Improved anti-aging compounds and their use in cancer treatment

Abstract

This invention provides compounds and methods for selectively inducing apoptosis in senescent cells. [Solution] The present invention relates to improved compounds used in the treatment of diseases or conditions in which the removal of senescent cells, scar cells, and / or cancerous cells is beneficial, such as cancer. The present invention also relates to a method for treating individuals who are suffering from or suspected of suffering from a disease or condition in which the removal of senescent cells, scar cells, and / or cancerous cells is beneficial.

Description

[Technical Field]

[0001] The present invention relates to improved compounds used in the treatment of diseases or conditions in which the removal of senescent cells, scarred cells, and / or cancerous cells is beneficial, such as cancer. The present invention also relates to methods for treating individuals who are suffering from or suspected of suffering from diseases or conditions in which the removal of senescent cells, scarred cells, and / or cancerous cells is beneficial. [Background technology]

[0002] Apoptosis (programmed cell death) results in the killing of damaged cells. Dying cells in the final stage of apoptosis present phagocytic molecules, which serve as a signal for these cells to be phagocytosed by cells with appropriate receptors, such as macrophages.

[0003] Senescent cells are thought to impair tissue function, and their genetic elimination may slow the onset of age-related characteristics. Senescent cells are permanently removed from the cell cycle and generally develop a persistent pro-inflammatory trait called senescence-associated secretory phenotype (SASP). SASP affects the cellular microenvironment, which may be beneficial in the early stages of life or in the acute phase of wound healing. However, unlike apoptotic cells that are permanently eliminated, senescent cells can proliferate over long periods and accumulate with age. Persistent senescent cells, due to their low but chronic SASP, are thought to accelerate the onset of aging and age-related diseases.

[0004] Senescent cells have been shown to accumulate with age and in areas of age-related pathological conditions. Furthermore, senescent cells may acquire mutations that allow them to re-enter the proliferative phase. Therefore, benign aging lesions retain the potential to become malignant. In fact, aging has been associated with a large number of (age-related) pathological conditions, and conversely, the genetic elimination of senescent cells may slow down the characteristics of aging.

[0005] In fact, recent studies have shown that genetically eliminating senescent cells in an aging-accelerated mouse model can significantly improve fitness and reduce aging parameters. These mice showed reduced signs of aging, measured by kyphosis (excessive bone curvature), muscle strength, fat deposition, and cataracts. This provides further evidence of a causal relationship between cellular senescence and SASP (Synthetic Aging Syndrome) and age-related traits and cancer (Non-Patent Literature 1). This proof-of-concept evidence was obtained through a genetic approach with limited therapeutic applicability.

[0006] Research has consistently revealed that FOXO (Forkhead Box O) transcription factors are important determinants of aging and longevity. FOXO proteins constitute a subfamily of transcription factors that act as crucial downstream regulators of longevity in insulin and insulin-like growth factor signaling, and these are conserved from Caenorhabditis elegans to mammals. Invertebrate genomes have one FOXO gene, while mammals have four: FOXO1, FOXO3, FOXO4, and FOXO6. In mammals, this subfamily is involved in a wide range of critical cellular processes that regulate stress tolerance, metabolism, cell cycle arrest, and apoptosis. Their roles in determining longevity are complex and not yet fully understood.

[0007] In Non-Patent Literature 2, Bourgeois and Madl identified both foxo and p53 as central players in aging over the past decade, disclosing that their misregulation is associated with numerous diseases, including cancer. However, many of the underlying molecular mechanisms, including the regulation of aging by foxo and p53, remain a mystery. It appears that foxo and p53 share multiple activities, including their central roles in regulating cellular senescence. In recent developments, Bourgeois and Madl have focused on the relationship between foxo and p53, particularly the foxo4-p53 axis and the foxo4 / p53 role in cellular senescence. The potential of strategies targeting the foxo4-p53 interaction to regulate cellular senescence is being explored as a drug target for the treatment and morbidity of age-related diseases.

[0008] Patent Document 1 relates to the use of agonists that inhibit Jun kinase and / or FOXO4 in the treatment of cancer and / or removal of senescent cells in an organism. In some embodiments, the agonist is a small molecule such as SP600125. In some embodiments, the agonist is a small molecule such as AS601245. In some embodiments, the agonist is a peptide as disclosed.

[0009] Patent Document 2 relates to activators that inhibit FOXO4 function, and their use in the treatment of cancer and / or removal of senescent cells in an organism. In some embodiments, the FOXO4-inhibiting activator is a peptide that inhibits FOXO4 function in cells, and the peptide comprises an amino acid sequence having at least 80% identity with a FOXO4 fragment as disclosed.

[0010] Patent Document 3 relates to a peptide comprising the amino acid sequence LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRP (Sequence ID 5), wherein the amino acids in the above amino acid sequence are D-amino acid residues, and to a method of using this peptide in the treatment of age-related disorders. This peptide exhibits apoptosis-inducing activity in senescent cells or in cells that express increased FOXO4 expression and Ser15 phosphorylated p53 (pSer15-p53) compared to control cells.

[0011] Patent Document 4 relates to a conditionally active protein that targets senescent cells, and a method for producing such a conditionally active protein.

[0012] As is evident from the above, there is still a demand in this technological field for compounds that selectively induce apoptosis in senescent cells. This is because cellular senescence is associated with degenerative (loss of function) diseases and cancer (gain of function). Currently, there are no suitable therapeutic compounds that can selectively induce apoptosis in senescent cells in vivo. [Prior art documents] [Patent Documents]

[0013] [Patent Document 1] International Publication No. 2013 / 152038 [Patent Document 2] International Publication No. 2013 / 152041 [Patent Document 3] International Publication No. 2016 / 118014 [Patent Document 4] International Publication No. 2018 / 129007 [Non-patent literature]

[0014] [Non-Patent Document 1] Baker et al., 2011. Nature 479(7372):232-6

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0015] Therefore, an object of the present invention is to provide a novel and improved compound that targets senescent cells for treatment and can be efficiently used for the treatment of diseases associated with cellular senescence.

Means for Solving the Problems

[0016] In a first aspect, the present invention provides: i) an amino acid sequence X3X2X4X5X7X5X4X4X6X 18 X8X3QNX9X8X 10 X 10 X 11 X 12 S * X 13 X 14 X 11 X 11 (SEQ ID NO: 1) (In the sequence, S * may be S or may not be present, X2 may not be present or may be selected from K, E, R, and H, X3 may not be present or may be selected from A, J, and S, X4 is selected from I, Z, and L, where Z is cyclo-hexyl-alanine, X5 is selected from A, G, S, E, and D, X6 is selected from E and D, X7 is selected from J, G, Q, A, S, and P, X8 is selected from B, W, Y, and F, where B is 2-methyl-tryptophan, X9 may not be present or may be selected from A and G, X 10 It does not exist, or it is selected from A and N. X 11 It is selected from R and K, X 12 It does not exist, or it is selected from R. X 13 It does not exist, or it is selected from G and S. X 14 It does not exist, or it is selected from A and C. X 18 It is selected from A and E, However, the J that forms the staple is either absent or present in two parts) and the peptide contains an amino acid sequence that is identical in at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably more than 95%. ii) peptides according to i) comprising non-natural amino acids and / or D-amino acids, preferably containing at least 80%, preferably at least 90%, more preferably at least 95% D-amino acids, or peptides according to i) consisting of at least 80%, preferably at least 90%, more preferably at least 95% D-amino acids, and iii) Retroinverse peptides of the peptides described in i) or ii), that is, peptides in which the amino acid sequence is reversed and the D-isoform is positioned instead of the L-isoform when compared to the native protein sequence. and its pharmaceutically acceptable salts, A compound selected from, Induces apoptosis in senescent cells, scar cells, and / or cancer cells. This problem is solved by providing a compound.

[0017] The above peptide is an amino acid sequence X1X 17 K * X2X 16 X3X3X2X4X5X7X5X4X4X6AX8X3QNX9X8X 10 X 10 X 11 X12 S * X 13 X 14 X 11 X 11 X 15 (Sequence 2) (In the array, Z, B, J, S * ,X2,X3,X4,X5,X6,X7,X8,X9,X 10 , X 11 , X 12 , X 13 , X 14 , and X 18 As stated above, K * This means that K is possible or does not exist. X1 is either absent or represents the LTL amino acid sequence. X 15 It does not exist, or it is selected from A and C. X 16 It is selected from A and P, X 17 It comprises an amino acid sequence that is identical to (or selected from R and S) by at least 70%, preferably at least 80%, more preferably at least 90%, and is not present. The compounds described above are preferably those according to the present invention that induce apoptosis in senescent cells, scar cells, and / or cancer cells.

[0018] More preferably, the compound according to the present invention comprises an amino acid motif X3QNX9X8 (for example, starting from position 18 of SEQ ID NO: 2) selected from SQNAW (SEQ ID NO: 43), SQNGW (SEQ ID NO: 44), and SQN-W (SEQ ID NO: 45), where "-" in the sequence indicates the absence of an amino acid. Even more preferably, the compound according to the present invention comprises an amino acid motif X2X4X5X7X5 (for example, starting from position 8 of SEQ ID NO: 2) selected from KIAAA (SEQ ID NO: 46), KIEAA (SEQ ID NO: 47), KIAAE (SEQ ID NO: 48), and KIEAE (SEQ ID NO: 49).

[0019] More preferably, the compound according to the present invention is general formula IX5X7X5ILX6AYX3QNX9W (Sequence ID 4) (in array) X3 is either nonexistent or selected from A, J, and S. X5 is selected from A, G, S, E, and D. X6 is selected from E and D. X7 is selected from J, G, Q, A, S, and P. X9 contains an amino acid motif X4X5X7X5X4X4X6AX8X3QNX9X8 (starting from position 9 of SEQ ID NO: 3, SEQ ID NO: 2) which is either absent or selected from A and G.

[0020] Preferably, the compound according to the present invention consists of a D-amino acid. The designation of the amino acid follows the standard single-letter notation.

[0021] Most preferably, the compound according to the present invention is LTLRKEASSEIAQSILDAYSQNGWANRRSSCKRP (Sequence ID 7), LTLRKKASSKIAQSILDAFSQNGWANRRSSCKRP (Sequence ID 8), LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRP (Sequence ID 9), RKKASSKIAQSILDAFSQNGWANRRSSCKRP (Sequence ID 10), RKKASSKIAAAILDAFSQNGWANRRSSCKRP (Sequence ID 11), RKKASSKIAAAILDAFSQNAWANRRSSCKRP (Sequence ID 12), RKKASSKIAAAILDAFSQNWRRKR (Sequence ID 13), RKKASSKIEAAILDAFSQNWRRKR (Sequence ID 14), RKKASSKIAAEILDAFSQNWRRKR (Sequence ID 15), RKKASSKIEAEILDAFSQNWRRKR (Sequence ID 16), RKKSKIAAAILDAFSQNWRRKR (Sequence ID 17), RKKSKIEAEILDAFSQNWRRKR (Sequence ID 18), AKIAAAILDAFSQNWRRKR (Sequence ID 19), AKIEAAILDAFSQNWRRKR (Sequence ID 20), LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRPPPRRRQRRKKRG (Sequence ID 21), RKKASSKIAAAILDAFSQNGWANRRSSCKRPPPRRRQRRKKRG (Sequence ID 22), RKKASSKIAAAILDAFSQNAWANRRSSCKRPPPRRRQRRKKRA (Sequence ID 23), RKKASSKIAAAILDAFSQNWRRKRPPRRRQRRKKRG (Sequence ID 24), RKKASSKIEAAILDAFSQNWRRKRPPRRRQRRKKRG (Sequence ID 25), RKKASSKIAAEILDAFSQNWRRKRPPRRRQRRKKRG (Sequence ID 26), RKKASSKIEAEILDAFSQNWRRKRPPRRRQRRKKRG (Sequence ID 27), RKKSKIAAAILDAFSQNWRRKRRRRQRRKKRG (Sequence ID 28), RKKSKIEAEILDAFSQNWRRKRRRRQRRKKRG (Sequence ID 29), AKIAAAILDAFSQNWRRKRRRRQRRKKRG (Sequence ID 30), AKIEAAILDAFSQNWRRKRRRRQRRKKRG (Sequence ID 31), RKKASSKIEAEILDAFSQNWRRKRPPRRRQRRKKRG (Sequence ID 53), RKKSKIEAEILDAFSQNWRKRRRRQRRKKRG (Sequence ID 54), AKIEAAILDAFSQNWRKRRRRQRRKKRG (Sequence ID 55), AKIEAEILDAFSQNWRKRRRRQRRKKRG (Sequence ID 56), AKIEAAILDEFSQNWRKRRRRQRRKKRG (Sequence ID 57), RKKASJKIAJAILDAFSQNWRRKRPPRRRQRRKKRG (Sequence ID 58), RKKASSKIAAAZLDAFSQNAWANRRSSCKRPPPRRRQRRKKRA (Sequence ID 59), AKIEAAILDAFSQNBRKRRRRQRRKKRG (Sequence ID 60), AKIEAEILEAFSQNBRKRRRRQRRKKRG (Sequence ID 61), AKIEAAZLDAFSQNBRKRRRRQRRKKRG (Sequence ID 62), RKKASSKIEAEILDAFSQNBRRKRPPRRRQRRKKRG (Sequence ID 63), RKKASSKIEAEZLDAFSQNBRRKRPPRRRQRRKKRG (Sequence ID 64), RKKASSKIEAEIZDAFSQNBRRKRPPRRRQRRKKRG (Sequence ID 65), RKKSKIAAAILDAFSQNWRKRRRRQRRKKRG (Sequence ID 67), and, RKKASSKIEAAILDAFSQNWRRKRPPRRRQRRKKRG (Sequence ID 68), (In the formula, J, B, and Z are defined as above) A peptide comprising an amino acid sequence selected from amino acid sequences that are at least 80%, preferably at least 90%, more preferably at least 95%, identical to any one of SEQ ID NOs: 1-4, 7-31, 53-65, and 67 and 68, A peptide comprising the amino acid sequence described in any one of SEQ ID NOs: 1-4, 7-31, 53-65, and 67 and 68, and a pharmaceutically acceptable salt thereof, These are peptides containing amino acid sequences, selected from the following. These peptides are, in particular, D-peptides.

