Peptide inhibitor for removing senescent cells, and use thereof

Abstract

The present invention relates to: a peptide inhibitor, which targets FOXO4-p53 interactions, for removing senescent cells; and a use thereof. The peptide inhibitor according to the present invention is designed on the basis of a p53 transactivation domain (TAD) sequence, thus exhibiting strong selectivity for senescent cells, has a synthesis cost due to being short and having an L-type amino acid configuration, and has the effect of efficiently interfering with FOXO4-p53 interactions despite being short and having an L-type amino acid configuration. Therefore, the peptide inhibitor according to the present invention is expected to be widely used in various health / medical fields requiring the removal of senescent cells.

Description

Peptide inhibitors for removing senescent cells, and uses thereof

[0001] The present invention relates to peptide inhibitors targeting the FOXO4-p53 interaction for eliminating senescent cells, and uses thereof.

[0002] After adulthood, organisms lose their bodily functions and reproductive capacity over time, ultimately leading to death. This is the result of accumulated DNA damage and various stressors both internal and external to the cell. In normal cells, irreversible and severe DNA damage is typically eliminated through the apoptotic pathway. However, if senescent cells are not eliminated and remain in a state of prolonged cell cycle arrest, they secrete the senescence-associated secretory phenotype (SASP), composed of inflammatory cytokines and chemokine growth factors, into surrounding tissues. This creates an inflammatory environment and promotes cell cycle arrest, inducing senescence in surrounding tissues. Cellular senescence occurs not only in normal cells but also in cancer cells. Commonly used cancer treatments, such as chemotherapy and radiation therapy, function to eliminate cancer cells by inducing DNA damage. However, insufficient treatment can induce senescence in cancer cells instead of apoptosis. For example, the topoisomerase inhibitor doxorubicin induces DNA damage in cancer cells by interfering with topoisomerase, which unwinds supercoiled DNA. However, low concentrations of doxorubicin do not induce apoptosis in cancer cells, but rather promote cellular senescence. Senescent cancer cells induced after chemotherapy enhance the invasiveness and stemness of cancer cells, significantly contributing to cancer recurrence. Therefore, effective chemotherapy should aim to eliminate both cancer cells and senescent cancer cells, minimizing the risk of recurrence.

[0003] Various drugs are being developed to eliminate senescent cells. Because senescent cells are generally characterized by resistance to apoptosis, drugs targeting EPHB1, PI3K / AKT, and Bcl-2 / Bcl-xL, which are involved in the apoptosis-resistant pathway, have been developed. However, drugs such as navitoclax and dastinib also affect normal cells, causing side effects such as thrombocytopenia, neutropenia, and pulmonary hypertension. Recently, the forkhead box O4 (FOXO4)-p53 interaction has begun to attract attention as a target for senescent cells. FOXO4 is a forkhead box O transcription factor that mediates various signals to maintain cellular homeostasis. FOXO4 is expressed in senescent cells and promyelocytic leukemia (PML) cells and interacts with p53 to inhibit its nuclear export. Consequently, this interaction inhibits p53-induced apoptosis in senescent cells in the mitochondria. Furthermore, the FOXO4-p53 interaction contributes to the maintenance of senescent cells by activating the transcription of p21cip1, a common target gene that suppresses apoptosis. FOXO4, in particular, is a promising target for the removal of senescent cells. FOXO4 expression is low in normal cells, but its expression is significantly increased in senescent cells. Several protein-protein interaction inhibitors have been developed using this characteristic. The previously developed FOXO4-D-Retro-Inverso (DRI) inhibits the FOXO4-p53 interaction and induces selective apoptosis in senescent cells. However, FOXO4-DRI has the problem of being expensive to synthesize due to its long 46 amino acids and D-form amino acids, and it targets p53, a tumor suppressor that can cause various side effects in clinical trials. Another drug, ES2, targets FOXO4 conserved region 3 (CR3), but has significantly lower selectivity than FOXO4-DRI.

[0004] Accordingly, the present invention has been made to solve the above problems, and relates to a novel peptide inhibitor targeting the FOXO4-p53 interaction for eliminating senescent cells, and uses thereof. The peptide inhibitor of the present invention is designed based on the p53 TAD (transactivation domain) sequence, exhibits strong selectivity for senescent cells, is economical to synthesize due to its short length and composition of L-type amino acids, and has the effect of efficiently interfering with the FOXO4-p53 interaction despite its short length and composition of L-type amino acids. Therefore, the peptide inhibitor of the present invention is expected to be widely utilized in various health / medical fields where senescent cell removal is required.

[0005] The present invention has been devised to solve the above-mentioned problems in conventional technology.

[0006] To this end, as one aspect of the present invention, a modified peptide is provided in which any one of the amino acids at positions 42 to 57 from the N-terminus within the p53 transactivation domain region is substituted with alanine.

[0007] In another aspect of the present invention, a vector is provided comprising a nucleic acid molecule encoding a modified peptide in which any one of the amino acids at positions 42 to 57 from the N-terminus within the p53 transactivation domain region is substituted with alanine.

[0008] In another aspect of the present invention, a transformant is provided that is transformed with a vector comprising a nucleic acid molecule encoding a modified peptide in which any one of the amino acids at positions 42 to 57 from the N-terminus in the p53 transactivation domain region is substituted with alanine.

[0009] In another aspect of the present invention, a pharmaceutical composition for preventing or treating an aging disease is provided, which comprises as an active ingredient a modified peptide in which any one of the amino acids at positions 42 to 57 from the N-terminus in the p53 transactivation domain region is substituted with alanine.

[0010] In the present invention, preferably, the modified peptide may include SEQ ID NO: 15.

[0011] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0012] Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, various specific details, such as specific configurations, compositions, and processes, are set forth to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and configurations. In other instances, well-known processes and manufacturing techniques have not been described in specific detail so as not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the appearances of "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0013] Unless otherwise specifically defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0014] Throughout the specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise stated.

