FOXL2 wild-type or truncated mutant expression plasmids were transfected into 293T cells. Cell lysates were immunoprecipitated with an anti-Myc antibody, and then immunoblotting was performed with the designated antibody.

[0057] Figures 1d and 1e show experimental results confirming that most FOX proteins colocalize with PARP1 within the nucleus. The subcellular localization and colocalization of PARP1 and FOX proteins were observed using confocal microscopy in SW480 cells transformed with a FLAG-tagged FOX protein expression plasmid.

[0058] Figures 2a to 2c show experimental results confirming that the FOX protein inhibits the binding of PARP1 to DNA.

[0059] Figure 2a shows that to determine whether the interaction between PARP1 and FOXL2 under normal or DNA damage conditions depends on the presence of DNA, SW480 cells were transformed with a control or FLAG-FOXL2 plasmid and then treated with H₂O₂ (100 μM, 1 hour) or MMS (1 mM, 30 min). Depending on whether DNase I (10 U) was used for treatment, cell lysates were immunoprecipitated (IP) with FLAG antibody, and then Western blotting was performed using the specified antibody.

[0060] Figure 2b shows that the DNA binding affinity of PARP1 was confirmed in FOXL2-deficient (FOXL2-KO) cells and FOXL2-stable KGN cells through chromatin immunoprecipitation (ChIP) analysis. Cells were transformed with the DSB repair reporter plasmid (3 μg), and after performing ChIP with the PARP1 antibody, quantitative PCR (qPCR) analysis was conducted using primers targeting the region indicated by the black arrow in the schematic. Data are expressed as the mean ± standard error (mean ± SEM) of three independent experiments, indicating significance of ***p < 0.001.

[0061] Figure 2c shows that the binding of PARP1 to DNA was evaluated through protein fractionation analysis in SW480 cells overexpressed with FOXL2. Cells were treated with 40 μM of Olaparib or Veliparib, and histone H3 and β-actin were used as fraction markers for chromatin and soluble soluble protein, respectively.

[0062] Figures 2d to 2f show experimental results confirming that the PARylation of PARP1 is regulated by interaction with FOXL2.

[0063] In Figure 2d, the degree of PARylation of PARP1 was analyzed after treatment with the specified reagents, Olaparib (40 μM), Veliparib (40 μM), or PARG inhibitor (PARGi-ADP-HPD, 10 μM), in control or FOXL2-transformed cells.

[0064] In Figure 2e, PARylation of PARP1 was compared in control cells and FOXL2 mutant transformed cells.

[0065] In Figure 2f, DNA binding of PARP1 was confirmed by protein fractionation analysis in SW480 cells overexpressing FOXL2 wild-type or mutant. Histone H3 and β-actin were used as fraction markers for chromatin and soluble protein, respectively.

[0066] Figures 2g and 2h show experimental results confirming that the FOX protein induces PARylation of PARP1 and inhibits DNA binding of PARP1.

[0067] Figure 2g confirmed the changes in PARP1 PARylation following the overexpression of FOX protein. SW480 cells were transformed with a specified FOX protein expression plasmid for 24 hours, then immunoprecipitation was performed using an anti-PARP1 antibody, and immunoblotting analysis was performed using the specified antibody.

[0068] In Figure 2h, the binding of PARP1 to DNA was confirmed through protein fractionation analysis in SW480 cells overexpressing FOX protein. Cells were analyzed under normal conditions or DNA damage conditions (1 mM MMS, 30 min treatment), and histone H3 (H3) and β-actin (β-Actin) were used as fraction markers for chromatin and soluble protein, respectively.

[0069] Figures 3a and 3b show experimental results confirming that the FOX protein promotes homologous recombination (HR) repair activity.

[0070] In Figure 3a, to analyze the effect of FOX protein on HR repair, cells stably expressing pDR-GFP were transformed with an I-SceI expression plasmid and a designated FOX protein expression plasmid. Data were presented as the mean ± standard error (mean ± SEM) of three independent experiments, showing statistically significant differences at the levels of *p < 0.05, **p < 0.01, and ***p < 0.001.

[0071] In Fig. 3b, Rad51 foci were observed in cells transformed with FOX protein depending on the presence or absence of H₂O₂ treatment (100 μM, 1 hour treatment followed by 1 hour recovery).

[0072] Figure 4a shows the experimental results identifying the intermediate region of the FH domain essential for FOXL2 function. A Myc-FOXL2 construct was designed to identify the residue required for binding with DNA repair proteins. Cell viability was measured in SW480 cells transformed with each FOXL2 mutant plasmid. Data are expressed as the mean ± standard error (mean ± SEM) of three independent experiments, and different letters indicate statistically significant differences (p < 0.05).

[0073] Figure 4b shows the amino acid sequence alignment and structural features of the forkhead domain of the FOX protein. The forkhead box motifs of all human FOX proteins were compared. Structurally, the FH domain has a helix-turn-helix core structure consisting of three helices, with two wings positioned on each side. Scanning of the forkhead box domain motif of the FOX protein revealed a conserved motif (12AA-Helix3) composed of 12 amino acids within the FOX protein.

[0074] Figures 4c and 4d show experimental results confirming that the Helix 3 region within the FH domain of FOXL2 is the major binding site of PARP1.

[0075] Figure 4c shows the schematic structures of the wild-type (WT) and Helix 3 deletion (ΔHelix3) mutants of FOXL2. Figure 4d compares the interaction affinity with DNA repair proteins using the WT and ΔHelix3 forms of FOXL2.

[0076] Figures 4e and 4f show experimental results confirming that FH-Helix 3 modulates the function of PARP1.

[0077] In Figure 4e, the effect of FH-Helix 3 on DNA binding of PARP1 was analyzed through protein fractionation in wild-type (WT) and ΔHelix3 mutant overexpressing cells of FOXD3 or FOXF1.

[0078] In Figure 4f, the PARylation level of PARP1 was confirmed by immunoblotting using a designated antibody after anti-PARP1 immunoprecipitation, based on changes in WT or ΔHelix3 mutant expression of FOX protein.