[0022] In a second embodiment, the present invention relates to a method for identifying improved compounds that bind to p53, or preferably bind to p53 to inhibit the interaction between FOXO4 and p53 in cells, a) A step of preparing at least one compound according to the present invention as described herein, b) a step of appropriately modifying the above compound a), A step of identifying at least one of the following: the binding of the at least one compound described in c)b) above to p53 or a fragment thereof, the stability of the at least one compound described above, and the binding of FOXO4 or a fragment thereof to p53 or a fragment thereof in the presence of the at least one compound described above, compared with the binding of FOXO4 or a fragment thereof to p53 or a fragment thereof in the absence of the compound described above. d) Based on what was identified in step c), a step of identifying an improved compound that binds to p53, or preferably binds to p53 and inhibits the interaction between FOXO4 and p53 in cells, compared to the compound provided in step a), This includes methods.

[0023] A preferred method according to the present invention is one in which the above-mentioned binding of at least one of the above-mentioned compounds is specific to p53 or the above-mentioned fragment thereof. A preferred method further comprises the step of testing the compound as identified for its activity to induce apoptosis in senescent cells, scar cells, and / or tumor cells and / or for its activity to kill these cells, preferably including the identification of caspase activity.

[0024] In a third embodiment, the present invention relates to a screening tool for screening compounds that bind to p53, preferably compounds that bind to p53 and inhibit the interaction between FOXO4 and p53 in cells, the screening tool comprising isolated cells expressing FOXO4 and / or cells expressing its p53-binding fragment, the cells optionally expressing p53 and / or cells expressing its FOXO4-binding fragment.

[0025] The above fragment of FOXO4 is an array The peptide is described in PRKGGSRRNAWGNQSYAELISQAIESAPEKRLTLAQIYEWMVRTVPYFKDKGDSNSSAGWKNSIRHNLSLHSKFIKVHNEATGKSSWWMLN (complete forkhead domain) (SEQ ID NO: 32) or PRKGGSRRNAWGNQSYAELISQAIESAPEKRLTL (SEQ ID NO: 33), and the above fragment of p53 is sequence A screening tool according to the present invention is preferred, which is the peptide described in AMDDLMLSPDDIEQWFTEDPGP (SEQ ID NO: 34).

[0026] In a fourth aspect, the present invention relates to a method for producing a pharmaceutical composition for treating or preventing senescent cells, scar cells, and / or cancer cells in a subject, comprising the steps of: formulating a compound according to the present invention to make a suitable pharmaceutical composition, or carrying out a method according to the present invention; and formulating the compound as identified to make a suitable pharmaceutical composition. The pharmaceutical composition according to the present invention may contain pharmaceutically acceptable excipients, such as stabilizers, bulking agents, buffers, carriers, diluents, vehicles, solubilizers, and binders, without particular limitation. As will be apparent to those skilled in the art, the selection of suitable excipients depends on the route of administration and dosage form, as well as the active ingredient and other factors. The pharmaceutical composition according to the present invention is preferably suited for parenteral administration.

[0027] In a fifth aspect, the present invention relates to a pharmaceutical composition obtained by a method according to the present invention for treating or preventing senescent cells, scar cells, and / or cancer cells in a subject.

[0028] In a sixth aspect, the present invention relates to at least one compound or pharmaceutical composition according to the present invention for use in pharmaceuticals. Preferably, at least one compound or pharmaceutical composition for use in accordance with the present invention, wherein the disease or condition to be prevented and / or treated is selected from conditions caused by senescent cells, scar cells, age-related diseases, kidney diseases, osteoarthritis, COPD, musculoskeletal diseases, cognitive impairment, and cancer.

[0029] In a seventh aspect, the present invention relates to a method for treating or preventing diseases or symptoms caused by senescent cells, scar cells, and / or cancer cells, age-related diseases, kidney diseases, osteoarthritis, COPD, musculoskeletal diseases, cognitive impairment, or cancer, comprising administering at least one compound according to the present invention, or a pharmaceutical composition according to the present invention, in an effective amount to the subject.

[0030] Other embodiments and advantages can be easily derived by reading the following description and examples, which are not limited thereto. [Brief explanation of the drawing]

[0031] [Figure 1] This figure shows the sequence of human FOXO4 (Homo sapiens), with the TP53 interaction domain sequence underlined. [Figure 2] This figure shows that the exemplary peptide CL04009 (SEQ ID NO: 11) is potent in binding to recombinant TP53-TAD2 and full-length TP53 isolated from cells, and is superior to the reference compound (FOXO4-DRI). A) Relative perturbations of NMR chemical shifts for 15N-labeled TP53-TAD2 alone (bottom panel), in the presence of CL04009 (SEQ ID NO: 11) (top panel), or with the reference compound (middle panel). The perturbation of NMR chemical shifts induced by the reference compound is set to 100%. B) Endogenous TP53 pulldown from HEK293T cell lysate using streptavidin beads is shown in the absence of any peptide, with biotinylated CL04009 (SEQ ID NO: 11), or with the biotinylated reference peptide (FOXO4-DRI). [Figure 3]This figure shows that peptide CL04009 (SEQ ID NO: 11) is potent and selective in senescent IMR90 lung fibroblasts, but not in healthy control IMR90 lung fibroblasts. This figure demonstrates that modified CL04009 (SEQ ID NO: 11), with improved binding to TP53, selectively induced apoptosis via caspase 3 / 7 activation in senescent IMR90 lung fibroblasts, but not in healthy control IMR90 lung fibroblasts. Furthermore, this peptide was effective at lower concentrations, indicating improved efficacy. The upper graph shows caspase activity, and the lower graph shows cell viability. FOXO4-DRI was used as a control. [Figure 4] This figure shows that CL04022 (SEQ ID NO: 12) binds to TP53-TAD more strongly than the reference compound FOXO4-DRI. The fluorescence polarization of FITC-labeled TP53-TAD2 (250 nM) is measured as the peptide concentration increases. [Figure 5] This figure shows that peptide CL04022 (SEQ ID NO: 12) is more stable than CL04009 (SEQ ID NO: 11) over a long period of several days, based on alanine exchange in the peptide. Both peptides were dissolved in H2O and maintained at room temperature. The peptide concentrations were measured at the indicated time points. [Figure 6] This figure shows that peptide CL04022 (SEQ ID NO: 12) initiates the induction of caspase 3 / 7 activation, an indicator of classical apoptosis, 24 hours after administration. Total caspase 3 / 7 activation is completed after 60 hours. The top row is the control. [Figure 7] This figure shows that modifications to peptide CL04009 (SEQ ID NO: 11) to improve selective TP53 binding induce apoptosis in senescent IMR90 cells (upper blue line), but not in healthy control IMR90 cells (lower black line), demonstrating a very potent and selective ability. The lower figure shows that peptide CL04088 (SEQ ID NO: 13) is superior to peptide CL04022 (SEQ ID NO: 12), but peptide CL04022 is still superior to the control. [Figure 8] This figure shows that modification of peptide CL04009 (SEQ ID NO: 11, shown as CL05055 in this figure) can further improve its efficacy in eliminating senescent cells in different organs. RPE = retinal pigment epithelial cells. CL03001 is the same as FOXO4-DRI used as a control. [Figure 9-1] This figure shows that CL04022 (SEQ ID NO: 12) is superior to FOXO4-DRI in inducing apoptosis (apoptosis) in glioblastoma cells. A) CL04022 (SEQ ID NO: 12) is more potent than FOXO4-DRI in inducing caspase 3 / 7 activation (apoptosis), and B) LDH (cell death) is reduced in GBM8 glioblastoma cells. C) shows that CL04022 (SEQ ID NO: 12) induces apoptosis in GBM8 glioblastoma cells but not in healthy WI38 cells. CL04022 (SEQ ID NO: 12) is also selective in inducing apoptosis when comparing GBM8 glioblastoma cells with healthy WI38 cells. [Figure 9-2] Same as above [Figure 10] This figure shows that CL04022 (SEQ ID NO: 12) is effective against colon cancer. A) Peptide CL04022 (SEQ ID NO: 12) is effective against two types of microsatellite-unstable (MSI) colon cancer 3D organoid cultures. The peptide is ineffective against one type of microsatellite-stable (MSS) strain. B) This shows that CL04022 (SEQ ID NO: 12) is selective in comparison between patient-derived colorectal cancer and healthy control samples. CL04022 (SEQ ID NO: 12) is less potent against normal wild-type colon organoids (compared to A above). This peptide is effective when such organoids are depleting APC. [Figure 11]This figure shows the superior stability of CL04183 (SEQ ID NO: 55) and CL05114 (SEQ ID NO: 60) in solution compared to FOXO4-DRI (CL03001). In the table, CL02001 specifies the "L" form of peptide CL03001, and CL02015 specifies the "L" form of peptide CL04183 (SEQ ID NO: 55). [Figure 12-1] A) This figure shows that peptide CL04022 (SEQ ID NO: 12) is more effective in colon cancer 3D organoids that survive chemotherapy (5'-fluorouracil or oxaliplatin), and B) This figure shows that CL04022 (SEQ ID NO: 12), in contrast to the standard treatment 5FU, not only causes cell cycle arrest but also induces cell death. [Figure 12-2] Same as above [Figure 13] This figure shows that no further tumor growth was observed in 3D colon organoids 14 days after exposure to CL04009 (SEQ ID NO: 11). This indicates that CL04009 (SEQ ID NO: 11) is not merely cell proliferation inhibitory, but cytotoxic, to colon organoids, as shown in the example of colon cancer in this figure. [Figure 14] In a single experiment, peptide CL04183(B) (SEQ ID NO: 55) demonstrated superior selectivity and efficacy compared to the original FOXO4-DRI (CL03001)(A) when comparing aging human RPE1 cells with control human RPE1 cells. [Figure 15] This figure shows that colorectal cancer-associated mutations in colon organoids are sensitized to FOXO4-based peptides in a p53-dependent manner. WT colon organoids and organoids from different tumor-progressed (TPO) groups were treated with CL04124 (SEQ ID NO: 68) or CL04183 (SEQ ID NO: 55) at increasing concentrations. Cell viability was measured after 5 days. [Figure 16]This figure shows that CL04183 (SEQ ID NO: 55) strongly induces apoptosis in patient-derived colorectal cancer organoids. A) Patient-derived colorectal cancer organoid strain CRC29 was treated with either 5'-fluorouracil (5-FU) or CL04183 (SEQ ID NO: 55). After 3 days, the cells were treated with calcein AM and propidium iodide. These stain live and dead cells, respectively. The organoids were imaged using an EVOS imaging system. B) CRC29 organoids were incubated with calcein AM and caspase 3 / 7 red dye for apoptosis, and then treated with CL04183 (SEQ ID NO: 55). Live imaging was immediately started using an LSM880 microscope, and images were taken every hour. C) CRC29 was treated with CL04183 (SEQ ID NO: 55) at increasing concentrations. After 5 days, cell viability was measured. [Figure 17] This figure shows that peptide CL04009 (SEQ ID NO: 11) induces apoptosis in aging human mammary epithelial cells more potently than the reference compound FOXO4-DRI / CL03001. MCF10 cells were treated with CL04009 (SEQ ID NO: 11) and FOXO4-DR at increasing concentrations, and after 2 days, caspase assays were performed to identify the induction of apoptosis. [Figure 18] This figure shows that CL04183 (SEQ ID NO: 55) reduces gray hair in naturally aged mice. 24-month-old C57BL / 6J mice were photographed before being administered either PBS or CL04183 (SEQ ID NO: 55) in three doses via IV injection, and photographed again 4 weeks after the first treatment. [Figure 19]This figure shows that peptide CL04183 (SEQ ID NO: 55) reduces luciferase-expressing colorectal cancer cells in vivo. A) The colorectal cancer organoid strain CRC29, which contains firefly luciferase, was transplanted into the cecum of immunodeficient mice. Two weeks after transplantation, bioluminescence levels were identified, and the mice were treated with three doses of CL04183 (SEQ ID NO: 55) or PBS. Two days after the final dose, all animals were imaged again, and luminescence values ​​were compared to baseline. B) Mice were injected with luciferin and then sacrificed. Individual organs were imaged, and luciferase signals were quantified using M3 Vision software. [Figure 20] This figure shows that peptide CL04183 (SEQ ID NO: 55) reduces colorectal cancer metastases to the liver and lung. The colorectal cancer organoid strain CRC29 was transplanted into the cecum of immunodeficient mice. Two weeks after transplantation, the animals were treated with three doses of CL04183 (SEQ ID NO: 55) or PBS. A) Lungs and livers were stained for human nucleoli to detect human cancer cells in the mouse organs. B) The amount of metastatic cells in the lungs was quantified using CellProfiler software. Six tile scans were analyzed per mouse. All counts were compared to the mean of the PBS-treated group. The number of liver metastases per stained section was visually counted. [Figure 21] This figure shows that CL04183 (SEQ ID NO: 55) induces apoptosis in metastatic cancer cells in vivo. Apoptosis induction was identified by TUNEL assay in CRC29 lung metastases treated with PBS or CL04183 (SEQ ID NO: 55). Each group consisted of three mice, and the percentage of TUNEL-positive cancer cells was quantified using FIJI. [Figure 22] This figure shows that three-negative breast cancer cell lines exhibit an open configuration of p53, which correlates with the sensitivity of CL04183 (SEQ ID NO: 55). The displayed human breast cancer cell lines were treated with CL04183 (SEQ ID NO: 55) at increasing concentrations, and cell viability was determined by MTS assay after 2 days. EC50 values ​​were calculated using GraphPad Prism. [Figure 23]This figure shows the efficacy of CL04183 (SEQ ID NO: 55) against NRAS-mutated and BRAF-mutated melanoma. See also Figure 14. [Figure 24] This figure shows NMR peptide binding data. It is a magnified view of the 1H,15N crossover peak of p53-TAD2 T55 extracted from the 1H,15N HSQC NMR spectra obtained for p53-TAD2 alone (bottom circle, red), with the addition of the reference peptide FOXO4-DRI / CL03001 (black), or with the addition of the test peptide (top circle, blue). A) Peptide SEQ ID NO: 22, B) Peptide SEQ ID NO: 23, C) Peptide SEQ ID NO: 24, D) Peptide SEQ ID NO: 25, E) Peptide SEQ ID NO: 26, and F) Peptide SEQ ID NO: 27. [Figure 25] This figure shows NMR peptide binding data. The enlarged view shows the 1H,15N crossover peak of p53-TAD2 T55 extracted from the 1H,15N HSQC NMR spectra obtained for p53-TAD2 alone (bottom circle, red), with the addition of the reference peptide FOXO4-DRI / CL03001 (black), or with the addition of the test peptide (top circle, blue). A) Peptide CL04022, SEQ ID NO: 12; B) Peptide CL04121, SEQ ID NO: 67; C) Peptide CL04183, SEQ ID NO: 55; D) Peptide CL04180, SEQ ID NO: 54; E) Peptide CL04230, SEQ ID NO: 56; and F) Peptide CL04231, SEQ ID NO: 57. [Figure 26] This figure shows NMR peptide binding data. The enlarged view shows the 1H,15N crossover peak of p53-TAD2 T55 extracted from the 1H,15N HSQC NMR spectra obtained when p53-TAD2 alone (bottom circle, red), when the reference peptide FOXO4-DRI / CL03001 (black) was added, or when the test peptide (top circle, blue) was added. A) Peptide CL04235, SEQ ID NO: 61, B) Peptide CL05114, SEQ ID NO: 60. [Modes for carrying out the invention]