[0015] In one aspect of the present invention, the present invention provides a modified peptide in which any one of the amino acids at positions 42 to 57 from the N-terminus within the p53 transactivation domain region is substituted with alanine.

[0016] In the present invention, the peptide refers to a polymer of amino acids, in which the linkage between amino acids is formed by an amide bond or a peptide bond.

[0017] In the present invention, the modified peptide refers to a case where one or more amino acids in a natural peptide are substituted with other amino acids, deleted, inverted, or inserted through artificial manipulation. Alternatively, the modified peptide may be a fragment of a certain portion extracted from a natural peptide through artificial manipulation. The modified peptide in which any one of the amino acids at positions 42 to 57 from the N-terminus in the p53 transactivation domain region of the present invention is substituted with alanine may be represented by any one of SEQ ID NOs: 1 to 15, but is not limited thereto. Alternatively, the modified peptide in which any one of the amino acids at positions 42 to 57 from the N-terminus in the p53 transactivation domain region of the present invention is substituted with alanine may be a peptide in which any one of the amino acids at positions 42, 48, 49, 51, 56, and 57 from the N-terminus in the p53 transactivation domain region is substituted with alanine, and this may preferably be represented by any one of SEQ ID NOs: 2, 6, 7, 8, 13, 14, and 15, and most preferably may be represented by SEQ ID NO: 15, but is not limited thereto. Alternatively, the modified peptide, in which any one of the amino acids at positions 42 to 57 from the N-terminus in the p53 transactivation domain region of the present invention is substituted with alanine, may additionally include a hydrophobic sequence or a cell-penetrating peptide (CPP) sequence at the N-terminus or the C-terminus. In this case, the cell-penetrating peptide refers to a peptide composed of 3 to 30 amino acids that binds to various substances such as proteins, peptides, and DNA and allows the substances to move into cells without a special receptor.Preferably, it may be a sequence as described in Table 1 below, but is not limited to a domain that can promote cell membrane penetration or intercellular movement of the desired peptide.

[0018] Definition sequencePFVPFVYLICPPGRKKRRQRRRPPANTPRQIKIWFQNRRMKWKKVp22DAATATRGRSAASRPTERPRAPARSASRPRRPVEMTSAAVALLPAVLLALLAPAAADQNQLMPPep-1KETWWETWWTEWSQPKKKRKVPep-2KETWFETWFTEWSQPKKKRKVTranscriptional transactivator (TAT)YGRKKRRORRRArginine tailFour or more arginines linked together (e.g., RRRR, RRRRRRRRRR, etc.)

[0019] In the present invention, a modified peptide in which any one of the amino acids at positions 42 to 57 from the N-terminus in the p53 transactivation domain region additionally including a hydrophobic sequence or a CPP sequence at the N-terminus or C-terminus is substituted with alanine may preferably be represented by SEQ ID NO: 16 or 17, but is not limited thereto.

[0020] In another aspect of the present invention, the present invention provides a vector comprising a nucleic acid molecule encoding a modified peptide in which any one of the amino acids at positions 42 to 57 from the N-terminus within the p53 transactivation domain region is substituted with alanine.

[0021] The definition of modified peptide herein is as described above in the present specification.

[0022] In the present invention, the nucleic acid refers to a polymer organic substance in which nucleotides are polymerized in a long chain shape, and refers to genetic information encoding the peptide of the present invention.

[0023] In the present invention, the vector is a means for introducing into a cell to express a protein, and a known recombinant vector such as a plasmid vector, a cosmid vector, a bacteriophage vector, etc. can be used, and can be easily produced by a person skilled in the art using DNA recombination technology according to any method known in the field to which the present invention pertains. Specific examples of the vector in the present invention may be selected from the group consisting of commercially widely used pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, Ti vectors, cosmids, lambda, lambdoid, M13, Mu, p1 P22, Qμ, T-even, T2, T3, T7, etc. phages, and plant viruses, but are not limited thereto. For the purpose of the present invention, a suitable recombinant vector can be selected depending on the properties of the host cell (prokaryotic cell or prokaryotic cell).

[0024] In another aspect of the present invention, the present invention provides a transformant transformed with a vector comprising a nucleic acid molecule encoding a modified peptide in which any one of the amino acids at positions 42 to 57 from the N-terminus within the p53 transactivation domain region is substituted with alanine.

[0025] Definitions of modified peptides and vectors herein are as described above in the present specification.

[0026] In the present invention, the transformation can be performed by any method known to those skilled in the art for introducing a nucleic acid molecule into a cell, preferably a eukaryotic cell such as a mammalian cell. Such methods include, for example, electroporation, lipofection based on cationic lipids and / or liposomes, calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection, or cationic polymer-based transfection such as DEAE-dextran or polyethyleneimine. Alternatively, the transformation can be performed using a virus, for example, a lentivirus. An introduced subject whose original trait has been changed by being transformed by the above method is called a transformant. The transformant may be, but is not limited to, a virus, a bacterium, a plant cell, or an animal cell.

[0027] In another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating an aging disease, comprising as an active ingredient a modified peptide in which any one of the amino acids at positions 42 to 57 from the N-terminus in the p53 transactivation domain region is substituted with alanine.

[0028] The definition of modified peptide herein is as described above in the present specification.