[0079] Figure 4g shows experimental results confirming that FH-Helix 3 of the FOX protein plays a key role in HR repair. After transforming cells stably expressing pDR-GFP with the I-SceI expression plasmid and the designated FOX protein expression plasmid, the effects of WT and ΔHelix3 mutants of the FOX protein on HR repair were analyzed. Data are expressed as the mean ± standard error (mean ± SEM) of the results from three independent experiments, indicating statistical significance at the levels of *p < 0.05, **p < 0.01, and ***p < 0.001.

[0080] Figure 5a shows the in vitro interaction between the FH-Helix3 peptide and the DNA repair protein. Direct interactions between the FH-Helix3 peptide and PARP1 were detected by performing in vitro immunoprecipitation (IP) using the His-tagged FH-Helix3 peptide and the recombinant protein. Immunoprecipitation was performed using IgG or a designated antibody.

[0081] Figure 5b shows the experimental results confirming the intracellular delivery of 12AA-Helix3. Effective intracellular delivery of the 12AA-Helix3 peptide was confirmed by confocal microscopy following immunofluorescence analysis using an 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).

[0082] Figure 5c shows the experimental results analyzing the function of the 12AA-Helix3 peptide. The effect of the 12AA-Helix3 peptide on HR repair was analyzed by transforming cells stably expressing pDR-GFP with an I-SceI expression plasmid and treating them with or without the 12AA-Helix3 peptide using a gold nanoparticle (AuNP) delivery system. Data were expressed as the mean ± standard error (mean ± SEM) of three independent experiments, and statistically significant differences were observed at the p < 0.001 level.

[0083] Figure 6a shows somatic and germline mutations within the forkhead domain. In Figure 6a, the amino acid sequences of the forkhead domain of the human FOX protein are aligned and mutations are indicated. Germline mutations are indicated in red, somatic mutations in blue, and residues containing both types of mutations in green. Germline mutation information was collected 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 / ).

[0084] Figures 6b to 6d show experimental results confirming that somatic mutations within the Helix3 region of the FOX protein affect HR repair activity.

[0085] In Figure 6b, 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.

[0086] In Figure 6c, 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.

[0087] In Figure 6d, the difference in PARP1 interaction between FOX-WT and FOX-mutant was confirmed by immunoprecipitation (IP) using an anti-FLAG antibody.

[0088] Figures 6e to 6h show experimental results confirming that the expression of PARP1 is increased in cancer patients.

[0089] In Figures 6e and 6f, PARP1 mRNA expression data were obtained from expression comparison data between normal and tumor tissues via the GENT2 portal (http: / gent2.appex.kr / gent2 / ) on the U133Plus2 platform. The relative mRNA expression levels of cancer and normal tissues were displayed as a heatmap, where red indicates high expression and green indicates low expression. The number of samples for each tissue type is shown on the right.

[0090] RNA-seq data in Figures 6g and 6h were obtained via GEPIA2 multiplex gene analysis (http: / gepia2.cancer-pku.cn / #index). Differences in RNA expression levels between cancer tissue and normal tissue were shown as a heatmap, and the number of samples for each cancer type is indicated at the bottom.

[0091] Figure 7a shows the cell viability analysis of various peptides in HCT116 colorectal cancer cells. Viability analysis was performed on HCT116 colorectal cancer cells after treatment with each peptide for 24 hours, and data are presented as the mean ± standard error (SEM) of three independent experiments. Statistical significance is indicated by the letters a, b, and c (p < 0.05). 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).

[0092] Figures 7b to 7e show the synergistic effect of peptide-chemotherapy combination therapy in cancer cells.

[0093] Figure 7b shows the results of measuring cell viability of BRCA2 mutant HCT116 cells treated with 12AA-Helix3-derived peptide and paclitaxel (10 nM, A) or carboplatin (200 μM, B).

[0094] Figure 7c shows the results of measuring the cell viability of BRCA1 mutant HCC-1937 cells treated with 12AA-Helix3-derived peptide and paclitaxel (2 μM, C) or carboplatin (200 μM, D).

[0095] Figure 7d shows the results of measuring the cell viability of MCF-7 cells treated with 12AA-Helix3-derived peptide and paclitaxel (10 nM, E) or carboplatin (200 μM, F).

[0096] Figure 7e shows the results of measuring the cell viability of MDA-MB-231 cells treated with 12AA-Helix3-derived peptide and paclitaxel (1 μM, G) or carboplatin (200 μM, H).

[0097] Organisms have developed at least five distinct DNA repair pathways to manage DNA damage resulting from endogenous and exogenous stress in order to maintain genomic stability. The loss of this genomic stability is closely associated with the onset and progression of many diseases. Forkhead box (FOX) proteins are a group 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 of embryonic development, differentiation, proliferation, apoptosis, autophagy, immune regulation, and bone metabolism. However, the regulatory effects of FOX proteins on DNA repair pathways are not yet clear, and their mechanisms of action need to be elucidated.

[0098] In the present invention, PARP1 (poly [ADP-ribose] polymerase 1), an important DNA repair regulator, was identified as a common yet novel interaction factor with FOX protein. The DNA binding activity of PARP1 was inhibited through hyper-poly (ADP)-ribosylation (PARylation) induced by direct interaction with FOX protein, and as a result, a homologous recombination (HR)-based DNA repair pathway was promoted.

[0099] Scanning of the forkhead box domain motif revealed that a 12-amino acid sequence (12AA-helix3) within the helix 3 region serves as a key site mediating the binding of the FOX protein to PARP1. Intranuclear delivery of the 12AA-helix3 peptide using a gold nanoparticle-based peptide delivery system resulted in increased HR repair activity. Furthermore, FOX proteins with somatic mutations within the helix3 region found in cancer patients were shown to exhibit weakened promoterative activity toward the DNA repair pathway. In silico analysis of mRNA expression indicated that PARP1 is upregulated in cancer tissues. Taken together, these findings elucidate a novel functional role of the FOX protein in the HR DNA repair pathway and propose a new mechanism of action for the FOX protein through its binding to PARP1.