[0032] In the experimental setting as performed in relation to the present invention, the inventors were surprised to find that, after further careful analysis of the interaction between FOXO4 and p53, it was possible to achieve substantially substantial functional improvements by introducing modifications to the compound.

[0033] Therefore, the main aspects of the present invention are: i) Amino acid sequence X3X2X4X5X7X5X4X4X6X 18 X8X3QNX9X8X 10 X 10 X 11 X 12 S * X 13 X 14 X 11 X 11 (Sequence ID 1) (In the array, S * This means that S is possible or does not exist. X2 is either nonexistent or selected from K, E, R, and H. X3 is either nonexistent or selected from A, J, and S. X4 is selected from I, Z, and L, where Z is cyclohexyl alanine. X5 is selected from A, G, S, E, and D. X6 is selected from E and D. X7 is selected from J, G, Q, A, S, and P. X8 is selected from B, W, Y, and F, where B is 2-methyltryptophan. X9 is either non-existent or selected from A and G. X 10 It does not exist, or it is selected from A and N. X 11 It is selected from R and K, X 12 It does not exist, or it is selected from R. X 13 It does not exist, or it is selected from G and S. X 14 It does not exist, or it is selected from A and C. X 18 It is selected from A and E, However, the J that forms the staple is either absent or present in two parts) and the peptide contains an amino acid sequence that is identical in at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably more than 95%. ii) peptides according to i) comprising non-natural amino acids and / or D-amino acids, preferably containing at least 80%, preferably at least 90%, more preferably at least 95% D-amino acids, or peptides according to i) consisting of at least 80%, preferably at least 90%, more preferably at least 95% D-amino acids, and iii) Retroinverse peptides of the peptides described in i) or ii), that is, peptides in which the amino acid sequence is reversed and the D-isoform is positioned instead of the L-isoform when compared to the native protein sequence. and its pharmaceutically acceptable salts, An improved compound selected from, Induces apoptosis and / or cell death in senescent cells, scar cells, and / or cancer cells. This relates to a compound. Preferably, the compound consists of a D-amino acid.

[0034] Preferably, the compound is one that binds to p53, or more preferably, binds to p53 to inhibit the interaction between FOXO4 and p53 in cells, and the compound is detectable by state-of-the-art assays and / or assays as disclosed herein. These assays include NMR (in which the ability of the peptide to bind to TP53 (fragments) is measured); pull-down assay (in which the biotinylated peptide is attached to (streptavidin) beads, which are poured into a cell lysate containing TP53, and the proportion of TP53 that binds to the peptide is investigated); and FRET / HTRF assay (in which the ability of the peptide to perturb the interaction between TP53 (fragments) and FOXO4 is evaluated).

[0035] The evolutionary differences between FOXO1 / 3 / 6 and FOXO4 suggest that several amino acids can be further modified for the purpose of creating improved compounds that exhibit desired inhibitory activity.

[0036] FOXO4 is evolutionarily distinct from FOXO1 / 3 and 6, as well as from its homologous FOXO molecules in lower organisms, in several unique residues. The inventors have never found a FOXO1 or FOXO3 foci, nor do they have any evidence that FOXO1 and 3 are important for senescent cell viability. Therefore, they analyzed whether these amino acids at least partially explain the efficacy of the FOXO4 peptide in senescent cell elimination. It was confirmed that D-retroinverse peptides based on a domain similar to that of FOXO4 showed selectivity in senescent cell elimination, while those based on the sequences of FOXO1 or FOXO3 did not.

[0037] Analysis of the FOXO4-FH fragment and the TP53-TAD2 domain indicated that the negative charge of TP53, as well as the hydrophobic Tyr, mediates the binding to FOXO4. Therefore, mutations to positive amino acids, such as Glu→Lys and Tyr→Phe, were expected to be beneficial to the strength of the interaction. NMR also showed that the N-terminal amino acid LTL is not required for the interaction. Finally, NMR showed that the negatively charged amino acids of TP53 contribute to the interaction with FOXO4 around the SQ motif (site). The NG amino acid site can be modified to improve molecular stability. All of these modifications offer novel strategies, and any one or a combination of these modifications leads to the rational design of improved molecules used in the context of this invention.

[0038] It has also been found that the active peptides in the context of the present invention can be considerably shorter than those proposed in the current state of the art, while still maintaining potent activity. Nevertheless, longer and more active peptides are also possible, and therefore, preferred compounds according to the present invention have amino acid sequences. X1X 17 K * X2X 16 X3X3X2X4X5X7X5X4X4X6AX8X3QNX9X8X 10 X 10 X 11 X 12 S * X 13 X 14 X 11 X 11 X 15 (Sequence 2) (In the array, Z, B, J, S * ,X2,X3,X4,X5,X6,X7,X8,X9,X 10 , X 11 , X 12 , X 13 , X 14 , and X 18 This is as described in claim 1, K * This means that K is possible or does not exist. X1 is either absent or represents the LTL amino acid sequence. X 15 It does not exist, or it is selected from A and C. X 16 It is selected from A and P, X 17 It comprises an amino acid sequence that is identical to (or selected from R and S) by at least 70%, preferably at least 80%, more preferably at least 90%, and is not present. The above compound is a peptide that induces apoptosis in senescent cells, scar cells, and / or cancer cells.

[0039] Furthermore, it has been discovered that the specific active peptide according to the present invention comprises a certain specific amino acid motif, preferably the amino acid motif X3QNX9X8 (for example, starting from position 18 of SEQ ID NO: 2) selected from SQNAW (SEQ ID NO: 43), SQNGW (SEQ ID NO: 44), and SQN-W (SEQ ID NO: 45), where "-" in the sequence indicates the absence of an amino acid.

[0040] Unexpectedly, when X9 is alanine (A), the peptide exhibits improved stability in solution, as exemplified in the peptides of SEQ ID NO: 12 and SEQ ID NO: 23 (see Examples). Therefore, this substitution is preferable for the purpose of providing further stability to the compounds according to the present invention.

[0041] More preferably, the compound according to the present invention comprises an amino acid motif X2X4X5X7X5 (for example, starting from position 8 of SEQ ID NO: 2) selected from KIAAA (SEQ ID NO: 46), KIEAA (SEQ ID NO: 47), KIAAE (SEQ ID NO: 48), and KIEAE (SEQ ID NO: 49).

[0042] More preferably, the compound according to the present invention is a general formula IX5X7X5ILX6AFX3QNX9W (Sequence ID 4) (In the array, X3 is either nonexistent or selected from A, J, and S. X5 is selected from A, G, S, E, and D. X6 is selected from E and D. X7 is selected from J, G, Q, A, S, and P. X9 contains an amino acid motif X4X5X7X5X4X4X6AX8X3QNX9X8 (starting from position 9 of SEQ ID NO: 3, SEQ ID NO: 2) which is either absent or selected from A and G.