[0029] In the present invention, the aging disease refers to a disease caused by the accumulation of aging, and may include, for example, cancer, diabetes, hypertension, arthritis, osteoporosis, sarcopenia, glomerulonephritis, chronic renal failure, asthma, chronic emphysema, pulmonary embolism, pneumonia, chronic allergies, cataracts, glaucoma, retinal degeneration, hearing loss, aortic disease, arteriosclerosis, heart failure, ischemic heart disease, arrhythmia, peripheral vascular disease and valvular disease, depression, delirium, emotional and behavioral disorders, senile dementia, Parkinson's disease, skin aging disease, etc., and the skin aging disease may include, but is not limited to, wrinkles, freckles, liver spots, dry skin, actinic keratosis, sagging skin, etc., and most preferably, cancer. The cancer refers to a disease characterized by uncontrolled cell growth, and refers to a cell mass called a tumor formed by such abnormal cell growth, which infiltrates surrounding tissues and, in severe cases, metastasizes to other organs of the body. Academically, it is also called a neoplasm. Cancer is an incurable chronic disease that often fails to achieve a fundamental cure even with surgery, radiation, and chemotherapy, causing suffering to patients and ultimately leading to death. There are various factors that cause cancer, but they are divided into intrinsic and extrinsic factors. The exact mechanism by which normal cells transform into cancer cells is not yet known, but it is known that a significant number of cancers are caused by external factors such as environmental factors. Intrinsic factors include genetic factors and immunological factors, while extrinsic factors include chemicals, radiation, and viruses. Genes involved in the development of cancer include oncogenes and tumor suppressor genes. Cancer occurs when the balance between these genes is disrupted by the internal or external factors described above.The cancer may preferably be breast cancer, uterine cancer, esophageal cancer, stomach cancer, brain cancer, rectal cancer, colon cancer, lung cancer, skin cancer, ovarian cancer, cervical cancer, kidney cancer, blood cancer, pancreatic cancer, prostate cancer, testicular cancer, laryngeal cancer, oral cancer, head and neck cancer, thyroid cancer, liver cancer, bladder cancer, osteosarcoma, lymphoma, melanoma, leukemia, etc., but is not limited thereto if the type of cancer is such.

[0030] In the present invention, the pharmaceutical composition refers to a composition administered for a specific purpose. For the purposes of the present invention, the pharmaceutical composition of the present invention is used for the prevention or treatment of aging diseases, specifically, and the cancer treatment may be to inhibit the proliferation of cancer or to inhibit the metastasis of cancer. The pharmaceutical composition may be formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, and aqueous suspensions, as well as topical preparations, suppositories, and sterile injectable solutions, according to conventional methods, but is not limited thereto. In addition, the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, and fragrances for oral administration, and buffers, preservatives, analgesics, solubilizers, isotonic agents, and stabilizers may be mixed and used for injections, and bases, excipients, lubricants, and preservatives may be used for topical administration. The formulation of the pharmaceutical composition of the present invention can be prepared in various ways by mixing it with the pharmaceutically acceptable carriers described above. For example, the pharmaceutical composition of the present invention may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. for oral administration, and the injection may be prepared in the form of unit dose ampoules or multiple doses. In addition, the composition may be formulated as a solution, suspension, tablet, capsule, sustained-release preparation, etc. Meanwhile, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil.Additionally, fillers, anti-coagulants, lubricants, humectants, fragrances, emulsifiers, preservatives, etc. may be included.

[0031] In the present invention, the routes of administration of the pharmaceutical composition include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal administration. Oral or parenteral administration is preferred.

[0032] In another aspect of the present invention, the present invention provides a method for treating an aging disease by administering to a subject in need of treatment for an aging disease a modified peptide in which any one of the amino acids at positions 42 to 57 from the N-terminus in the p53 transactivation domain region is substituted with alanine, or a pharmaceutical composition comprising the peptide.

[0033] Definitions of modified peptides, aging diseases, and pharmaceutical compositions herein are as described above in the present specification.

[0034] In another aspect of the present invention, the present invention provides a modified peptide in which any one of the amino acids at positions 42 to 57 from the N-terminus in the p53 transactivation domain region is substituted with alanine, or a use of a pharmaceutical composition comprising the peptide, for treating an aging disease.

[0035] Definitions of modified peptides, aging diseases, and pharmaceutical compositions herein are as described above in the present specification.

[0036] Hereinafter, the present invention will be described in detail based on examples.

[0037] The peptide inhibitor for eliminating senescent cells of the present invention is designed based on the p53 TAD (transactivation domain) sequence, exhibits strong selectivity for senescent cells, is economical to synthesize due to its short length and L-form amino acid composition, and is effective in efficiently interfering with the FOXO4-p53 interaction despite its short length and L-form amino acid composition. Therefore, the peptide inhibitor of the present invention is expected to be widely utilized in various health / medical fields where senescent cell removal is required.

[0038] Figure 1 shows the domain structures of the FOXO4 and p53 proteins. The primary sequence of the p53 TAD is shown in Figure 1. Arrows connect domains known to interact. The peptide sequence of the p53 TAD PEP is shown in red. The abbreviations in Figure 1 are as follows:

[0039] TAD: transactivation domain;

[0040] PRD: a proline-rich domain;

[0041] DBD: DNA binding domain;

[0042] TD: tetramerization domain;

[0043] NRD: negative regulatory domain;

[0044] CR1 and CR3: transactivation domains;

[0045] FHD: forkhead domain;

[0046] NLS: nuclear localization sequence;

[0047] NES: nuclear export sequence.

[0048] Figure 2 shows the observation of the interaction between FOXO4 FHD and p53 TAD. In Figure 2, the intensity ratio (I) of the FHD signal to the residue number for FHD complexed with TAD modified with S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate (MTSL) at S46C ox / I red ) plot is displayed. Figure 2a shows the I according to the area ox / I red and each error bar, with an asterisk (*) indicating the expanded region due to the influence of MTSL. Figure 2b is an experimental model of paramagnetic relaxation enhancement (PRE) of FOXO4 FHD (PDB ID: 1E17), I ox / I red Less than 0.2 is red, I ox / I red Hydrophobic residues with a value less than 0.2 are shown in blue. Figure 2c shows the hetNOE values ​​of the p53 TAD apo and the p53 TAD-FOXO4 FHD complex. Figure 2d shows the difference in hetNOE values ​​between the p53 TAD-FOXO4 FHD complex and the p53 TAD apo.