[0100] Accordingly, the present invention relates to a FOX (Forkhead Box) protein-derived peptide characterized by promoting the homologous recombination (HR) repair pathway of DNA by inhibiting the binding of PARP1 (Poly (ADP-ribose) polymerase 1) protein to DNA, wherein

[0101] The above FOX protein-derived peptide comprises a peptide represented by the following Formula I:

[0102] GWX3NSX6RHNLX 11 X 12 [Formula I]

[0103] In Formula I,

[0104] X3 is any one selected from the group consisting of Gln, Asn, Glu, His, Lys, and Arg;

[0105] X6 is any one selected from the group consisting of Ile, Leu, and Met;

[0106] X 11 is any one selected from the group consisting of Ser, Thr, Asn, Gly, Ala, and Tyr; and

[0107] X 12 is one selected from the group consisting of Leu, Ile, Val, Met, and Ala.

[0108] In the entire specification including the following claims, “peptide derived from FOX protein” may mean a peptide comprising an amino acid sequence derived from the helix 3 region of the FOX protein. Accordingly, “peptide derived from FOX protein” may 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 mean a peptide derived from an amino acid sequence region that binds to the PARP1 protein among the structurally corresponding sites of the helix 3 of the FOX protein, and which exhibits inhibitory activity on the binding of PARP1 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 DNA homologous recombination repair pathway promoting 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.”

[0109] In the present invention, by observing that PARP1 competitively binds between DNA and FOXL2, it was confirmed that the FOX protein, the helix 3 region of the FOX protein, or a peptide derived from the helix 3 region of the FOX protein regulates the binding of PARP1 to DNA. Accordingly, as per the entire claim below, the FOX protein, the helix 3 region of the FOX protein, or a peptide derived from the helix 3 region of the FOX protein may function as an inhibitor of PARP1, but is not limited thereto.

[0110] 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. 51, which consists of 12 amino acids, the DNA homologous recombination 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.

[0111] In addition, in Formula I of the present invention,

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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. In that each peptide did not exhibit significant inhibition of DNA homologous recombination repair pathways or anticancer activity, X3 of the FOX protein-derived peptide may not contain Lys; X6 may not contain Val or Thr, and X 11 may not include Cys, and X 12 It may not include Phe, but is not limited to this.

[0119] Including all claims below, 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 entire specification including the following claims, the His tag is not necessarily limited to six histidine residues, and the number and arrangement of histidine residues may be adjusted according to the expression efficiency, solubility, or purification efficiency of the protein.

[0120] Including all claims below, the his tag may comprise 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.

[0121] 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. 51 and SEQ ID NO. 52, but is not limited thereto. In the entire specification including the following claims, the FOX protein-derived peptide may comprise a sequence having sequence homology with one or more amino acid sequences selected from the group consisting of SEQ ID NO. 51 and SEQ ID NO. 52 at a level capable of performing the same function.

[0122] In all claims below, the nucleic acid molecule of a polynucleotide sequence (used interchangeably with a base sequence) is a concept that 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.

[0123] 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.

[0124] In addition, in the entire claim below, the amino acid sequence may be applied with the same concept as the polynucleotide sequence. That is, the amino acid sequence is a concept that includes variants capable of performing the same functional action as, that is, 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.

[0125] In one embodiment of the present invention,

[0126] The above FOX protein-derived peptide may comprise, but is not limited to, an amino acid sequence commonly derived from the helix 3 region of any one or more proteins selected from the group consisting of the following:

[0127] 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.

[0128] Including all claims below, the FOX protein-derived peptide may comprise, but is not limited to, 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.

[0129] In an embodiment of the present invention, amino acid sequences commonly derived from the helix 3 regions of FOXL2, FOXC1, FOXI1, FOXK1, and FOXS1 proteins 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 PARP1 protein. Furthermore, it was confirmed that when treated with this, inhibitory activity of the DNA homologous recombination repair pathway is exhibited, and therapeutic effects are shown against various types of cancer. Accordingly, in the present specification including the entire claims below, the FOX protein-derived peptide may include amino acids commonly derived from the helix 3 regions of FOXL2, FOXC1, FOXI1, FOXK1, and FOXS1 proteins, but is not limited thereto.

[0130] Meanwhile, in the present specification including the entire claim below, when the FFOX 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 it has an amino acid sequence 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 DNA homologous recombination repair inhibitory activity is also found to be poor. Therefore, the FOX protein-derived peptide may be characterized by being derived from the helix 3 region of a specific family protein of the FOX protein.

[0131] In one embodiment of the present invention, the FOX protein-derived peptide may promote a homologous recombination repair pathway of DNA by being characterized by one or more selected from the group consisting of the following, but is not limited thereto:

[0132] Interacting with the PARP1 protein to regulate PARP1's DNA recognition ability by inhibiting PARP1's binding to DNA damage sites; and

[0133] Induces poly(ADP-ribosyl)ization (PARylation) of PARP1.

[0134] 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.

[0135] In this specification, including all claims below, “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 can improve cell membrane recognition and binding efficiency. Additionally, aptamer-bound gold nanoparticles enable selective delivery to target cells while minimizing cytotoxicity, thereby increasing the intracellular delivery efficiency of therapeutic peptides. In this case, in this specification, including all claims below, the anti-DNA aptamer of the DNA aptamer may include the nucleotide sequence of SEQ ID NO. 57, but is not limited thereto.

[0136] In the present invention, it was confirmed that the 12AA-Helix3 peptide exhibits excellent inhibitory activity against the DNA homologous recombination repair pathway when delivered in combination with a gold nanoparticle-DNA aptamer. However, the 12AA-Helix3 peptide alone may have limitations in delivery via AuNPs. Therefore, as per the entire claim below, 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.

[0137] 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.