[0043] In the context of the present invention, it has been found that the amino acid motif LTLRKEASSE (SEQ ID NO: 35) is unnecessary for the overall functional improvement of the peptide, and this motif can be reduced to a single Ala, replaced with Ala, a "neutral" amino acid, or a positively charged amino acid. In contrast, the "core motif" X4X5X7X5X4X4X6AX8X3QNX9X8 (SEQ ID NO: 3, starting from position 9 of SEQ ID NO: 2) is the most important, and this core motif can be selected from the general formula IX5X7X5ILX6AFX3QNX9W (SEQ ID NO: 4). That being said, within this motif, the amino acid "SQNG" (SEQ ID NO: 50) appears to function as a structural linker, functioning to return the "W" to the hydrophobic core through folding. Several residues can be deleted, and one or two "G"s are sufficient; the peptide will also function using AANG (SEQ ID NO: 51) or SQ-G or SQAG (SEQ ID NO: 52). The terminal W is essential and difficult to replace with natural AA. The first "I" is essential and must be a hydrophobic amino acid, preferably I / L, but non-natural ones are also acceptable. In the IX5X7X5IL (SEQ ID NO: 36, e.g., IAQSIL, SEQ ID NO: 37) portion, the interval between "I"s must be two to three residues. Several modifications are acceptable as long as they do not disrupt the helix or hydrophobic core. Similarly, the terminal "IL" is essential and must be a hydrophobic amino acid, preferably I / L, but non-natural ones are also acceptable. Furthermore, the interval between the following "L" and "Y" is preferably two residues, but single amino insertions are acceptable. Again, modifications are acceptable as long as they do not disrupt the helix or hydrophobic core. "Y" is essential and must be a hydrophobic amino acid, preferably F / Y, but non-natural ones are also acceptable. Similar to the N-terminal region described above, the motif ANRRSSCKRP (SEQ ID NO: 38) is not necessary for the overall functional improvement of the peptide, and this motif can be reduced to at least three positively charged residues, but even with fewer residues, the peptide remains active.

[0044] Most preferably, the compound according to the present invention is LTLRKEASSEIAQSILDAYSQNGWANRRSSCKRP (Sequence ID 7), LTLRKKASSKIAQSILDAFSQNGWANRRSSCKRP (Sequence ID 8), LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRP (Sequence ID 9), RKKASSKIAQSILDAFSQNGWANRRSSCKRP (Sequence ID 10), RKKASSKIAAAILDAFSQNGWANRRSSCKRP (Sequence ID 11), RKKASSKIAAAILDAFSQNAWANRRSSCKRP (Sequence ID 12), RKKASSKIAAAILDAFSQNWRRKR (Sequence ID 13), RKKASSKIEAAILDAFSQNWRRKR (Sequence ID 14), RKKASSKIAAEILDAFSQNWRRKR (Sequence ID 15), RKKASSKIEAEILDAFSQNWRRKR (Sequence ID 16), RKKSKIAAAILDAFSQNWRRKR (Sequence ID 17), RKKSKIEAEILDAFSQNWRRKR (Sequence ID 18), AKIAAAILDAFSQNWRRKR (Sequence ID 19), AKIEAAILDAFSQNWRRKR (Sequence ID 20), LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRPPPRRRQRRKKRG (Sequence ID 21), RKKASSKIAAAILDAFSQNGWANRRSSCKRPPPRRRQRRKKRG (Sequence ID 22), RKKASSKIAAAILDAFSQNAWANRRSSCKRPPPRRRQRRKKRA (Sequence ID 23), RKKASSKIAAAILDAFSQNWRRKRPPRRRQRRKKRG (Sequence ID 24), RKKASSKIEAAILDAFSQNWRRKRPPRRRQRRKKRG (Sequence ID 25), RKKASSKIAAEILDAFSQNWRRKRPPRRRQRRKKRG (Sequence ID 26), RKKASSKIEAEILDAFSQNWRRKRPPRRRQRRKKRG (Sequence ID 27), RKKSKIAAAILDAFSQNWRRKRRRRQRRKKRG (Sequence ID 28), RKKSKIEAEILDAFSQNWRRKRRRRQRRKKRG (Sequence ID 29), AKIAAAILDAFSQNWRRKRRRRQRRKKRG (Sequence ID 30), AKIEAAILDAFSQNWRRKRRRRQRRKKRG (Sequence ID 31), RKKASSKIEAEILDAFSQNWRRKRPPRRRQRRKKRG (Sequence ID 53), RKKSKIEAEILDAFSQNWRKRRRRQRRKKRG (Sequence ID 54), AKIEAAILDAFSQNWRKRRRRQRRKKRG (Sequence ID 55), AKIEAEILDAFSQNWRKRRRRQRRKKRG (Sequence ID 56), AKIEAAILDEFSQNWRKRRRRQRRKKRG (Sequence ID 57), RKKASJKIAJAILDAFSQNWRRKRPPRRRQRRKKRG (Sequence ID 58), RKKASSKIAAAZLDAFSQNAWANRRSSCKRPPPRRRQRRKKRA (Sequence ID 59), AKIEAAILDAFSQNBRKRRRRQRRKKRG (Sequence ID 60), AKIEAEILEAFSQNBRKRRRRQRRKKRG (Sequence ID 61), AKIEAAZLDAFSQNBRKRRRRQRRKKRG (Sequence ID 62), RKKASSKIEAEILDAFSQNBRRKRPPRRRQRRKKRG (Sequence ID 63), RKKASSKIEAEZLDAFSQNBRRKRPPRRRQRRKKRG (Sequence ID 64), RKKASSKIEAEIZDAFSQNBRRKRPPRRRQRRKKRG (Sequence ID 65), RKKSKIAAAILDAFSQNWRKRRRRQRRKKRG (Sequence ID 67), and, RKKASSKIEAAILDAFSQNWRRKRPPRRRQRRKKRG (Sequence ID 68), (In the formula, J, B, and Z are defined as above) A peptide comprising an amino acid sequence selected from amino acid sequences that are at least 80%, preferably at least 90%, more preferably at least 95%, identical to any one of SEQ ID NOs: 1-4, 7-31, 53-65, and 67 and 68, A peptide comprising the amino acid sequence described in any one of SEQ ID NOs: 1-4, 7-31, 53-65, and 67 and 68, preferably a peptide comprising the amino acid sequence described in any one of SEQ ID NOs: 55, 60, 63, 64, or 65, The pharmaceutically acceptable salt, Selected from, It is a peptide containing an amino acid sequence.

[0045] In the compounds of the present invention, according to the one-letter notation of amino acids, X1 is absent or represents the amino acid LTL. This is because the N-terminal amino acid LTL is not essential for the interaction. X2 is a positively charged amino acid, preferably selected from K, E, R, and H. X3 is a small non-polar amino acid, preferably selected from A and S, and can form a staple using J as defined herein. X4 is selected from hydrophobic amino acids, preferably I and L, and the modified amino acid Z, where Z is cyclo-hexyl-alanine. X5 is a small positively or negatively charged amino acid, preferably selected from A, G, S, E, and D. X6 is a negatively charged amino acid, preferably selected from E and D. X7 is a small or polar amino acid, preferably selected from G, Q, A, S, and P, and can form a staple using J as defined herein. X8 is selected from aromatic amino acids, preferably W, Y, and F, and B, where B is 2-Me-tryptophan. X9 is a small polar or non-polar amino acid, preferably selected from A and G. X 10 is selected from A and N and charge-neutral polar amino acids, preferably Q. X 11 and X 12 are selected from positively charged amino acids, preferably R and K. X 13 is selected from G and S, X 14 and / or X 15 is selected from A and C, X 16 is selected from A and P, X 17 is selected from R and S, X 18 is selected from A and E. Preferably, the compound can have non-naturally occurring amino acids having the same or substantially the same desired properties as the natural amino acids as shown herein at the relevant positions of the above compounds as shown in the formula herein. It is also possible to introduce modified amino acids, such as linker amino acids, staples, disulfide bridges, glycosylation sites, ubiquitination, and / or pegylation sites, etc., as desired. [[ID=二十]]

[0046] [[ID=二十一]] Stapled peptides are short peptides bound by synthetic fixatives ("staples"), typically exhibiting an alpha-helical structure. Staples are formed by covalent bonds between two amino acid side chains, creating a macrocyclic peptide molecule. Therefore, staples generally represent the covalent bond between two previously independent entities. Among its applications, peptide stapling is particularly used to enhance the pharmacological properties of peptides.

[0047] The peptides of the present invention can be of any length, as long as the compound has the function of binding to and / or binding to p53 to inhibit or substantially inhibit the interaction between FOXO4 and p53 in cells. Preferably, the compounds are compounds with a length of 100 to 19 amino acids, more preferably 75 to 19, and even more preferably 50 to 19 amino acids. Particularly preferred are the peptides of the present invention having lengths selected from the lengths given by SEQ ID NOs: 1 to 4, 7 to 31, 53 to 65, and one of 67 and 68.

[0048] The compounds according to the present invention are preferably retroinverse peptides containing an amino acid sequence that is at least 80%, more preferably at least 90%, and most preferably at least 95% identical to any one of SEQ ID NOs: 1-4, 7-31, 53-65, and 67 and 68.

[0049] When used herein, the term "peptide" preferably refers to a synthetically synthesized peptide, preferably a peptide mimetic, and more preferably a D-peptide. The term "peptide" encompasses peptide analogs, which may have modifications that enhance the stability of the peptide while it is in the body or enhance its permeability into cells. Such modifications include, but are not limited to, N-terminal modifications, C-terminal modifications, peptide bond modifications, and skeletal modifications. Examples of peptide bond modifications include, but are not limited to, CH2-NH, CH2-S, CH2-S=O, O=C-NH, CH2-O, CH2-CH2, S=C-NH, CH=CH, or CF=CH. Methods for preparing peptide mimetic compounds are known in the art and are disclosed, for example, in Quantitative Drug Design, CA Ramsden Gd., Chapter 17.2, F. Choplin, Pergamon Press (1992).

[0050] Further preferred modifications relate to the amino acids themselves and include introducing amino acids that do not occur naturally, preferably those having the same or substantially the same properties as the desired properties of the natural amino acids, preferably at the same or substantially the same position as the above compound. Preferred examples are 2-Me-tryptophan or cyclohexylalanine. Further modifications include modified amino acids, such as linker amino acids, staples, cysteine ​​crosslinks, glycolation sites, ubiquitination, and / or pegylation sites. Staple formation can be achieved by introducing two α-pentenylglycine residues or α,α'-pentenylalanine residues to crosslink i and i+4 residues or i and i+7 residues within the peptide. These residues are used to create a total hydrocarbon, alkene-containing, desmethyl-restricted, i.e., staple. Preferably, this is Fmoc-L-2-(4'-pentenyl)alanine and ring-closing metathesis (RCM) (see, for example, Kim YW, Kutchukian PS, Verdine GL. Introduction of all-hydrocarbon i,i+3 staples into alpha-helices via ring-closing olefin metathesis. Org Lett. 2010 Jul 2;12(13):3046-9. doi: 10.1021 / ol1010449). Other strategies that can be used to crosslink or restrict peptides include lactam crosslinking, hydrogen bond substitutes, photoswitches, thioethers, and triazoles introduced by "click" chemical reactions.

[0051] In one preferred embodiment, the compound according to the present invention further comprises a peptide sequence that confers cell penetrating properties, organelle-targeting properties, nuclear localization, mitochondrial localization, blood-brain barrier permeability, cell membrane localization, and / or peptidase cleavage. Examples of cell penetrating sequences include the HIV TAT sequence (GRKKRRQRRRPP, SEQ ID NO: 41) or ARKKRRQRRRPPP (SEQ ID NO: 66). Other preferred sequences are known from the literature and can be adopted by referring, for example, Ramaker et al. (Ramaker et al. (2018) Cell penetrating peptides: a comparative transport analysis for 474 sequence motifs, Drug Delivery, 25:1, 928-937). For nuclear localization, see, for example, Kim YH, Han ME, Oh SO. The molecular mechanism for nuclear transport and its application. Anat Cell Biol. 2017;50(2):77-85. doi:10.5115 / acb.2017.50.2.77). The above peptide sequence can preferably be condensed at the N and / or C-terminal portion of the peptide.

[0052] When used herein, the term “D-isoform” refers to an amino acid sequence in which at least a portion of the amino acid residues in the sequence have a molecular spatial arrangement referred to as “D” (Latin: dexter; right). The compounds of the present invention contain at least 80%, more preferably at least 90%, more preferably at least 95%, most preferably 99%, or 100% D-amino acids (i.e., “all D”). The peptides of the present invention preferably have at least 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more D-amino acid residues. Similarly, the peptides of the present invention preferably contain at least 80%, more preferably at least 85%, even more preferably at least 90%, 95%, 98%, or 99%, most preferably 100% D-amino acid residues.