[0049] Figure 3 shows the results of the verification of the inhibitory ability of p53 TAD PEP on the binding of FOXO4 FHD and p53 TAD. Figure 3a shows the results of a fluorescence polarization anisotropy experiment performed using a fluorescently labeled peptide by measuring the FPA value of fluorescein isothiocyanate (FITC)-labeled p53 TAD PEP as the concentration of FHD increases. Figure 3b 1 H- 15 N heteronuclear single quantum coherence (HSQC) NMR spectra of p53 TAD incubated with increasing stoichiometric equivalents of PEP (0, 100, and 200 μM, respectively). 15 N-labeled FOXO FHD- 14N was obtained for the p53 TAD complex.

[0050] Figure 4 shows the results of a competitive FPA inhibition assay of an alanine-substituted peptide. Figure 4 shows that increasing the concentration of the alanine-substituted peptide inhibits binding of FITC-labeled p53 TAD to FOXO4 FHD.

[0051] Figure 5 shows the results of a competitive FPA inhibition assay of peptide candidates with substituted residues. Figure 5a shows that FITC-labeled p53 TAD PEP binding to FOXO4 FHD was inhibited by increasing the concentration of WT and sequence-optimized peptide candidates (CAND). Figure 5b shows the IC at pH 7.4 for WT and CAND. 50 Indicates the value and net charge value.

[0052] Figure 6 shows the validation results of candidate peptides in a doxorubicin-induced senescence model. Figure 6a shows images of dividing and senescent A375 cells stained for senescence-associated beta-galactosidase (SA-b-gal). Figure 6b shows the viability assay results of senescent and control A375 cells cultured with increasing doses of CPP-CAND and FOXO4-DRI (μM). Figure 6c shows the selectivity index (SI) of FOXO4-DRI and CPP-CAND. SI 50 , and SI 75 IC obtained from nonlinear regression analysis for both groups 50 , IC 75 Reflects changes in value.

[0053] Figure 7 shows the viability analysis results of senescent and control A375 cells cultured with increasing doses of p53 TAD PEP and CAND (μM) as additional validation results of candidate peptides in a doxorubicin-induced senescence model.

[0054] Figure 8 shows that inhibition of FOXO4-p53 binding in CPP-CAND induces apoptosis via a caspase-dependent pathway. Figure 8a shows foci images of FOXO4 and TP53BP1 (53BP1) in mitotic and doxorubicin-induced senescent A375 cells, with the nuclear outlines indicated by yellow lines. Figure 8b is a diagram showing the distribution of FOXO4 and p53bp1 foci per nucleus in A375 cells. Figure 8c shows FOXO4 foci images in senescent A375 cells treated with 30 μM CPP-CAND for 24 hours. Figure 8d is a bar graph of FOXO4 foci / nucleus in A375 cells. Significance was determined relative to the DMSO control group, and **** p-value < 0.0001. Figure 8e shows the results of caspase 3 / 7 activity in A375 cells treated with 20 μM CPP-CAND for 24 hours and in senescent A375 cells. The percentage of green fluorescent cells per cell is indicated by the panel figures. The scale bar in Figures 8a and 8c is 10 μm, and the scale bar in Figure 8e is 100 μm.

[0055] Figure 9 shows the verification results of induction of caspase 3 / 7 activity by DMSO as a control.

[0056] We treated dividing and senescent cells with 20 μM CPP-CAND and evaluated caspase 3 / 7 activity to confirm caspase-dependent apoptosis. As a result, despite treating each cell type with the same concentration of CPP-CAND, caspase 3 / 7 activity was observed in 44% of senescent cells, whereas almost no activity was observed in dividing cells. Caspase 3 / 7 activity was also barely detectable in the DMSO-treated control group of dividing and senescent cells. This suggests that caspase-dependent apoptosis was specifically induced in senescent cells. In summary, CPP-CAND inhibits the FOXO4-p53 interaction in the nucleus, enabling the nuclear export of p53, and selectively inducing apoptosis in senescent cells.

[0057] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0058] Throughout the specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise stated.

[0059] [Research Methods]

[0060] 1. Sample preparation

[0061] FOXO4 FHD(95-195) was subcloned into pET His6 Tobacco Etch Virus (TEV) LIC cloning vector (2B-T, obtained from Scott Gradia, Addgene plasmid #29666) and transformed into BL21(DE3). p53 TAD(13-61) was subcloned into pET His6 glutathione-S-transferase (GST) TEV LIC cloning vector (2G-T, obtained from Scotta Gradia, Addgene plasmid #29707) and transformed into BL21(DE3) pLysS. Cells were grown to an OD600 of 0.7–0.8 in lysogenic medium (LPS solution LB-05) at 37°C, then 0.5 mM isopropyl-1-thio-β-D-galactopyranoside (LPS solution IPTG025) was added and cultured overnight at 18°C. The overexpressed protein was purified on a Ni-NTA column (Cytiva) using an elution buffer containing 50 mM NaH2PO4, 300 mM NaCl, and 300 mM imidazole. His6- and GST-tagged protein samples were repeatedly purified on the Ni-NTA column after digestion with TEV protease. For further purification of FOXO4 FHD and p53 TAD, gel filtration chromatography was performed using Hi-Load 16 / 600 superdex 200 pg and 75 pg (Cytiva) on AKTA pure and AKTA prime systems (Cytiva), respectively, and the columns were pre-equilibrated with a buffer containing 20 mM HEPES (pH 7.0) and 150 mM NaCl. For NMR studies, cells were 15 Grown in M9 medium containing NH4Cl 15 N-labeled proteins were produced. All peptides, including FITC-labeled p53 TAD(42-57), were manufactured at 95% purity by Dandicure Co., Ltd. (Ochang, Korea).