[0138] In all claims below, the terms “protein,” “polypeptide,” or “peptide” are used interchangeably and refer to polymers of amino acid residues, for example, as commonly found in proteins in their natural state.

[0139] 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.

[0140] In one embodiment of the present invention, the cancer may be one or more selected from the group consisting of colorectal cancer, breast cancer, 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, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, and thyroid cancer, but is not limited thereto.

[0141] In all claims below, the breast cancer may include BRCA1-deficient triple-negative breast cancer, estrogen receptor-positive (hormone-dependent) breast cancer, and triple-negative breast cancer (highly invasive and metastatic). In one embodiment of the present invention, excellent anticancer activity of the FOX protein-derived peptide of the present invention was confirmed against HCC1937, MCF-7, and MDA-MB-231 as respective breast cancer cell lines. Since the breast cancer cell lines include both BRCA mutation and normal characteristics, the breast cancer is not limited to the presence of a BRCA mutation.

[0142] 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.

[0143] In one embodiment of the present invention,

[0144] 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.

[0145] In the entirety of the following claims, “combined administration” in this specification 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. A combined therapeutic regimen may be defined as one that provides a synergistic effect, but is not limited thereto, wherein the efficacy, measured by, for example, the degree of response, the rate of response, the time to disease progression, or the survival period, is therapeutically superior to the efficacy obtainable by administering one or the rest of the components of the combined therapeutic regimen at a normal dose.

[0146] In the present specification, including all claims below, “chemical anticancer agent” may mean a first-generation anticancer agent also referred to as a “cytotoxic anticancer agent” or “chemical drug anticancer agent.”

[0147] In one embodiment of the present invention, the pharmaceutical composition for cancer prevention or treatment is,

[0148] 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

[0149] 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.

[0150] 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 it or the conjugate; and the anticancer agent.

[0151] 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 containing 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 containing 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.

[0152] 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, 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.

[0153] Including all claims below, as set forth herein, 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, ophthalmic preparations, oleic acid preparations, emulsions, suspensions, ethanol preparations, troches, aromatic preparations, limonene adrenergic preparations, 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, respectively, according to conventional methods, and said external preparations may be used as creams, gels, patches, sprays, ointments, warning agents, lotions, liniments, pastes, or cataplasms, etc. It can have a formulation.

[0154] 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.

[0155] When formulating, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0160] 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.

[0161] 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-analizing 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.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] Including all claims below, the pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, “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.

[0166] 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.

[0167] 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, intravenous injection, intramuscular injection, paraspinal (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.

[0168] 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.

[0169] 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.

[0170] In the present invention, “administration” means providing a predetermined composition of the present invention to an individual by any appropriate method.

[0171] 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.

[0172] In this specification, including all claims below, the term “kit” refers to a tool that additionally includes a formulation or a substance for the function, storage, etc., of the kit claimed in the present invention, which enables 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., which are typically required for the storage and processing methods thereof. In this case, 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.

[0173] Including all claims below, 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 may be fully or partially detachable, and may also be equipped with a stopper that allows access to the contents by a needle. The kit may include an outer package, and the outer package may include instructions regarding the use of the components.

[0174] 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.

[0175] Meanwhile, as per the entire claim below, 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. As per the entire claim below, “enhancing the anticancer effect” may include all effects that can consequently strengthen the function of the anticancer drug. For example, it is a concept that includes 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 consequently by inhibiting the formation of resistance or tolerance in cancer cells to the anticancer drug. That is, as per the entire claim below, the active ingredient of the present invention may be used as a compound for combination administration with a known anticancer drug for the purpose of enhancing the anticancer effect. That is, as per the entire claim below, the active ingredient of the present invention may be used for combination administration with an anticancer drug to enhance the anticancer effect of said anticancer drug.

[0176] 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.

[0177] In the present specification, including all claims below, the pharmaceutical composition for enhancing the anticancer effect of the anticancer agent may be in the form of a mixture of the FOX protein-derived peptide and the chemical anticancer agent, but is not limited thereto.

[0178] 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.

[0179] In all claims below, when the term “comprising” is used in this specification, it means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0180] Terms such as “about,” “substantially,” as used herein, including in all claims below, are used to mean at or near the stated value when inherent manufacturing and material tolerances are presented in the said meaning, 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.

[0181] In all claims below, the term “combination thereof” included in the Markush-type expression means one or more mixtures or combinations selected from the group consisting of the components described in the Markush-type expression, and means including one or more selected from the group consisting of said components.

[0182] The terms used in this specification, including all claims below, have been selected to be as widely used as possible while considering their functions in the present 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, terms used in this invention should be defined not merely by their names, but based on their meanings and the overall content of the present invention.

[0183] 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.

[0184]

[0185] [Example]

[0186]

[0187] Plasmids

[0188] pcMV-Myc-FOXL2 wild-type and ΔAla, ΔFH, and FH mutant 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.). FOX protein expression plasmids were constructed by performing polymerase chain reaction (PCR) using specified templates and primers. The PCR products were cleaved with specified restriction enzymes (Takara Bio, Shiga, Japan) and ligated into the p3XFLAG-CMV-10 (Sigma-Aldrich, St. Louis, MO, USA) vector. The restriction enzymes, primers, and templates used are shown in Table 1.

[0189] The mutants pCMV-Myc-FOXL2 ΔFH1, ΔFH2, ΔFH3 and ΔHelix3, p3XFLAG-CMV10-FOXD3-ΔHelix3, and p3XFLAG-CMV10-FOXF1-ΔHelix3 were constructed by recombinant PCR, and the primer pairs used were FOXL2-ΔFH1-F / R, FOXL2-ΔFH2-F / R, FOXL2-ΔFH3-F / R, FOXL2-ΔHelix3-F / R, FOXD3-ΔHelix3-F / R, and FOXF1-ΔHelix3-F / R. The oligonucleotide sequences of the recombinant PCR primers are shown in Table 2.