[0053] When used herein, the abbreviation "DRI" refers to a D-retroinverse isoform or a peptide fragment thereof, in which the amino acid sequence is inverted, particularly with respect to the sequence of the human FOXO4 protein, and the D-isoform is positioned in place of the L-isoform. As those skilled in the art will know, the DRI peptide according to the present invention may include a combination of L-amino acid residues and D-amino acid residues, or it may consist entirely of D-amino acid residues (all-D), as disclosed above, for example.

[0054] The terms “sequence identity %” or “% identical sequence” are defined herein as the percentage of nucleotides in a nucleic acid sequence or amino acids in an amino acid sequence that are identical to each nucleotide or amino acid in the nucleic acid or amino acid sequence of interest, after sequence alignment has been performed and, optionally, gaps have been introduced as necessary to achieve the maximum sequence identity percentage. Alignment methods and computer programs are known in the art. Sequence identity is calculated over substantially the entire length, preferably the full length, of the amino acid sequence of interest. As will be obvious to those skilled in the art, consecutive amino acid residues in one amino acid sequence are compared to consecutive amino acid residues in the other amino acid sequence. Preferably, the stereochemistry of the amino acid residues, e.g., D or L, is irrelevant to the determination of amino acid sequence identity. For example, D-Ala (DA) exhibits sequence identity with L-Ala (LA) in the context of this invention.

[0055] Those skilled in the art will also be aware of the existence of standard in vitro assays to identify the degree of apoptosis in cells and / or cell cultures, such as tests to assess the levels of cytoplasmic cytochrome C (a marker of apoptosis) and TUNEL (a marker of apoptosis). Using these standard assays, those skilled in the art can easily evaluate and compare the apoptosis-inducing activity of various compounds for different cell types or different developmental stages of cells, for example, comparing senescent and non-senescent cells. Other standard apoptosis assays are the annexin V assay and cleavage caspase 3 staining. Cell viability is essentially the opposite of apoptosis, and to detect it, MTT assays (colorimetric assays to assess cell viability), ATP detection assays, real-time cell density (e.g., xCELLigence) assays, or colony formation assays are available. Further information on the assays used can be found in the literature, for example, Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007;35(4):495-516. The assay preferably includes identification of decreased viability, such as increased caspase 3 / 7 activity, decreased mitochondrial cytochrome C, increased TUNEL positivity, increased extracellular annexin V positivity, and / or increased propidium iodide inclusion by cells, and / or decreased calcein AM incorporation, or an MTS viability assay.

[0056] Typically, and preferably, senescent cells are characterized by the presence / decreased staining of the markers LMNB1 and / or HMGB1, and p21 Cip1 and / or p16 INK4a Cells can be identified as having an increase in p21, and / or the presence of the DNA-SCARS markers yH2AX and / or 53BP1 and / or PML, and / or the presence of the SASP markers IL1 and / or IL6 and / or MMP1. The cells do not need to show the complete absence of TP53 or its variants. Typically, cancer cells are preferably p21 Cip1Cells can be identified as those with increased levels of TP53 and / or the presence of DNA-SCARS markers yH2AX and / or 53BP1 and / or PM, and / or the presence of SASP markers IL1 and / or IL6 and / or MMP1. Cells do not necessarily need to show the complete absence of TP53 or its variants. Typically, and preferably, scar cells can be identified as those with elevated levels of DNA-SCARS: 53BP1 and / or yH2AX, and / or enlarged PML / FOXO4 intranucleolus clusters.

[0057] In another embodiment, the present invention provides nucleic acids encoding the peptide according to the present invention (optionally contained in a vector such as an expression vector, to the extent biologically feasible). The nucleic acid can be DNA, RNA, cDNA, PNA, or a combination thereof. In a further embodiment, the present invention provides a host cell containing the nucleic acid or vector according to the present invention. The host cell can be any suitable prokaryotic or eukaryotic cell, such as cancer cells, senescent cells, scar cells, human non-embryonic stem cells, yeast cells, bacterial cells, and recombinant host cells expressing FOXO4 and / or its p53 binding fragment, and optionally selected from the group of recombinant host cells expressing p53 and / or its FOXO4 binding fragment.

[0058] As also described above, the present invention relates to improved inhibitory compounds designed because evolutionary differences between FOXO1 / 3 / 6 and FOXO4 have shown that some amino acids can still be modified or altered for the purpose of creating improved inhibitory compounds. All of these modifications offer novel strategies, and any one or a combination of these modifications ultimately leads to the "rational design" of the improved molecule used in the context of the present invention. The present invention also includes strategies aimed at further improving compounds that have only partially undergone "directional evolution" or "directional mutagenesis."

[0059] Therefore, another important aspect of the present invention is a method for identifying improved compounds that bind to p53, or preferably bind to p53 to inhibit the interaction between FOXO4 and p53 in cells, a) A step of preparing at least one compound according to the present invention, b) a step of appropriately modifying the above compound a), A step of identifying at least one of the following: the binding of the at least one compound described in c)b) above to p53 or a fragment thereof, the stability of the at least one compound described above, and the binding of FOXO4 or a fragment thereof to p53 or a fragment thereof in the presence of the at least one compound described above, compared with the binding of FOXO4 or a fragment thereof to p53 or a fragment thereof in the absence of the compound described above. d) Based on what was identified in step c), a step of identifying an improved compound that binds to p53, or preferably binds to p53 and inhibits the interaction between FOXO4 and p53 in cells, compared to the compound provided in step a), This method includes [something].

[0060] A preferred method according to the present invention is one in which the above-mentioned bond of at least one of the above-mentioned compounds is identified / detected as specific to and / or specific to p53 or the above-mentioned fragment.

[0061] More preferably, the method according to the present invention further comprises testing the compound as identified for its ability to induce apoptosis in senescent cells, scar cells, or cancer cells or tumor cells and / or kill these cells, preferably comprising identifying caspase activity.

[0062] In the context of the present invention, the above method can be applied to a compound several times in succession for the purpose of "rational design" of an improved molecule, and for further improving a compound that has only partially undergone "directional evolution" or "directional mutagenesis."

[0063] Following the provision of a compound (e.g., a peptide), the compound can be modified, for example, after appropriate chemical synthesis of the compound. In general, many methods for modifying the compounds of the present invention are known to those skilled in the art and are disclosed in the literature. Modification of compounds is usually classified into several categories, for example, a) mutation / change of amino acids to different amino acids, b) chemical modification of amino acids, for example, through the addition of further chemical groups, c) modification of the structure of amino acids (e.g., from L-type to D-type) or modification of their bonds (e.g., introduction of retroinverse bonds), d) alteration of the length of the compound, and e) addition of further groups (including marker groups, labels, linkers, or carriers, such as chelators) to the molecule.

[0064] Evolutionary differences between FOXO1 / 3 / 6 and FOXO4 suggest that several amino acids can still be modified to create improved inhibitory compounds exhibiting desired binding activity and ultimately inhibitory activity. Analysis of the FOXO4-FH fragment and the TP53-TAD2 domain showed that the negative charge of TP53 and the hydrophobic Tyr intervene in the binding to FOXO4. Therefore, the amino acids of the peptide according to this invention can be mutated to positive amino acids to benefit the strength of the interaction. Examples include lysine (K), arginine (R), and histidine (H). NMR experiments also showed that the negatively charged amino acid of TP53 contributes to the interaction with FOXO4 around the SQ motif (site). This site can be phosphorylated, and to prevent this, it can be mutated to a small amino acid such as alanine (A), glycine (G), or serine (S). Methods for introducing such mutations are known to those skilled in the art, and such methods include introducing changes during the chemical synthesis of peptides, or modifying the peptide encoding the nucleotide sequence by genetic methods, such as oligonucleotide-based mutagenesis or mutagenesis including PCR. Random mutagenesis is also possible. Further guidance regarding predicted amino acid changes is provided above for X1-X 12found in the context of the definitions relating to these, which are the remaining X 13 ~X 18 are also readily applicable to the definitions of.

[0065] Furthermore, amino acids can be modified by chemical modification, for example, through the addition of further chemical groups during synthesis or via post-translational or post-synthetic modifications. Methods for modifying amino acids are known in the current state of the art and are summarized, for example, in Christopher D. Spicer & Benjamin G. Davis. Selective chemical protein modification Nature Communications volume 5, Article no.: 4740 (2014) or Sakamoto S, Hamachi I. Recent Progress in Chemical Modification of Proteins. Anal Sci. 2019 Jan 10;35(1):5-27.

[0066] Similarly, it is possible to modify the amino acid structure (e.g., from L-form to D-form) or the bonds between amino acids (e.g., introducing retro-inverse bonds during synthesis). NMR also showed that the three N-terminal amino acids LTL are not necessary for the interaction. Thus, the length of the compound can be modified, for example, by removing these three amino acids.

[0067] Finally, chemical and / or functional groups added to the molecule, such as marker groups, labels, linker amino acids, staples, cysteine ​​crosslinks, glycolation sites, ubiquitination and / or pegylation sites, linkers, or carriers, such as chelators. Similarly, the introduction or addition of amino acids (staples) that enable the formation of FOXO4-derived DRI peptide dimers or polymers is included. Furthermore, stabilization modifications aimed at improving the stability and / or efficacy of the FOXO4-derived DRI peptide in eliminating senescent cells may be included (e.g., so-called end-capping). The stabilization of the FOXO4 peptide can also be achieved by mutating the site(s) where the peptide is normally cleaved, such as the NG site. Staple formation can be achieved by introducing two α-pentenylglycine or α,a'-pentenylalanine residues to crosslink the i-residue with the i+4 residue or the i-residue with the i+7 residue within the peptide. These residues are used to create a total hydrocarbon, alkene-containing, desmethyl-restricted, i.e., staple. Other strategies that can be used to crosslink or restrict peptides include lactam crosslinking, hydrogen bond substitutes, photoswitches, thioethers, and triazoles introduced by "click" chemical reactions.

[0068] In the next step, the modified compound is tested for at least one of the bindings between the at least one of the above compounds and p53 or its fragment. As discussed herein, the properties of the compound that binds to p53 or its (such as TP53) binding fragment are essential for all applications of the compound, for them to be therapeutic or diagnostic compounds. In the context of the present invention, “improved” binding encompasses both scenarios in which the modified compound binds to the same extent as the unmodified (i.e., starting) compound despite being modified (e.g., by dimerization or by the addition of a marker or other group). Preferably, the compound is modified to exhibit stronger binding to the target, i.e., p53 or its binding fragment. Also preferably, the compound exhibits longer binding to the target, i.e., p53 or its binding fragment, for example, because the in vitro or in vivo stability of the modified compound is improved. Even more preferably, the compound exhibits reduced phosphorylation of the modified compound. Also included are combinations of at least two of these properties.

[0069] Assays for detecting the binding of a compound to a target (i.e., p53 and / or its binding fragments) are known to those skilled in the art, and such assays preferably include mass spectrometry, NMR assays, pull-down assays, and the like.

[0070] Furthermore, the test includes a competitive assay, which compares the properties of the modified compound in the presence of at least one of the above compounds with the binding of FOXO4 or its fragment to p53 or its fragment in the absence of the above compound.

[0071] A more preferable method according to the present invention is one in which the above-mentioned binding of at least one of the above-mentioned compounds is identified / detected as specific to and / or specific to p53 or its fragment. As described above, the property of a compound to bind to p53 or its binding fragment (e.g., TP53) is essential for all uses of the compound, for them to be therapeutic or diagnostic compounds. Ideally, the binding is specific to the intended target, namely p53 or its binding fragment, or at least substantially or essentially specific. This reduces or avoids any undesirable side effects in the pharmaceutical use of the compound, and reduces or avoids any background or false-positive results in the diagnostic use.

[0072] In the final step of this method, the improved compound is identified, in a preferred embodiment of the present invention, as binding to p53, or preferably binding to p53 to inhibit the interaction between FOXO4 and p53 in cells, based on the above identification in step c), compared to the compound provided in step a). Although this function appears essential for the therapeutic function of the compounds according to the present invention by eliminating senescent cells, such properties / functions may not be strictly required for the improved diagnostic molecules according to the present invention.