[0062] 2. NMR spectroscopy

[0063] NMR experiments were performed using a Bruker 900 MHz NMR spectrometer equipped with a cryogenic probe (KBSI, Ochang, Korea) and a Bruker 600 MHz NMR spectrometer equipped with a cryogenic probe (GIST, Gwangju). All experiments were performed at 25°C in a buffer containing 20 mM HEPES (pH 7.0), 150 mM NaCl, and 1 mM DTT. The amide nitrogens and protons of FOXO4 FHD and p53 TAD were previously assigned. NMR experimental data were processed with Topspin (Bruker) and analyzed using the POKY script. 1 H- 15 N heteronuclear Overhauser effect (hetNOE) measurements were recorded using a Bruker 900 MHz NMR spectrometer with a cryogenic probe (KBSI, Ochang, Korea), and proton ( 1 The saturation period of H) was set to 5 s, and the hetNOE values ​​were derived by comparing the peak heights between the spectrum sets. The PRE experiment was performed using a Bruker 600 MHz NMR spectrometer equipped with a cryogenic probe (GIST, Gwangju), and the paramagnetic probe MTSL was labeled at cysteine ​​residue 46 of p53 TAD. 300 μM of MTSL-labeled p53 TAD was added to 150 μM of 15 N-labeled FOXO4 FHD was added and the experiment was performed in a buffer containing 20 mM HEPES (pH 7.0) and 150 mM NaCl. Peak intensities were measured and the reduced state (I red , diamagnetic, with ascorbate) on the strength of oxidation state (I ox , paramagnetic, without ascorbate) were plotted. I ox / I red The standard error for the values ​​was calculated by propagating the signal-to-noise ratio of each spectrum by substituting it into Equation 1 below.

[0064] [Formula 1]

[0065]

[0066] In the above equation 1, δI ox / I red is I ox / I red is the calculated error of the ratio, δI ox is the noise level of the oxidized spectrum, and δI red is the noise level of the reduced spectrum.

[0067] 1 H- 15 For N HSQC titration experiments, 14 With N-FOXO4 FHD 15 The concentration of p53 TAD PEP was increased while maintaining the same ratio of N-labeled p53 TAD.

[0068] 3. Fluorescence polarization anisotropy analysis

[0069] Fluorescence polarization analysis was performed at 25°C using a Cytation 5 cell imaging multimode reader (BioTek) equipped with a green FP filter using 96-well microplates (SPL 30496). Each assay was repeated three times, and the results were averaged. Experiments were performed in a buffer containing 20 mM HEPES (pH 7.0), 150 mM NaCl, 1 mM DTT, and 5% DMSO, and the dissociation constants (K) of FHD and p53 TAD PEP d ) values ​​were determined by mixing 10 nM FITC-labeled p53 TAD PEP with increasing concentrations of FHD. The plates were then equilibrated at room temperature for 2 h. FPA values ​​were determined using excitation at 485 nm and emission at 528 nm and calculated by substituting the following equation (2).

[0070] [Formula 2]

[0071]

[0072] In competitive inhibition experiments, increasing concentrations of inhibitor peptides were titrated with a mixture of 120 nM FHD and 10 nM FITC-p53 TAD PEP, and IC, which represents the half maximal concentration of a substance that inhibits a specific biological function in vitro, was determined. 50 The values ​​were determined through analysis.

[0073] 4. Cell culture

[0074] Human malignant melanoma (A375) cells were obtained from the American Type Culture Collection (ATCC, CRL-1619IG-2) and used within passage 10 for all experiments. Cells were cultured in DMEM (Welgene, LM001-05) supplemented with 10% FBS (Gibco 16000-044) and 1% penicillin / streptomycin (P / S, Gibco 15140-122) at 5% CO2 and 37°C in a humidified atmosphere. TrypLE™ enzyme (Express Enzyme, 1X) containing phenol red (Gibco 12605010) was used to detach adherent cells, and the cell lines were regularly tested for mycoplasma contamination using a mycoplasma detection kit (MycoStrip™, InvivoGen, rep-mys-10). Cell senescence was induced using doxorubicin. A375 cells were cultured at 30% to 50% confluency and treated with complete growth medium containing 0.1 μM doxorubicin (Sigma D1515) for 2 days. Cells were then allowed to recover in fresh growth medium for 4 days before further experiments. Senescence β-galactosidase assays were performed using a senescence β-galactosidase staining kit (Cell Signaling Technology, 9860) according to the manufacturer's instructions, and stained cells were imaged using a ZEISS Primovert inverted microscope (Carl Zeiss Microscopy).

[0075] 5. Cell viability

[0076] Cells were seeded in triplicate in 96-well plates, containing approximately 12,000 senescent cells and 6,000 non-senescent cells per well. After 1 day, the plates were treated with various concentrations of peptides and replenished daily for 2 days. Cell proliferation was quantified using the Promega CellTiter 96™ AQueous One Solution Cell Proliferation Assay (MTS) according to the manufacturer's instructions (Promega G3580) and measured 3 hours later using a microplate reader (Tecan).

[0077] 6. Immunofluorescence

[0078] For immunofluorescence analysis, cells (typically 15,000) were seeded in 8-well plates (SPL 30408). After each experiment, the cells were washed once with PBS (LPS solution CBP007B) and fixed with 10% formalin solution (Sigma HT501128-4L) for 10 min at room temperature. The cells were then washed with PBS and permeabilized with PBS containing 0.1% Triton X-100 (Daejung 8566-4405) for 10 min at room temperature. The cells were then washed again with PBS and treated with PBST (PBS + 0.05% Tween20, LPS Solution CBP007T) containing 1% bovine serum albumin (BSA, Sigma A9418) and 22.52 mg / mL glycine for 1 h. Primary antibodies FOXO4 (Cell Signaling Technology 9472) and TP53BP1 (BD Biosciences 612523) were diluted in PBST containing 1% BSA and treated overnight at 4°C. After primary antibody incubation, cells were washed with PBST and treated with secondary antibodies (Goat Anti-Rabbit IgG H&L Alexa Fluor 488, Abcam ab150077; Goat Anti-Mouse IgG H&L Alexa Fluor 594, Abcam ab150116) diluted in PBST containing 1% BSA for 1 h at room temperature. After secondary antibody incubation, cells were washed again with PBST and mounted using Fluoroshield Mounting medium containing DAPI (Abcam ab104139). For experiments involving caspase-3 / 7 staining, CellEvent Caspase-3 / 7 green detection reagent (Thermo Scientific C10723) and CPP-CAND were added to live cells 24 h prior to imaging using an Eclipse Ti-U inverted microscope (NIKON).