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196] Mammalian Cell Culture and Transfection

[0197] Human embryonic kidney cell line (293T; ATCC, Manassas, VA, USA) and human colon cancer cell line (SW480) were cultured in Dulbecco's modified Eagle's medium (DMEM; Caisson, North Logan, UT, USA) with 10% fetal bovine serum (FBS; Caisson) and 1% penicillin-streptomycin (P / S; Caisson) added at 37°C and 5% CO₂. FOXL2 KO cells (Jin et al., 2020) and FOXL2 stable expression 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 each prepared according to known methods and cultured in DMEM / F12 medium supplemented with 10% FBS and 1% P / S. 293T cells were transformed using polyethyleneimide (polyethylenimine, PEI; Polysciences Inc., Warrington, PA, USA), and SW480 cells were transformed with Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

[0198]

[0199] Reagents and Antibodies

[0200] Anti-PARP1 (sc-74469) and Anti-β-actin (sc-47778) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-H3 (05-928) was purchased from Upstate, and Anti-FLAG (#14793) and Anti-Myc (#2276S) were purchased from Cell Signaling Technology (Danvers, MA, USA). Goat anti-mouse IgG for Alexa Fluor 546 staining and goat anti-rabbit IgG for Alexa Fluor 488 staining were purchased from Invitrogen (Waltham, MA, USA). Anti-PAR (ALX-804-220-R100) antibody was purchased from Enzo Life Sciences (New York, USA).

[0201]

[0202] Immunoprecipitation and Immunoblot Analysis

[0203] Cells were transformed with a specified plasmid or treated with chemical reagents. Immunoprecipitation and immunoblot analysis were performed according to known methods (Jin et al., 2020). Briefly, after cell lysis, the cells were stirred overnight at 4°C with the specified antibody and Dynabeads Protein G (Thermo Scientific, Rockford, IL). Subsequently, the immunoprecipitated proteins were eluted, denatured at 100°C, separated by SDS-PAGE, and transferred to a PVDF membrane (Millipore, Bedford, MA, USA). After the antibody reaction, protein bands were visualized using an Amersham Imager 600 (GE Healthcare Life Sciences, Amersham, UK), and band intensity was quantified using OptiQuant software (Bio-Rad Laboratories, Hercules, CA, USA).

[0204]

[0205] Confocal Microscopy and Immunofluorescence Analysis

[0206] Cells (4 × 10^4) were seeded onto circular coverslips inserted in a 24-well plate. After overnight incubation, the cells were transformed with the designated plasmid and fixed with 4% paraformaldehyde for 15 minutes. Subsequently, the cells were permeabilized with 0.2% Triton X-100 for 15 minutes and blocked with a blocking buffer containing 1% BSA for 1 hour at room temperature. The designated primary antibodies were diluted in 1% BSA solution and incubated overnight at 4°C, followed by three washes with PBS-T (0.1% Tween-20 in PBS). Alexa Fluor 546 goat anti-mouse IgG (1:1000; Invitrogen) and Alexa Fluor 488 rabbit anti-goat IgG (1:1000; Invitrogen) were incubated for 1 hour at room temperature. After washing three times with PBS-T, the images were attached to slides using Fluoroshield™ with DAPI mounting solution (ImmunoBioScience Crop, Mukilteo, WA, USA). Fluorescence images were observed using a Zeiss LSM 800 confocal laser scanning microscope (Carl Zeiss, Gottingen, Germany).

[0207]

[0208] Chromatin-enriched Protein Fractionation

[0209] Cells were seeded into 60 mm culture dishes and treated with the specified plasmid or reagent. Subsequently, the cells were washed with PBS and lysed in 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 (15,000 rpm, 20 min, 4°C), the supernatant was collected as water-soluble protein, and the pellet was resuspended in 1× SDS loading buffer and sonicated for 10 seconds. All samples were denatured at 100°C for 10 minutes and used for immunoblot analysis.

[0210]

[0211] Homologous Recombinant DNA Repair Assay (HR DNA Repair Assay)

[0212] HR DNA repair analysis was performed according to a known method (Jin et al., 2020). 293T-DR-GFP cells incorporating pDR-GFP (Addgene plasmid #26475) onto the chromosome were transformed with the designated plasmid for 24 hours. Subsequently, the pCBASceI DNA plasmid was additionally transformed for 24 hours, after which GFP fluorescence was measured using FlexStation 3.

[0213]

[0214] Preparation of Gold Nanoparticle-DNA Aptamer-Peptide Complex

[0215] His-tag attached 12AA-Helix3 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.). The anti-His aptamer (5'-GCT-ATG-GGT-GGT-CTG-GTT-GGG-ATT-GGC-CCC-GGG-AGC-TGG-C-A₁0-Thiol-3') was bound to gold nanoparticles (AuNP) according to the description of a known method (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, aptamer-bound AuNPs were treated at 80°C for 5 minutes, and 1 nM AuNP-AptHis and 10 μM His-tag peptide were reacted in AMP buffer (200 mM Tris-HCl pH 8.8, 1 mM MgCl₂, 200 mM NaOH) for 10 minutes, after which 0.5 μg Lipofectamine 3000 was added. After equilibrating in PBS for 10 minutes, the cells were treated and incubated at 37°C for 24 hours.

[0216]

[0217]

[0218]

[0219] Cell Viability Assay

[0220] Cells (1 × 10^4) were seeded into a 96-well plate and transformed with a specified plasmid or treated with a reagent. After 24 hours, cell viability was measured using the CellTiter-Glo Assay Kit (Promega, Madison, WI, USA), which detects metabolically active cells based on ATP content.

[0221]

[0222] Statistical Analysis

[0223] Statistical analysis was performed using GraphPad PRISM (San Diego, CA, USA), and the unpaired, two-tailed Student's t-test was used for comparison with the control group. A p-value of less than 0.05 was considered statistically significant.

[0224]

[0225] Example 1. Identification of PARP1, a Novel Interacting Protein with FOX Protein

[0226] In this example, the possibility of FOX proteins interacting with endogenous PARP1 was investigated. Specifically, a total of 19 FLAG-tagged FOX protein expression plasmids were constructed, one selected from each subgroup from FOXA to FOXS, and 293T cells were transformed and then immunoprecipitation (IP) was performed.