[0073] The identification of such improved compounds may involve detecting the efficacy of these compounds (e.g., FOXO4-peptide) in binding to TP53 and eliminating senescent cells, and optionally identifying the structural requirements that determine this property; and / or detecting the selectivity of these compounds (e.g., FOXO4-peptide) in eliminating senescent cells, and likewise optionally identifying the structural requirements that determine this property. FOXO4 is evolutionarily distinct from FOXO1 / 3 and 6, as well as from its homologous FOXO species, in several unique residues. We have never found any FOXO1 or FOXO3 focus, nor do we have any evidence that FOXO1 and 3 are important in senescent cell viability. Therefore, we analyzed whether these amino acids at least partially explain the efficacy of FOXO4-peptide in eliminating senescent cells. It was confirmed that D-retroinverse peptides based on a domain similar to that of FOXO4 showed selectivity in eliminating senescent cells, while those based on the sequences of FOXO1 or FOXO3 did not.

[0074] More preferably, the method according to the present invention further comprises testing the identified compounds for their activity in inducing apoptosis in senescent cells, scar cells, cancer cells, or tumor cells and / or killing these cells, preferably including the identification of caspase activity. As already described above, those skilled in the art will also be aware of the existence of in vitro standard assays for identifying the degree of apoptosis in cell cultures, such as tests for evaluating the levels of cytoplasmic cytochrome C (a marker of apoptosis) and TUNEL (a marker of apoptosis). Using such standard assays, those skilled in the art can easily evaluate and compare the apoptosis-inducing activity of various compounds for different cell types or different developmental stages of cells, for example, between senescent and non-senescent cells. Other standard apoptosis assays are the annexin V assay and cleavage caspase 3 staining. The assays preferably include identification of decreased viability, such as increased caspase 3 / 7 activity, decreased mitochondrial cytochrome C, increased TUNEL positivity, increased extracellular annexin V positivity, and / or increased propidium iodide inclusion by cells, and / or decreased calcein AM incorporation, or MTS viability assays. Cell viability is essentially the opposite of apoptosis, and to detect it, MTT assays (colorimetric assays to assess cell viability), ATP detection assays, real-time cell density (e.g., xCELLigence) assays, or colony formation assays are available. Further information on the assays used can be obtained from the literature, for example, from Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007;35(4):495-516. Preferably, if the peptide or improved peptide according to the present invention causes killing, elimination, removal, or reduction of cell viability in at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of cells in a senescent cell culture, the peptide is considered to exhibit apoptosis-inducing activity in senescent cells.Preferably, the peptide according to the present invention selectively exhibits apoptosis-inducing activity in senescent cells, i.e., not in non-senescent cells. The peptide according to the present invention preferentially induces apoptosis in senescent cells over apoptosis in non-senescent cells by at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3, 4, 5, or higher multiples.

[0075] The peptides according to the present invention are preferably isolated. The peptides according to the present invention are preferably produced using appropriate peptide synthesis.

[0076] In one embodiment of the method according to the present invention, the above-mentioned FOXO4 or its p53-binding fragment, p53 or its FOXO4-binding fragment, and / or compound (peptide) are labeled, for example, by fluorescent labeling, detectable labeling, stable isotope labeling, and / or mass labeling. Such labels are known in the literature and include FITC, Alexa, Dylight, FAM, and others. Detectable labels may be recognized by antibodies, streptavidin / avidin, or are based on enzymatic reactions that yield a detectable product. Mass labeling can be achieved by adding isotopes or other suitable mass markers. In principle, any label available for experimental and therapeutic / imaging purposes is also available.

[0077] A more preferable method according to the present invention is one in which the above-mentioned FOXO4 or its fragment, and / or p53 or its fragment, are expressed in cells by recombination. Methods for expressing peptides by recombination and for isolating and purifying the products produced by the above recombination are known in the art and are described in the respective literature. In general, any suitable cells can be used to produce the above-mentioned FOXO4 or its fragment, and / or p53 or its fragment by recombination, preferably cells selected from the group of cancer cells, senescent cells, human non-embryonic stem cells, yeast cells, bacterial cells, and recombinant host cells expressing FOXO4 or its p53-binding fragment, wherein the recombinant host cells optionally express p53 or its FOXO4-binding fragment, i.e., express both or more of the constructs to be used.

[0078] Next, yet another aspect of the present invention relates to a screening tool for screening compounds according to the present invention that bind to p53, preferably by binding to p53 and inhibiting the interaction between FOXO4 and p53 in cells, the screening tool comprising isolated cells expressing FOXO4 and / or cells expressing its p53-binding fragment, the cells optionally expressing p53 and / or cells expressing its FOXO4-binding fragment.

[0079] The screening tool of the present invention is an important part for carrying out the method according to the present invention and can also be used as a first step to verify the pharmaceutically active properties of a compound in vivo. Preferably, the above FOXO4 or its fragment and / or p53 or its fragment are expressed by recombination in the above cells. Methods for expressing peptides by recombination and isolating and purifying the products produced by the above recombination are known in the art and are described in the respective literature. In general, any suitable cells can be used to produce the above FOXO4 or its fragment and / or p53 or its fragment by recombination, preferably cells selected from the group of cancer cells, scar cells, senescent cells, human non-embryonic stem cells, yeast cells, bacterial cells, and recombinant host cells expressing FOXO4 or its p53 binding fragment, wherein the recombinant host cells optionally express p53 or its FOXO4 binding fragment, i.e., express both or more of the constructs to be used. In one embodiment of the tool according to the present invention, the above-mentioned FOXO4 or its p53-binding fragment, p53 or its FOXO4-binding fragment, and / or compound (peptide) are labeled, for example, by fluorescent labeling, detectable labeling, and / or mass labeling. As described above, such labels are known in the literature and include FITC, Alexa, Dylight, FAM, and others. Detectable labels may be recognized by antibodies, streptavidin / avidin, or based on enzymatic reactions that yield a detectable product. Mass labeling can be achieved by adding isotopes or other suitable mass markers.

[0080] Preferably, the screening tool according to the present invention comprises a peptide described in sequence RKK PRKGGSRRNAWGNQSYAELISQAIESAPEKRLTLAQIYEWMVRTVPYFKDKGDSNSSAGWKNSIRHNLSLHSKFIKVHNEATGKSSWWMLN (SEQ ID NO: 32) or PRKGGSRRNAWGNQSYAELISQAIESAPEKRLTL (SEQ ID NO: 33), preferably a fragment of FOXO4 which is the whole D-peptide, and a fragment of p53 which is the peptide described in sequence AMDDLMLSPDDIEQWFTEDPGP (SEQ ID NO: 34).

[0081] Another important aspect of the present invention relates to the use of the compounds according to the present invention in diagnostics, for example, as diagnostic compounds. Clearly, the compounds according to the present invention are identified / used for the purpose of binding to p53 or p53 fragments, preferably to further inhibit the interaction between FOXO4 and p53 in cells. These properties are also utilized in diagnostic contexts. More preferably, the binding of at least one of the above compounds is specific to and / or specific to p53 or p53 fragments. This reduces or avoids any background or false-positive results in diagnostic applications. The compounds (peptides) according to the present invention are preferably labeled, for example, by fluorescent labeling, detectable labeling, and / or mass labeling. As described above, such labels are known in the literature and include FITC, Alexa, Dylight, FAM, and others. Detectable labels may be recognized by antibodies, streptavidin / avidin, or based on enzymatic reactions that result in detectable products. Mass labeling can be achieved by adding isotopes or other suitable sample markers. However, diagnostic methods can be performed without labeling, and binding can be detected directly or indirectly, for example, using NMR.

[0082] One specific example of a diagnostic use or diagnostic method is a method for detecting the expression of p53 or its fragments in cells, which involves detecting p53 or its fragments using the compound according to the present invention, and if the binding is increased compared to control cells that do not express p53 or its fragments, it indicates that p53 or its fragments are expressed in the cells. Due to the high specificity of the compound according to the present invention, the compound provides an improved molecule for diagnostic use. The diagnosis may further include detecting p53-related diseases or conditions, such as cancer, or other conditions caused by conditions resulting from senescent cells, such as age-related diseases, kidney diseases, non-alcoholic steatohepatitis (NASH) / non-alcoholic fatty liver disease (NAFLD), hepatic fibrosis, idiopathic pulmonary fibrosis (IPF), amyotrophic lateral sclerosis (ALS), osteoarthritis, COPD, musculoskeletal diseases, and cognitive decline.

[0083] In the context of the present invention, the term "about" means including ±20%, preferably ±10%, of a given amount, unless otherwise specified.

[0084] Preferably, the method according to the present invention is suitable for automation and, preferably, is carried out in an automated and / or high-process manner. Typically, this involves the use of chips and their respective machines, such as robots. Automation is particularly preferred in the case of identification and / or screening of improved compounds. Similarly, the use of the screening tool of the present invention can also be included in automation.

[0085] Next, yet another important aspect of the present invention relates to a method for producing a pharmaceutical composition for treating or inhibiting senescent cells in a subject, the method comprising the steps of: formulating compounds according to the present invention to make a suitable pharmaceutical composition; or performing a method according to the present invention to identify an improved compound that preferably inhibits the interaction between FOXO4 and p53 in cells; and formulating the identified compound to make a suitable pharmaceutical composition. The present invention also relates to a pharmaceutical composition for treating or inhibiting senescent cells in a subject, obtained by the above method according to the present invention.

[0086] When used herein, the term "pharmaceutical composition" refers to a composition made under conditions that make it suitable for administration to mammals, preferably humans, for example, a pharmaceutical composition made under GMP conditions. The pharmaceutical composition according to the present invention may contain pharmaceutically acceptable excipients, which include, without particular limitation, stabilizers, bulking agents, buffers, carriers, diluents, vehicles, solubilizers, and binders. As will be obvious to those skilled in the art, the selection of appropriate excipients depends on the route of administration and dosage form, as well as the active ingredient and other factors. The pharmaceutical composition according to the present invention is preferably suitable for parenteral administration.

[0087] Preferably, the pharmaceutical composition according to the present invention comprises a mixture of compounds and / or a mixture of at least one of the above compounds and a further pharmaceutically active ingredient. The term "pharmaceutically active ingredient," as used herein, refers to a compound that inhibits or prevents cell viability and / or function, and / or causes cell destruction (cell death), and / or exerts an antitumor / antiproliferative effect, for example, directly or indirectly preventing the development, maturation, or spread of tumor cells. This term also includes agents that cause only a cell division arrest effect and not merely a cytotoxic effect. This term also includes alkylating agents, such as platinum drugs (e.g., cisplatin, carboplatin, and oxaliplatin), antimetabolites, such as 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (Xeloda), cladribine, clofarabine, cytarabine (Ara-C), phloxuridine, fludarabine, gemcitabine (Gemzar), hydroxyurea, and methotrexate, antitumor antibiotics, preferably doxorubicin, topoisomerase inhibitors, mitotic inhibitors, corticosteroids, angiogenesis inhibitors, tyrosine kinase inhibitors, protein kinase A inhibitors, members of cytokine families, and radioisotopes. In the context of the present invention, taxanes, such as paclitaxel or docetaxel, are also included as chemotherapeutic agents.

[0088] In another embodiment, the present invention provides at least one peptide, pharmaceutical composition, or nucleic acid according to the present invention, which is used in pharmaceuticals, or used as a pharmaceutical, or used in the treatment of a pathological condition, disease, or disorder.

[0089] When used herein, the term “disease or condition caused by senescent cells, scar cells, and / or cancer cells” refers to any disease or condition in a mammal, preferably a human subject, in which the presence of senescent cells, i.e., cellular senescence, in the mammal, preferably a human subject is associated with the disease or condition in the subject.

[0090] Normal and pathological degenerative age-related traits (loss of function) and cancer (gain of function) have been established to be causally related to cellular senescence. Senescent fibroblasts have been linked to decreased breast milk production, senescent pulmonary artery smooth muscle cells to pulmonary hypertension, senescent skin cells to epidermal thinning and decreased collagen content, senescent astrocytocytes to Alzheimer's disease and Parkinson's disease, and senescent chondrocytes to osteoarthritis.