[0079] [Research Results]

[0080] 1. The p53 TAD2 domain plays a critical role in the interaction with FOXO4 FHD.

[0081] The interaction between FOXO4 and p53 in the nucleus has been reported to promote cellular senescence. This interaction is mediated by a double binding between the FOXO4 forkhead domain (FHD) and the p53 TAD and the FOXO4 CR3 to the p53 DNA-binding domain (DBD) (Fig. 1). The inventors of the present invention performed a pre-exposure recombinant (PRE) experiment to further investigate the interaction between FOXO4 FHD and p53 TAD. For the PRE experiment, we 15 We used N-labeled FOXO4 FHD and p53 TAD, which were MTSL-labeled at cysteine ​​46. MTSL induces peak broadening near the labeled residue, allowing us to identify which region of FOXO4 FHD is close to the p53 TAD. Our results showed a significant decrease (<0.2) in binding intensity observed at the N terminus and helices 3 and 4 of FOXO4 FHD ( Figures 2A and 2B ). It is noteworthy that approximately half of the residues showing significant intensity decreases are hydrophobic amino acids. This finding is consistent with previous investigations of the interaction between FOXO4 FHD and p53 TAD using chemical shift perturbation (CSP) and suggests the importance of hydrophobic interactions in the interaction between FOXO4 FHD and p53 TAD.

[0082] Previous studies have investigated the interaction between p53 TAD and FOXO4 FHD using CSP analysis. 15After titration of FOXO4 FHD against N-labeled p53 TAD, significant perturbations of more than 2 sigma were observed in many residues within the p53 TAD2 region. This indicates the importance of the p53 TAD2 region for FOXO4 FHD binding. CSP analysis is a technique used to observe interaction sites based on changes in the surrounding chemical environment, which means that even regions not directly involved in the interaction may experience changes in the chemical environment if they are adjacent to regions important for the interaction. Therefore, by observing protein-protein interactions through hetNOE, which provides information on protein flexibility, we can identify key residues directly involved in the interaction. To design peptide templates, we investigated the interaction between FOXO4 FHD and p53 TAD using hetNOE analysis to identify important residues involved in binding within the p53 TAD2 region. Initially, we used hetNOE analysis to determine the binding sites in the apo state. 15 A hetNOE experiment was performed on the N-labeled p53 TAD. In general, a hetNOE value closer to 1 indicates a stronger structure in the corresponding region. The results showed that regions with secondary structure generally exhibited high values ​​(over 0.6), while most residues in the p53 TAD apo state exhibited low values ​​(below 0.6) (Fig. 2c). This result indicates a disordered protein lacking secondary structure, suggesting that the p53 TAD exists in a flexible conformation without secondary structure. Subsequently, 15We performed hetNOE experiments on the complex formed by N-labeled p53 TAD and unlabeled FOXO4 FHD. In contrast to the p53 TAD apo results, the hetNOE values ​​of most residues increased compared to those observed in the apo state of p53 TAD (Fig. 2c). This indicates that p53 TAD and FOXO4 FHD are bound to each other. In particular, residues corresponding to TAD2 and the C-terminus were observed to have hetNOE values ​​increased by more than 0.2 (Fig. 2d). This result is consistent with previous studies and supports the notion that the TAD2 region plays a key role in the interaction with FOXO4 FHD. Conversely, residues within the range of 45-52 did not show a significant increase in hetNOE values, except for the residue at position 49. Taken together, these results suggest that the anterior and posterior regions of TAD2 and the C-terminal region of p53 TAD appear to be important for the interaction with FOXO4 FHD.

[0083] 2. Hydrophobic residues of p53 TAD2 are important for interaction with FOXO4 FHD.

[0084] Based on the importance of the p53 TAD2 region and the C terminus in interacting with FOXO4 FHD, we aimed to develop an inhibitor targeting the FOXO4-p53 interaction. We designed a prototype peptide inhibitor, p53 TAD PEP, consisting of 16 amino acids corresponding to residues 42-57 of the p53 TAD sequence. We selected the peptide sequence by considering hetNOE and CSP analyses, which included regions showing significant binding or a significant increase in hetNOE values. To confirm the binding function of p53 TAD PEP to FOXO4 FHD, we performed FPA assay. The FPA assay was performed by increasing the concentration of unlabeled FOXO4 FHD and keeping the concentration of FITC-labeled p53 TAD PEP constant. The test results showed that p53 TAD PEP had a K of 0.11 μM. d It effectively bound to FOXO4 FHD (Fig. 3a). In addition, the present inventors confirmed whether p53 TAD PEP effectively inhibits the interaction between FOXO4 FHD and p53 TAD. 1 H- 15 N HSQC NMR experiments were performed. Specifically, as the concentration of p53 TAD PEP increased, 15 Titration experiments were performed on the complex of N-labeled p53 TAD(13-61) and unlabeled FOXO4 FHD. As a result, the addition of p53 TAD PEP resulted in the dissociation of p53 TAD(13-61) from FOXO4 FHD. 15We observed that the N-p53 TAD-FOXO4 FHD complex chemically shifted to the unbound p53 TAD state (Fig. 3b). This indicates that p53 TAD PEP effectively disrupts the interaction between p53 TAD and FOXO4 FHD, suggesting that p53 TAD PEP may effectively function as a peptide inhibitor targeting the FOXO4-p53 interaction.

[0085] Furthermore, to improve the p53 TAD PEP sequence, it is necessary to identify residues important for FOXO4 FHD binding. The present inventors identified residues important for their binding by testing the replacement of acidic residues and residues within the p53 TAD2 region known to be important for FOXO4 FHD binding with alanine residues in previous studies (Table 2). Table 2 below shows the IC of wild-type (WT) and alanine-substituted peptides. 50 Indicates a value.