[0227]

[0228] As a result, it was confirmed that all FOX proteins, excluding FOXM1, FOXN3, FOXP3, and FOXR2, interact with PARP1 with different binding affinities (Fig. 1a). Domain mapping using the truncated FOXL2 protein confirmed that the binding domain of PARP1 is the forkhead domain (Figs. 1b and 1c).

[0229]

[0230] In addition, the intracellular localization of FOX protein and PARP1 was confirmed through immunofluorescence analysis using anti-FLAG and anti-PARP1 antibodies and confocal microscopy.

[0231] As a result, it was observed that most FOX proteins, excluding FOXM1 and FOXR2, were colocalized with PARP1 within the nucleus (Figs. 1d and 1e).

[0232]

[0233] Taken together, these results suggest that PARP1 acts as a general nuclear substrate for FOX proteins.

[0234]

[0235] Example 2. Confirmation of the inhibitory effect of FOX protein on PARP1 and DNA binding activity according to PARylation regulation

[0236] Example 2-1. Inhibitory activity of FOX protein on PARP1 DNA binding

[0237] When DNA is damaged within a cell, PARP1 acts as a first sensor that recognizes and binds to DNA damage sites, including single-strand breaks and double-strand breaks. Therefore, the interaction between the FOX protein and PARP1 may inhibit PARP1's binding to DNA. To determine whether the interaction between PARP1 and FOXL2 under normal or DNA damage conditions depends on the presence of DNA, SW480 cells were transformed with a control or FLAG-FOXL2 plasmid and treated with a reagent that induces DNA damage.

[0238]

[0239] As a result, DNase I treatment significantly increased the interaction between PARP1 and FOXL2 (Fig. 2a), which suggests that the presence of DNA interferes with the binding of PARP1 and FOXL2, and that the interaction with FOXL2 can inhibit the DNA binding activity of PARP1.

[0240]

[0241] The DNA binding affinity of PARP1 was further investigated through chromatin immunoprecipitation (ChIP) analysis in FOXL2-deficient (Foxl2-KO) KGN cells and FOXL2-stable KGN cells.

[0242]

[0243] As a result, binding of PARP1 to DNA damage sites increased in FOXL2-deficient cells but decreased in FOXL2-stabilized expression cells (Fig. 2b). This implies that FOXL2 inhibits the recruitment of PARP1 to DNA damage sites. Additionally, protein fractionation analysis confirmed the binding of PARP1 to DNA; consequently, PARP1 binding to DNA was inhibited upon FOXL2 overexpression, while treatment with a PARP1 inhibitor increased PARP1 binding to chromatin DNA compared to the untreated group (Fig. 2c).

[0244]

[0245] These results indicate that FOXL2 inhibits the DNA binding affinity of PARP1, and that this effect can be reversed by treatment with a PARP1 inhibitor, suggesting that the enzymatic activity of PARP1 is important for the DNA binding of PARP1 regulated by FOXL2.

[0246]

[0247] Execution 2-2. Confirmation of Regulation of PARP1 Paraylation by FOXL2

[0248] Since PARylation of PARP1 is known as a major post-translational modification in response to DNA damage, changes in PARylation in FOXL2-transformed cells were analyzed.

[0249] As a result, hyper-PARylation of PARP1 was observed in cells transformed with FOXL2 (Fig. 2d).

[0250]

[0251] Next, to determine whether the PARylation of PARP1 depends on interaction with FOXL2, a FOXL2 mutant containing only the FH domain (FH domain-only mutant) and an FH domain deletion (ΔFH) plasmid were used.

[0252] As a result, it was confirmed that the FH domain, identified as the binding domain with PARP1, is sufficient for the PARylation of PARP1 (Fig. 2e).

[0253]

[0254] Since auto-PARylated PARP1 cannot bind to DNA and interaction with FOXL2 can induce excessive PARylation of PARP1, the DNA binding activity of PARP1 was analyzed through chromatin-enriched protein fractionation.

[0255] As a result, both the wild-type (WT) and FH domain-only FOXL2 capable of binding to PARP1 reduced the DNA binding activity of PARP1 (Fig. 2f).

[0256]

[0257] These results indicate that the PARylation of PARP1 is regulated by interaction with FOXL2, and as a result, the DNA binding activity of PARP1 is inhibited.

[0258]

[0259] Example 2-3. Confirmation of the inhibitory activity of FOX protein binding to PARP1 and DNA through the regulation of PARP1 PARylation.

[0260] To extend the experimental results for FOXL2 to the FOX protein group, the effect of FOX protein on the PARylation of PARP1 was analyzed.

[0261] As a result, it was confirmed that PARP1 is PARylated when most FOX proteins are overexpressed (Fig. 2g), which suggests that the PARylation of PARP1 can generally be induced by FOX proteins.

[0262]

[0263] Next, the effect of FOX protein on PARP1-DNA binding under normal and DNA damage conditions was confirmed through protein fraction analysis.

[0264] As a result (Fig. 2h), FOXA1, FOXC1, FOXF1, and FOXO3 inhibited PARP1's DNA binding under both normal and DNA damage conditions. In particular, under DNA damage conditions, PARP1 bound more to DNA, but FOXF1 and FOXO3 significantly inhibited this binding. Therefore, it can be seen that other FOX proteins can also inhibit PARP1's DNA binding by regulating PARP1's PARylation.

[0265]

[0266] Example 3. Confirmation of the homologous recombination repair pathway-promoting activity of FOX protein

[0267] PARP1 is involved in various DNA repair pathways, including homologous recombination (HR) repair, base excision repair (BER), and single-strand break repair (SSBR), and also partially contributes to the non-homologous end joining (NHEJ) pathway. Unlike the NHEJ pathway, which is a relatively simple mechanism involving the direct joining of damaged DNA ends, HR repair utilizes DNA sequence homology to precisely repair double-strand breaks (DSBs) and constitutes an error-free repair pathway. Since the FOX protein inhibits the DNA binding activity of PARP1, the FOX protein may affect the DNA repair pathways in which PARP1 is involved.