[0091] Conditions and diseases that have been associated with cellular senescence include, but are not limited to, atherosclerosis, pulmonary emphysema, diabetic ulcers, kidney disease (see, e.g., Valentijn FA, et al. Cellular senescence in the aging and diseased kidney. J Cell Commun Signal. 2018 Mar; 12(1): 69-82), kyphosis, osteoporosis, macular degeneration, COPD and insulin resistance, diabetes, obesity, laminopathy (e.g., Hutchinson-Gilford progeria), hernia, sarcopenia and cachexia, arthritis, scoliosis, and cancer. Therefore, genetically eliminating senescent cells significantly improves fitness and reduces aging parameters, as demonstrated in aging-accelerated mouse models (Baker et al., 2011. Nature 479(7372):232-6). This proof-of-concept evidence was actually obtained in a genetic manner that has little therapeutic applicability. The present invention provides novel and improved compounds that target senescent cells for therapeutic purposes and can be used to treat diseases associated with cellular senescence, as well as methods for identifying such compounds.

[0092] Another embodiment relates to at least one compound or pharmaceutical composition used for prevention or treatment according to the present invention, wherein the conditions to be prevented and / or treated are selected from conditions caused by senescent cells, age-related diseases, kidney diseases, non-alcoholic steatohepatitis (NASH) / non-alcoholic fatty liver disease (NAFLD), hepatic fibrosis, idiopathic pulmonary fibrosis (IPF), amyotrophic lateral sclerosis (ALS), arthritis, osteoarthritis (like osteoarthritis), COPD, musculoskeletal diseases, cognitive impairment, atherosclerosis, pulmonary emphysema, diabetic ulcers, kyphosis, osteoporosis, macular degeneration, COPD and insulin resistance, diabetes, obesity, laminopathy, such as Hutchinson-Gilford progeria, hernia, sarcopenia and cachexia, scoliosis, and cancer.

[0093] Preferably, the above treatment is a combination therapy with an anticancer chemotherapy agent or other standard therapeutic agents for the respective disease or condition, or the above treatment is applied to senescent cells, scar cells, and / or cancer cells that have survived the above treatment after being pretreated with an anticancer chemotherapy agent or other standard therapeutic agents for the respective disease or condition.

[0094] As a result, another aspect relates to a method for treating or preventing diseases or symptoms caused by senescent cells, scar cells, age-related diseases, kidney diseases, non-alcoholic steatohepatitis (NASH) / non-alcoholic fatty liver disease (NAFLD), hepatic fibrosis, idiopathic pulmonary fibrosis (IPF), amyotrophic lateral sclerosis (ALS), arthritis, osteoarthritis (like osteoarthritis), COPD, musculoskeletal diseases, cognitive decline, atherosclerosis, pulmonary emphysema, diabetic ulcers, kyphosis, osteoporosis, macular degeneration, COPD and insulin resistance, diabetes, obesity, laminopathy, such as Hutchinson-Gilford progeria, hernia, sarcopenia and cachexia, scoliosis, and cancer, in subjects requiring treatment or prevention, the method comprises administering at least one compound or pharmaceutical composition according to the present invention to the subject in an effective amount.

[0095] The method according to the present invention is preferable in which the above treatment is a combination therapy with an anticancer chemotherapy agent or other standard therapeutic agents for the respective disease or condition, or the above treatment is applied to senescent cells, scar cells, and / or cancer cells that have survived the above treatment after being pretreated with an anticancer chemotherapy agent or other standard therapeutic agents for the respective disease or condition.

[0096] "Treatment" means the reduction and / or remission of the symptoms of the disease. Effective treatment achieves, for example, the removal of senescent cells, and a reduction in tumor volume and the number of cancer cells. Treatment can also prevent (prevent) and reduce the number of senescent cells, reduce the spread of cancer, and, for example, affect metastasis and / or its formation. Treatment may be a naive treatment (when no other treatment for the disease has yet been initiated) or a treatment after initial treatment (for example, after surgery or recurrence). Treatment may also be a combination therapy, which may involve, for example, chemotherapy, surgery, and / or radiation therapy. Treatment may be given to subjects, particularly humans, who have or are suspected of having a disease or condition in which the removal of senescent cells would be beneficial.

[0097] In the context of the present invention, it has been unexpectedly discovered that the compounds according to the present invention can be used advantageously, as illustrated in the examples and drawings of the present invention, and are most effective in the case of treatment-resistant cancer. In addition to being effective, the compounds of the present invention have been shown to be not only cell division inhibitory but also cytotoxic to cancer cells. The peptide is most effective in cells that have been pretreated with a cytotoxic agent as part of chemoradiotherapy and have survived this treatment. Therefore, treatment using the peptide can be performed after / following a cytotoxic first treatment, such as chemo / radiotherapy, dietary stress, exogenous stress, or oxidative stress, or as a combination therapy, as described above, i.e., administered together rather than sequentially or to act together. This is particularly efficient when used in combination with small molecule inhibitors such as BRAF inhibitors.

[0098] The present invention further relates to a kit comprising a first container containing a compound according to the present invention and a second container containing a chemotherapeutic agent. The kit may appropriately include instructions for administration to mammals, preferably human subjects. Human subjects are preferably those suffering from or suspected of suffering from diseases or conditions caused by senescent cells, scar cells, age-related diseases, kidney diseases, non-alcoholic steatohepatitis (NASH) / non-alcoholic fatty liver disease (NAFLD), hepatic fibrosis, idiopathic pulmonary fibrosis (IPF), amyotrophic lateral sclerosis (ALS), arthritis, osteoarthritis (like osteoarthritis), COPD, musculoskeletal diseases, cognitive decline, atherosclerosis, pulmonary emphysema, diabetic ulcers, kyphosis, osteoporosis, macular degeneration, COPD and insulin resistance, diabetes, obesity, laminopathy, such as Hutchinson-Gilford progeria, hernia, sarcopenia and cachexia, scoliosis, and cancer. The compounds and chemotherapeutic agents of the present invention can be incorporated into a single dosage form, for example, a pharmaceutical composition, but preferably into multiple dosage forms, with the compounds of the present invention in one container and the chemotherapeutic agents in a separate container. In a kit for use according to the present invention, the compounds of the present invention and the chemotherapeutic agents are preferably administered simultaneously. Preferably, the peptide is administered after or following the administration of the chemotherapeutic agents. Preferably, the compounds of the present invention are administered as an adjuvant in a manner that can eliminate, kill, or reduce the viability of cells that have senescent as a result of treatment with chemotherapeutic agents. Unexpectedly discovered, the compounds of the present invention can reduce the off-target effects of existing chemotherapeutic agents. The kit according to the present invention preferably includes instructions for a dosing regimen to obtain the optimal combination effect of the compounds of the present invention and the chemotherapeutic agents.

[0099] In another embodiment of the present invention, the present invention then relates to a diagnostic kit, comprising materials for carrying out the method according to the present invention as described herein, together with optionally auxiliaries, in one or separate containers, and / or instructions for carrying out the above method according to the present invention. The kit may include the compound and / or the compound as identified herein. Furthermore, it may include dyes, antibodies, and other components for the detection assay as disclosed above. The kit may include the compound and / or the compound as identified herein in the labeled form as disclosed above.

[0100] The present invention will be further described here in the following embodiments with reference to the accompanying drawings, but the present invention is not limited thereto. For the purposes of the present invention, all references cited herein constitute part of this specification by reference.

[0101] Sequence IDs 1-4, 7-31, and 43-66 represent the peptides of the present invention, or parts thereof.

[0102] Sequence IDs 5, 6, and 42 show control peptides, e.g., FOXO4-DRI / CL03001.

[0103] Sequence IDs 39 and 40 show the amino acid sequences of human foxo4 and p53, respectively. [Examples]

[0104] In the context of the present invention, the terms FOXO4-DRI or CL03001, when relating to a peptide and / or a control peptide, refer to a peptide having the D-amino acid sequence LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRPPPRRRQRRKKRG (SEQ ID NO: 6) or the L-amino acid sequence PRKGGSRRNAWGNQSYAELISQAIESAPEKRLTL (SEQ ID NO: 42).

[0105] Cell culture: Human IMR90, WI38, and RPE cells were grown at 37°C in Dulbecco's modified Eagle medium (DMEM; Lonza) containing 10% FCS and 1% penicillin / streptavidin, using 3.0% O2 and 5.0% CO2. To induce senescence, the cells were irradiated with 10 Gy of ionizing radiation (Gammacell 1000) and left for at least 10 days to allow senescence to occur.

[0106] GBM8 cells were cultured in Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM F-12). Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 was high in glucose, contained L-glutamine, and was phenol red-free. It was supplemented with 1% (10 mM) HEPES, 1% penicillin / streptomycin, 2% B-27 (50X), 5 μg / ml porcine intestinal mucosa-derived heparin sodium salt grade IA, 20 ng / ml recombinant human fibroblast growth factor (rhFGF), and 20 ng / ml recombinant human epidermal growth factor (rhEGF). The cells were placed in plastic containers coated with Matrigel matrix (20-fold dilution in DMEM F-12) and cultured in a humidified incubator at 37°C, 5% CO2, and 3% O2.

[0107] To perform viability and apoptosis assays, cells were divided and placed in 96-well plates, and treated with peptides after 2 days. Non-senescent and cancer cells were seeded at 1500 cells / well, while senescent cells were seeded at 8000 cells / well.

[0108] Organoid culture: The organoids were grown in a humidified incubator at 37°C and 5% CO2. All organoids were cultured in Matrigel (Corning) droplets in advanced DMEM / F12 (Lonza). Advanced DMEM / F12 was supplemented with 1% glutamax, 1% penicillin / streptomycin, 1% (10 mM) HEPES, 10% Noggin-conditioned medium, 2% B-27 (50X; (Thermo / Life Technologies), N-acetylcysteine ​​((Sigma-Aldrich, 1.25 mM)), A83-01 (Tocris, 500 nM), and SB203580 (Invitrogen / Life Technologies, 3 μM). For viability and apoptosis assays, organoids were subcultured by resuspending them in ice-cold medium, followed by centrifugation in 15 ml test tubes at 4°C. The resulting pellet was trypsin-treated at 37°C for 5 minutes to obtain single cells, which were then washed twice with advanced DMEM / F12 medium. The cells were then resuspended on Matrigel and 5 Microwaves were seeded in 96-well plates using μl droplets. After 15 minutes, 100 μl of fresh medium was added to each well. Two days after seeding, the organoids were treated with peptides and chemotherapy.

[0109] Caspase assay: For the caspase assay, caspase-Glo3 / 7 assay reagent (Promega) was added to the culture medium two days after treatment. The plate was then wrapped in aluminum foil and incubated on a shaker at room temperature for 1 hour, followed by measurement with a luminescent plate reader.

[0110] MTS assay: Six days after treatment, cells were incubated with 10 μl of CellTiter 96® AQueous One Solution cell proliferation assay (Promega) at 37°C for 1 hour, and then subjected to an MTS assay. Subsequently, absorbance was measured at 490 nm using a Spectramax M5e.

[0111] Life / death assays using organoids: Live / death assays were performed on both larger organoids three days after peptide and / or chemotherapy treatment, or on organoids treated as single cells and grown for two weeks prior to the assay. To perform the assay, calcein AM (1:1000) and propidium iodide (PI; 1:100) were added to the organoid culture medium, and live and dead cells were detected, respectively. The organoids were then imaged using a Zeiss Cell Observer microscope, and the signal was quantified using FIJI.

[0112] Live filming: Organoids and IMR90 cells were grown in 96-well cell culture plates, and either 488 IncuCyte caspase 3 / 7 reagent for apoptosis (Sartorius) or IncuCyte caspase 3 / 7 red apoptosis assay reagent (Essen Bioscience) was added to each well. After adding the peptide directly, the plate was transferred to a Zeiss Cell Observer microscope equipped with thermal and CO2 control compartments. Live imaging was then initiated. During live imaging, images were recorded every 2 hours for 60 hours. The images were processed using Zen image processing software (Zeiss).

[0113] Alternatively, live imaging was performed using an LSM880 confocal microscope. In this experiment, organoids were seeded in glass-bottom cell culture dishes and incubated with calcein AM (Sigma-Aldrich) and IncuCyte caspase 3 / 7 red apoptosis assay reagent (Essen Bioscience) to measure cell viability. Imaging was started after adding the peptide directly.