[0086] Sequence number (No.) Peptide sequence IC 50(μM) SEQ ID NO: 1WTDLMLSPDDIEQWFTED7.1 ± 0.5 SEQ ID NO: 2D42AALMLSPDDIEQWFTED1.4 ± 0.1 SEQ ID NO: 3L43ADAMLSPDDIEQWFTEDN / D SEQ ID NO: 4M44ADLALSPDDIEQWFTED9.5 ± 4.8 SEQ ID NO: 5L45ADLMASPDDIEQWFTED5.7 ± 3.2 SEQ ID NO: 6D48ADLMLSPADIEQWFTED1.8 ± 0.2 SEQ ID NO: 7D49ADLMLSPDAIEQWFTED1.1 ± 0.2 SEQ ID NO: 8E51ADLMLSPDDIAQWFTED0.9 ± 0.1 SEQ ID NO: 9Q52ADLMLSPDDIEAWFTED1.3 ± 0.3 SEQ ID NO: 10W53ADLMLSPDDIEQAFTEDN / D SEQ ID NO: 11F54ADLMLSPDDIEQWATEDN / DSEQ ID NO: 12T55ADLMLSPDDIEQWFAED1.3 ± 0.5SEQ ID NO: 13E56ADLMLSPDDIEQWFTAD1.6 ± 0.3SEQ ID NO: 14D57ADLMLSPDDIEQWFTEA2.6 ± 0.4

[0087] Next, the inhibitory activity of alanine-substituted p53 TAD PEP was evaluated by titrating the concentration of FITC-labeled p53 TAD PEP upon binding to FOXO4 FHD (Fig. 4). The results showed that replacement of residues L43, W53, and F54 with alanine significantly impaired the inhibitory activity, indicating the importance of these residues for FOXO4 FHD binding (Table 2). Moreover, a slight decrease in the inhibitory activity was observed when residue M44 was replaced with alanine. This suggests that hydrophobic residues of the p53 TAD significantly contribute to FOXO4 FHD binding. Interestingly, alanine substitutions of sequences other than those mentioned did not decrease the inhibitory activity but rather enhanced it, suggesting that these residues are not critical for FOXO4 FHD binding and may represent candidates for substitution to enhance the inhibitory efficacy.

[0088] 3. Peptides with reduced acidity were designed for cell permeability.

[0089] Based on alanine screening, the inventors of the present invention aimed to optimize the sequence of p53 TAD PEP for enhanced cell permeability. The net charge of p53 TAD PEP at pH 7.4 is -6.2. Considering that cell membranes are negatively charged, peptide drugs are known to efficiently penetrate cell membranes when the overall charge is neutral or positive. Referring to the results in Table 2 above, the negatively charged residues of p53 TAD PEP do not appear to be critical for FOXO4 FHD binding. Therefore, the inventors designed a peptide by substituting negatively charged residues D42, D48, D49, E51, E56, and D57 within p53 TAD PEP with alanine to increase binding affinity for FOXO4 FHD and enhance membrane penetration. The inhibitory potency of the designed peptides was verified using an FPA competition assay, demonstrating a sustained effect in inhibiting the interaction between FOXO4 and p53 TAD (Fig. 5a). Consequently, the inhibitory potency was enhanced approximately threefold compared to the p53 TAD PEP (Fig. 5b). Therefore, the inventors were evaluated to have successfully designed a peptide (CAND; SEQ ID NO: 15) optimized for cellular applications.

[0090] 4. CPP-CAND exhibits selective senolytic activity.

[0091] Doxorubicin is known to induce cellular senescence in the A375 melanoma cell line. The present inventors induced senescence in A375 cells using doxorubicin and identified senescent cells through SA-b-gal staining (Fig. 6a). The cells were treated with the peptide of the present invention (CAND) and cell viability was measured using the MTS assay, and the IC 50The values ​​were determined. As a result, both p53 TAD PEP and CAND showed limited efficacy in inducing apoptosis in both dividing and senescent cells (Fig. 7). We hypothesized that the ineffectiveness of the peptides in inducing apoptosis in both cell types may be due to inadequate cellular uptake. To address this issue, we attempted to enhance cellular uptake of the peptides using various techniques, such as incorporating CPP sequences or lipid conjugation. Because CAND has a net charge of -0.2, making it overall neutral, it is difficult for CAND to effectively translocate to the nucleus on its own. Therefore, to enhance intracellular transport, we designed two CPP sequence-linked peptides to enhance cellular uptake and promote nuclear localization. Ultimately, one peptide incorporated a hydrophobic sequence at its N-terminus (PFVYLI-CAND; SEQ ID NO: 16), and the other peptide incorporated a positively charged hydrophilic HIV-TAT sequence at its N-terminus (CPP-CAND; SEQ ID NO: 17). Their testing revealed that the PFVYLI-CAND peptide containing the hydrophobic CPP sequence did not induce cell death in either cell type (Fig. 7). In contrast, CPP-CAND incorporating the positively charged HIV-TAT sequence demonstrated senolytic activity (Fig. 6b). Compared with the conventional drug FOXO4-DRI, SI 75 Despite its shorter length, it showed significant superiority with approximately 1.2 times higher selectivity, and SI 50 exhibited similar efficacy (Fig. 6c). Additionally, treatment of cells with a peptide containing only the HIV-TAT sequence and a peptide in which the HIV-TAT sequence was introduced at the N-terminus of the p53 TAD PEP resulted in minimal cell death (Fig. 7). These results suggest that the peptide developed in this invention exhibits potent intracellular functionality as a senolytic drug with selectivity for senescent cells.

[0092] 5. CPP-CAND inhibits FOXO4-p53 binding and induces apoptosis through a caspase-dependent pathway.

[0093] FOXO4 is known to be progressively concentrated in euchromatin foci after senescence induction and to be retained in the nucleus of senescent cells through interaction with p53, thereby contributing to the maintenance of the senescent state. Previously developed drugs, FOXO4-DRI and ES2 peptides, selectively induce apoptosis in senescent cells by disrupting the FOXO4-p53 interaction in the nucleus of these cells. Since the CPP-CAND of the present invention is designed to disrupt the FOXO4-p53 interaction, the disruption of FOXO4-p53bp1 foci in the nucleus of senescent cells after CPP-CAND treatment was investigated.