[0268]

[0269] Therefore, in order to elucidate the role of FOX protein in the HR repair pathway, in this example, FOX protein was overexpressed by transforming cells stably expressing pDR-GFP with an I-SceI expression plasmid.

[0270] As a result, ectopic expression of most FOX proteins promoted HR repair activity (Fig. 3a).

[0271]

[0272] In addition, the HR-promoting effect of the FOX protein was further confirmed by analyzing the localization of RAD51, which acts as a central strand exchange factor in the HR repair pathway.

[0273] As a result, RAD51 foci are known as alternative indicators of HR repair, and the accumulation of RAD51 foci increased in cells overexpressing FOX protein (Fig. 3b). This is consistent with the results of increased HR activity observed in HR repair systems.

[0274]

[0275] Example 4. Identification of the key interaction site mediating interaction with PARP1 protein: Helix3 region of FOX protein

[0276] To identify the precise binding site of the FOX protein, a mutant expression vector with a shorter truncation within the FH domain of FOXL2 was constructed. Cell viability analysis was performed to evaluate the functional effects of the FOXL2 mutant.

[0277] As a result, the decrease in cell viability induced by wild-type FOXL2 (WT-FOXL2) was lost in cells expressing ΔFH2 or ΔFH-FOXL2 (Fig. 4a), which suggests that the FH2 region, consisting of amino acids 93-117, is a key site for the function of FOXL2.

[0278]

[0279] Amino acid alignment of the forkhead box domain revealed that the Helix 3 region within the FH2 domain is highly conserved among the FOX protein family. This suggests that the Helix 3 region of FH may be a key site involved in interactions with DNA repair proteins (Fig. 4b).

[0280]

[0281] Accordingly, a Helix 3 deletion (ΔHelix3) FOXL2 expression vector was constructed (Fig. 4c), and immunoprecipitation (IP) analysis was performed to confirm the interaction with PARP1.

[0282] As a result, FOXL2 with a Helix 3 deletion mutation failed to bind to PARP1 (Fig. 4d). These results suggest that the Helix 3 region of the FOX protein is a key binding site mediating the interaction with PARP1.

[0283]

[0284] To evaluate the effect of the Helix 3 region on PARP1 function, the DNA binding affinity of PARP1 to wild-type (WT) FOX or ΔHelix3 FOX proteins was compared.

[0285] As a result, WT-FOXD3 and FOXF1 inhibited DNA binding of PARP1, but ΔHelix3-FOXD3 and ΔHelix3-FOXF1 did not exhibit this inhibitory effect (Fig. 4e). In addition, unlike the WT FOX protein, the Helix 3-deficient FOX protein failed to induce hyper-PARylation of PARP1 (Fig. 4f).

[0286]

[0287] Finally, to evaluate the effect of the Helix 3 region on HR DNA repair activity, an HR repair assay was performed after transfection with WT or ΔHelix3 FOXL2 / D3 / F1 expression plasmids.

[0288] As a result, the WT FOX protein effectively promoted HR repair, but this promoting effect was lost in the ΔHelix3 mutant (Fig. 4g).

[0289]

[0290] In summary, these results show that the FH-Helix3 region regulates DNA binding and PARylation of PARP1 and plays a key role in promoting the HR repair pathway.

[0291]

[0292] Example 5. Confirmation of HR repair regulating effect of gold nanoparticle-DNA aptamer complex loaded with 12AA-Helix3 peptide

[0293] By scanning the forkhead box domain motif of the FOX protein, 12 conserved amino acid motifs in the FOX protein were identified (Fig. 5a).

[0294] In addition, in vitro immunoprecipitation (IP) was performed using the synthesized His (6x)-tagged 12AA-Helix3 peptide 1, consisting of 12 amino acids, and recombinant PARP1 protein to detect direct interactions between the FH-Helix3 peptide and PARP1. As a result, it was found that the FH-Helix3 peptide binds to PARP1 (Fig. 5b).

[0295]

[0296] These data indicate that the conserved 12-amino acid motif site of the Helix3 region, particularly the FH domain, is the major interaction site of PARP1.

[0297]

[0298] In addition, after confirming the function of the FH-Helix3 region in vitro, a gold nanoparticle-based delivery system was used to deliver 12AA-Helix3 peptide 1 into cells.

[0299] As a result, the effective delivery of 12AA-Helix3 peptide 1 was confirmed by confocal microscopy (Fig. 5c), and the black dots in the bright-field image were the delivered nanoparticles.

[0300]

[0301] Next, an HR repair assay was performed.

[0302] As a result (Fig. 5d), 12AA-Helix3 peptide 1 was found to promote the HR repair pathway.

[0303]

[0304] Example 6. Confirmation of the dependence of somatic mutations and PARP1 expression levels on the promotion of HR DNA repair by FOX protein.

[0305] Example 6-1. Confirmation of the effect of somatic mutations in the Helix3 region on HR DNA repair activity

[0306] Since the Helix3 region of the FH domain has been identified as a key site for binding to PARP1, somatic mutations within the Helix3 region may affect DNA repair pathways. To confirm potential clinical relevance, in this example, information on cancer-related mutations within the 12-amino acid motif of 12AA-Helix3 peptide 1 of the Helix3 region was collected from cBioPortal (Fig. 6a).

[0307] As a result of calculating the mutation frequency, the 7th arginine (R) was found to be the residue with the most frequent mutations (Fig. 6b).

[0308]

[0309] In addition, several mutant FOX expression vectors were constructed, and DNA repair analysis was performed on cells expressing wild-type (WT) or mutant FOX proteins.

[0310] As a result (Fig. 6c), the HR repair promoting effect of the FOX protein with a mutation at the 7th arginine residue was reduced compared to the wild-type FOX. In addition, the HR repair promoting effect was also low in the FOX protein with mutations at the 2nd tryptophan, 5th serine, and 10th leucine residues.

[0311]

[0312] The interaction between the FOX mutant and PARP1 was further confirmed through immunoprecipitation (IP).

[0313] As a result (Fig. 6d), the FOX protein with a mutation in the 7th arginine residue did not exhibit binding affinity with PARP1. Additionally, mutants with the 5th serine (S>A) and 10th leucine (L>I) residues also failed to bind to PARP1. On the other hand, mutants with the 2nd tryptophan (W>C) and 5th serine (S>C) residues were still able to bind to PARP1.

[0314]

[0315] These results indicate that the Helix3 region, particularly the 7th arginine residue, plays a crucial role in HR DNA repair and interaction with PARP1.

[0316]

[0317] Example 6-2. Increase in PARP1 Expression in Cancer Tissue

[0318] In addition to mutations in the FOX protein, an analysis was performed to determine the clinical significance of PARP1 expression levels in cancer.

[0319]

[0320] As a result, the Affymetrix U133Plus2 microarray dataset was extracted and analyzed via the GENT2 portal, and PARP1 mRNA expression was increased in blood, brain, breast, cervical, colon, and liver cancer tissues compared to normal tissue (Figs. 6e and 6f).

[0321] In addition, analysis of RNA-seq data from GEPIA2 revealed that PARP1 mRNA levels were significantly increased in various cancer tissues, including cervical squamous cell carcinoma and adenocarcinoma (CESC), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), esophageal carcinoma (ESCA), head and neck squamous cell carcinoma (HNSC), hepatocellular carcinoma (LIHC), pheochromocytoma and paraganglioma (PCPG) (Figs. 6g and 6h).

[0322]

[0323] This upregulation of PARP1 expression may be associated with the development and progression of cancer, and inhibition of PARP1 in cancer cells has the potential to inhibit cancer growth. Therefore, the 12AA-Helix3 peptide has the potential to be developed as a novel and efficient PARP1 inhibitor and applied to cancer treatment.

[0324]

[0325] Example 7. Confirmation of broad antitumor effects by 12AA-Helix3 peptide

[0326] Example 7-1. Selection of 12AA-Helix3 peptide

[0327] To identify peptides with anticancer potential, peptide selection was first performed on the three types of peptides in Table 3 through cell viability analysis in HCT116 cancer cells.

[0328]

[0329] As a result (Fig. 7a), Peptide_1 significantly reduced cell viability to 0.75, Peptide_2 showed a relatively weak inhibitory effect (0.83), and Peptide_3 showed no toxicity (1.05). Peptide_1 (sequence: GWQNSIRHNLSL), derived from FOXL2 / C1 / I1 / K1 / S1, was used in subsequent experiments because it showed the best inhibitory activity.

[0330]

[0331] Example 7-2. Confirmation of excellent anticancer activity by 12AA-Helix3 peptide

[0332] In this example, considering that the 12AA-Helix3 peptide selected in Example 7-1 targets PARP1 and regulates the HR DNA repair pathway, the effects of peptide monotherapy and chemotherapy combination therapy were evaluated in BRCA mutant cell lines and BRCA wild-type cell lines by cell viability analysis. Paclitaxel and carboplatin were selected as chemotherapy drugs.

[0333]

[0334] The results were confirmed as shown in Figures 7b to 7e.

[0335] Specifically, in cells with a BRCA2 mutation (HCT116, BRCA2 insertion: p.I2675DfsTer6), Peptide_1 induced approximately 20% cell death, and apoptotic activity was significantly increased when combined with paclitaxel or carboplatin (particularly, a 69.7% decrease in survival rate compared to the control group when combined with paclitaxel) (Fig. 7b). In breast cancer cells with a BRCA1 mutation (HCC1937, BRCA1 insertion: p.Q1756PfsTer74), moderate cytotoxicity was observed with Peptide_1 alone, and antitumor activity was further increased when combined with paclitaxel or carboplatin (Fig. 7c).

[0336]

[0337] In contrast, breast cancer cells without BRCA mutations (MCF-7 and MDA-MB-231) showed a relatively low but statistically significant level of susceptibility. Treatment with Peptide_1 alone induced approximately 15% cell death in MCF-7 and MDA-MB-231 cells, and the cell death-inducing effect was further increased when combined with paclitaxel or carboplatin (Figs. 7d and 7e).

[0338]

[0339] These results show that the peptide of the present invention has broad anticancer efficacy, with enhanced antitumor activity, particularly when combined with chemotherapy agents in BRCA-mutated carcinomas.

[0340]

[0341] 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.

[0342] According to FOX protein as a PARP1 inhibitor in DNA repair, a novel interaction mechanism between FOX protein and PARP1 has been identified, revealing that FOX protein acts as a direct regulator in the DNA damage repair process. The 12AA-helix3 peptide of the present invention, composed of 12 amino acids, can improve DNA repair efficiency by promoting the homologous recombination (HR) repair pathway. Therefore, the present invention can be usefully applied to the development of novel DNA repair promoters and cancer treatment compositions based on the FOX protein-PARP1 interaction, and thus its industrial applicability is recognized.

Claims

1. A FOX (Forkhead Box) protein-derived peptide characterized by promoting the DNA homologous recombination repair pathway by inhibiting the binding of PARP1 (Poly (ADP-ribose) polymerase 1) protein to 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. 51 and SEQ ID NO.

52.

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 promotes a homologous recombination repair pathway of DNA by being characterized by one or more selected from the group consisting of the following: Interacting with the PARP1 protein to regulate PARP1's DNA recognition ability by inhibiting PARP1's binding to DNA damage sites; and Induces poly(ADP-ribosyl)ization (PARylation) of PARP1.

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 colorectal cancer, breast cancer, 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, 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. A composition for the prevention or treatment of cancer, comprising as an active ingredient any 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.

15. Use for manufacturing a preparation for the prevention or treatment of cancer, comprising as an active ingredient any 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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