[0094] Immunofluorescence results showed a significant increase in FOXO4 and p53bp1 foci in the nuclei of senescent cells compared to those of dividing cells (Figures 8a and 8b). We also observed frequent overlap between FOXO4 and p53bp1 foci, indicating interaction and colocalization between FOXO4 and p53 in the nuclei of senescent cells (Figure 8a), suggesting effective induction of FOXO4-p53-mediated senescence. After CPP-CAND treatment, a significant decrease in the number of FOXO4 foci in the nuclei of senescent cells was observed, approximately 7.1-fold. This suggests successful inhibition of the FOXO4-p53 interaction within the nucleus by CPP-CAND (Figures 8c and 8d). Furthermore, we treated dividing and senescent cells with 20 μM CPP-CAND and assessed caspase 3 / 7 activity to confirm caspase-dependent apoptosis. Our results showed that, despite treating each cell type with the same concentration of CPP-CAND, caspase 3 / 7 activity was observed in 44% of senescent cells, whereas almost no activity was observed in dividing cells (Fig. 8e). Caspase 3 / 7 activity was also barely detectable in the DMSO-treated control group of dividing and senescent cells (Fig. 9). This suggests that caspase-dependent apoptosis was specifically induced in senescent cells. In summary, CPP-CAND inhibits the FOXO4-p53 interaction in the nucleus, enabling the nuclear export of p53, and selectively inducing apoptosis in senescent cells.

[0095] The sequences of the peptide inhibitors of the present invention are shown in Table 3 below.

[0096] Sequence number (No.) Peptide sequence Sequence number 15 CANDALMLSPAAIAQWFTAA Sequence number 16 PFVYLI-CANDPFVYLIALMLSPAAIAQWFTAA Sequence number 17 CPP-CANDGRKKRRQRRRPPALMLSPAAIAQWFTAA

[0097] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0098] It is expected that the peptide inhibitor of the present invention will be widely utilized in various health / medical fields where removal of senescent cells is required.

Claims

1. A modified peptide in which any one of the amino acids at positions 42 to 57 from the N-terminus within the p53 transactivation domain region is substituted with alanine.

2. In paragraph 1, A modified peptide, wherein the peptide is represented by any one of SEQ ID NOs: 1 to 15.

3. In paragraph 1, The above peptide is a modified peptide in which any one of the amino acids at positions 42, 48, 49, 51, 56, and 57 from the N-terminus within the p53 transactivation domain region is substituted with alanine.

4. In paragraph 3, A modified peptide, wherein the peptide is represented by any one of SEQ ID NOs: 2, 6, 7, 8, 13, 14, and 15.

5. In paragraph 3, The above peptide is a modified peptide in which amino acids at positions 42, 48, 49, 51, 56, and 57 from the N-terminus within the p53 transactivation domain region are substituted with alanine.

6. In paragraph 5, The above peptide is a modified peptide represented by SEQ ID NO:

15.

7. In paragraph 1, A modified peptide, wherein the peptide further comprises a hydrophobic sequence or a cell-penetrating peptide (CPP) sequence at the N-terminus or the C-terminus.

8. In paragraph 7, The above peptide is a modified peptide represented by SEQ ID NO: 16 or 17.

9. A vector comprising a nucleic acid molecule encoding a first modified peptide.

10. A transformant transformed with the vector of Article 9.

11. A pharmaceutical composition for the prevention or treatment of aging diseases, comprising the peptide of paragraph 1 as an active ingredient.

12. In paragraph 11, A pharmaceutical composition, wherein the above aging disease is any one selected from the group consisting of cancer, diabetes, hypertension, arthritis, osteoporosis, sarcopenia, glomerulonephritis, chronic renal failure, asthma, chronic emphysema, pulmonary embolism, pneumonia, chronic allergies, cataracts, glaucoma, retinal degeneration, hearing loss, aortic disease, arteriosclerosis, heart failure, ischemic heart disease, arrhythmia, peripheral vascular disease and valvular disease, depression, delirium, emotional and behavioral disorders, senile dementia, Parkinson's disease, wrinkles, freckles, liver spots, dry skin, actinic keratosis, and sagging skin.

13. A method for treating an aging disease by administering the peptide of claim 1 or a pharmaceutical composition containing the peptide of claim 1 to a subject in need of treatment for an aging disease.

14. In paragraph 13, A method wherein the above aging disease is any one selected from the group consisting of cancer, diabetes, hypertension, arthritis, osteoporosis, sarcopenia, glomerulonephritis, chronic renal failure, asthma, chronic emphysema, pulmonary embolism, pneumonia, chronic allergies, cataracts, glaucoma, retinal degeneration, hearing loss, aortic disease, arteriosclerosis, heart failure, ischemic heart disease, arrhythmia, peripheral vascular disease and valvular disease, depression, delirium, emotional and behavioral disorders, senile dementia, Parkinson's disease, wrinkles, freckles, liver spots, dry skin, actinic keratosis, and sagging skin.

15. Use of the peptide of claim 1 or a pharmaceutical composition comprising the peptide of claim 1 for treating aging diseases.

16. In paragraph 15, The use of the above aging disease is any one selected from the group consisting of cancer, diabetes, hypertension, arthritis, osteoporosis, sarcopenia, glomerulonephritis, chronic renal failure, asthma, chronic emphysema, pulmonary embolism, pneumonia, chronic allergies, cataracts, glaucoma, retinal degeneration, hearing loss, aortic disease, arteriosclerosis, heart failure, ischemic heart disease, arrhythmia, peripheral vascular disease and valvular disease, depression, delirium, emotional and behavioral disorders, senile dementia, Parkinson's disease, wrinkles, freckles, liver spots, dry skin, actinic keratosis, and sagging skin.

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