[0114] mouse The mouse experiments were conducted after approval from the Dutch Animal Ethics Committee. These experiments included male NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ / J mice (Charles River) were used. Human CRC29 colorectal cancer organoids containing firefly luciferase were transplanted into the cecum to induce primary tumor formation at this site. The transplantation was performed under ketamine and dexmedetomidine anesthesia. The animals were also administered capprofen 30 minutes before surgery and the following day. Fourteen days after transplantation, the mice were sedated with isoflurane and imaged with a BioSpace Imager. Subsequently, the animals were treated with either PBS or 5'-fluorouracil (5-FU) at a dose of 50 mg / kg via intravenous injection. The initial dose of CL04183 (2.5 mg / kg) was administered intravenously one week after 5-FU treatment, and this treatment was repeated at two and four days later. Four weeks after transplantation, the mice were imaged again to identify the therapeutic effect, and then sacrificed. All organs were collected, re-imaged with BioSpace Imager, and luciferase signals were quantified using M3 Vision software.

[0115] immunohistochemistry Paraffin sections of liver and lung tissue were rehydrated with decreasing ethanol concentration, washed with TBS, and boiled in 10 mM sodium citrate buffer (pH 6) for 20 minutes to unmask the antigen. After allowing the slides to cool for 30 minutes, the tissue was permeabilized with 0.2% TX-100-containing TBS at room temperature for 5 minutes. Subsequently, the sections were washed with TBS and incubated for 1 hour in block buffer containing 2% w / v secondary antibody-compatible serum (e.g., donkey or goat) and 0.1% fish gelatin-containing 1% BSA. The sections were then surrounded with a water-repellent pen and incubated overnight at 4°C with primary antibody diluted in TBS / 1% BSA. The following day, the tissue was washed three times with TBS and incubated for 1 hour with fluorescently labeled secondary antibody diluted in block buffer (including nuclear staining with Hoechst 33342). Next, the slides were washed twice with TBS, incubated in Sudan Black solution for 20 minutes to reduce background, and then washed with desalted water. The sections were then mounted using Vectashield and imaged using an LSM880 Zeiss confocal microscope.

[0116] Apoptosis staining TUNEL assays were performed on lung metastases to identify apoptosis induction after treatment. Rehydrated sections were treated with 20 μg / ml ProtK in PBS for 15 minutes and then permeabilized with 0.1% sodium citrate containing 0.1% Triton X-100. Subsequently, the tissue was labeled with a 10% TUNEL enzyme-labeled solution (ROCHE) at 37°C for 1 hour. The nuclei were labeled with Hoechst 33342 (ThermoFisher), and the slides were mounted using soft set mounting medium (Vectashield). Images were acquired using an LSM880 confocal microscope (Zeiss), and the percentage of TUNEL-positive cells was analyzed using FIJI.

[0117] Protein expression and purification: Expression constructs of human p53 fragments 1-312 (p53-TADBD), 94-312 (p53-DBD), 1-94 amino acid fragment (p53-TAD), and 37-57 amino acid fragment (p53-TAD2) were generated by synthesizing the corresponding optimized p53 cDNA constructs and inserted into the pETM11-ZZ-His6 vector using the NcoI / BamHI restriction site (Genscript). The inventors also generated optimized cDNA expression constructs (Genscript) for human foxo4 fragments 86-208 (FOXO4-FH) that were inserted into the pETM11-ZZ-His6 vector. The inventors used three different chemically competent E. coli strains: E. coli BL21(DE3) and E. coli BL21-(DE3 Star) for protein expression, and E. coli TOP10 for plasmid DNA amplification.

[0118] Protein expression and purification were performed using a protocol known to those skilled in the art, as published.

[0119] Fluorescence polarization: A 116 μl solution of a specific peptide (in the range of 1 μM to 100 μM) was prepared. Subsequently, 4 μl of FITC-labeled p53 peptide (stock concentration 15 μM, final concentration 500 nM) was added. Then, 35 μl was transferred to each well of a 384-well plate. Measurements were performed in sets of three.

[0120] Data was obtained using a ClarioStar Plus plate reader. Endpoint measurements were performed by flashing 200 times per well. Gain and focus height adjustments were made for each measurement using a 482 nm excitation filter and a 530 nm fluorescence filter. Fluorescence intensity, parallel fluorescence polarization, and perpendicular fluorescence polarization were recorded.

[0121] Data analysis was performed using MARS Version 3.4 (BMG), Microsoft Excel, and GraphPad Prism Version 8.

[0122] NMR chemical shift mapping: Prepare a 5 mm NMR tube containing 500 μl of 100 μM p53-TAD2. 1 H, 15 N HSQC NMR spectra were recorded. Then, peptides were added in stepwise increasing amounts, followed by separate measurements after each step. 1 H, 15 N HSQC spectra were measured. NMR spectra were acquired using a 600 MHz Bruker Avance NOE NMR spectrometer equipped with a TXI 600S3 probe head. Data acquisition and processing were performed using Topspin 3.5 and Topspin 4.0 (Bruker). Data analysis, peak selection, and assignment were performed using ccpNMR.

[0123] Table 1: Selection of peptides according to the present invention [Table 1-1] [Table 1-2] [Explanation of Symbols]

[0124] Figure 2A) % p53 binding p53 binding % Unbound Figure 2B) HEK293T lysate HEK293T lysate Streptavidin beads Figure 3 % Caspase activity % Ctrl + FOXO4-DRI comparison + FOXO4-DRI Ctrl + CL04009 (Contact + CL04009) Sen + FOXO4-DRI Senescent cells + FOXO4-DRI Sen + CL04009 Senescent cells + CL04009 Concentration in μM (in μM units) % Viability (AqueousOne) Viability%(AqueousOne) Sen - FOXO4-DRI Senescent Cells-FOXO4-DRI Sen - CL04009 Senescent Cells - CL04009 Concentration in μM (in μM units) Figure 4 TAD2 peptide binding Signal (au) Signal (arbitrary unit) Figure 5 Time in days (in days) Figure 6 Start Start 24h 24 hours 60h 60 hours Red = Active Caspase-3 / 7 (=apoptosis) (=apoptosis) Figure 7 % Caspase activity % Ctrl + CL04022 (Contact + CL04022) Ctrl + CL04088 (Contact + CL04088) Sen + CL04022 Senescent cells + CL04022 Sen + CL04088 Senescent cells + CL04088 Concentration in μM (in μM units) % Viability (AqueousOne) Viability%(AqueousOne) Concentration in μM (in μM units) Figure 8 % Viability (AqueousOne) Viability%(AqueousOne) Concentration in μM (in μM units) % Viability (AqueousOne) Viability%(AqueousOne) Concentration in μM (in μM units) Figure 9A) % Caspase activity % Figure 9C) % Caspase activity % Figure 10A) MSI CRC line 1 MSI CRC stock 1 MSI CRC line 2 MSI CRC stock 2 MSI CRC line 3 MSI CRC stock 3 % Viability (AqueousOne) Viability%(AqueousOne) [CL04022 in uM] [CL04022 (in μM units)] Figure 10B) % Viability (AqueousOne) Viability%(AqueousOne) Figure 11A) Amount of Peptide [%] Time (h) Elapsed time (hours) (Figure 11B) Compound Matrix Matrix (Figure 12A) Oxaliplatin Caspase Figure 12B) (continued) 5'-Fluorouracil Alive raw Dead Figure 13 % Organoid outgrowth % [Compound (μM)] (Figure 14A) % Viability (AqueousOne) Viability%(AqueousOne) Non-senescent Senescent aging (Figure 14B) % Viability (AqueousOne) Viability%(AqueousOne) Non-senescent Senescent aging Figure 15 % Viability (AqueousOne) Viability%(AqueousOne) Viability (AqueousOne) Figure 16A Alive / Dead Untreated High dose '5-FU Figure 16B Relative expression Hours Calcein-AM Caspase Figure 16C % Viability (AqueousOne) [Compound] (μM) Figure 17 % Caspase activity [Compound] (uM) Figure 18 Before Side view Top view Crop Figure 19A CL04183 (1 wk) Tumor luminescence (Fold change vs. baseline) Figure 19B Lung % luminescence compared to PBS Figure 20A Lung Liver Figure 20B Lung % tumor load in lung (relative to PBS) Liver #Number of metastases Figure 21 % Apoptotic cells (TUNEL) % Apoptotic cells (TUNEL) Figure 23A) % Viability (AqueousOne) Viability%(AqueousOne) Figure 23B) % Viability (AqueousOne) Viability%(AqueousOne)

Claims

1. below i) Amino acid sequence X 3 X 2 X 4 X 5 X 7 X 5 X 4 X 4 X 6 X 18 X 8 X 3 QNX 9 X 8 X 10 X 10 X 11 X 12 S ＊ X 13 X 14 X 11 X 11 (Array No. 1) (In the array, S ＊ This means that S is possible or does not exist. X 2 It does not exist, or it is selected from K, E, R, and H. X 3 It does not exist, or it is selected from A, J, and S. X 4 It is selected from I, Z, and L, where Z is cyclohexyl alanine. X 5 It is selected from A, G, S, E, and D. X 6 It is selected from E and D, X 7 It is selected from J, G, Q, A, S, and P. X 8 is selected from B, W, Y, and F, where B is 2-methyltryptophan. X 9 It does not exist, or it is selected from A and G. X 10 It does not exist, or it is selected from A and N. X 11 It is selected from R and K, X 12 It does not exist, or it is selected from R. X 13 It does not exist, or it is selected from G and S. X 14 It does not exist, or it is selected from A and C. X 18 It is selected from A and E, However, a peptide containing an amino acid sequence that is identical by at least 70%, at least 80%, or at least 90% to (where J is either absent or present in two ways) that forms a staple. ii) The peptide described in i) containing non-natural amino acids and / or D-amino acids, or the peptide described in i) containing at least 80% D-amino acids or consisting of at least 80% D-amino acids, iii) Retroinverse peptide of the peptide described in i) or ii), and its pharmaceutically acceptable salts, A compound selected from, Induces apoptosis in senescent cells, scar cells, and / or cancer cells. compound.

2. The peptide has an amino acid sequence X 1 X 17 K ＊ X 2 X 16 X 3 X 3 X 2 X 4 X 5 X 7 X 5 X 4 X 4 X 6 AX 8 X 3 QNX 9 X 8 X 10 X 10 X 11 X 12 S ＊ [[ID=四十八]]X[[ID=四十九]] 13 [[ID=五十]]X[[ID=五十一]] 14 [[ID=五十二]]X[[ID=五十三]] 11 [[ID=五十四]]X[[ID=五十五]] 11 [[ID=五十六]]X[[ID=五十七]] 15 [[ID=五十八]](Array number 二) (In the array, Z, B, J, S ＊ , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , and X 18 This is as described in claim 1, K ＊ This means that K is possible or does not exist. X 1 This indicates that it does not exist, or represents the LTL amino acid sequence. X 15 It does not exist, or it is selected from A and C. X 16 It is selected from A and P, X 17 It includes an amino acid sequence that is identical to (non-existent or selected from R and S) by at least 70%, or at least 80%, or at least 90%, The compound according to claim 1, wherein the compound induces apoptosis in senescent cells, scar cells, and / or cancer cells.

3. The amino acid motif X 3 QNX 9 X 8 is selected from SQNAW (SEQ ID NO: 43), SQNGW (SEQ ID NO: 44), and SQN-W (SEQ ID NO: 45), and in the sequence, "-" indicates the absence of an amino acid. The compound according to claim 1 or 2

4. The aforementioned amino acid motif X 2 X 4 X 5 X 7 X 5 The compound is selected from KIAAA (SEQ ID NO: 46), KIEAA (SEQ ID NO: 47), KIAAE (SEQ ID NO: 48), and KIEAE (SEQ ID NO: 49), as described in any one of claims 1 to 3.

5. the amino acid motif X 4 X 5 X 7 X 5 X 4 X 4 X 6 AX 8 X 3 QNX 9 X 8 (SEQ ID NO: 3) is general formula IX 5 X 7 X 5 ILX 6 AFX 3 QNX 9 W (Sequence ID 4) (In the array, X 3 It does not exist, or it is selected from A, J, and S. X 5 It is selected from A, G, S, E, and D. X 6 It is selected from E and D, X 7 It is selected from J, G, Q, A, S, and P. X 9 (either does not exist, or is selected from A and G) The compound according to any one of claims 1 to 4.

6. The aforementioned compound, LTLRKEASSEIAQSILDAYSQNGWANRRSSCKRP (Sequence ID 7), LTLRKKASSKIAQSILDAFSQNGWANRRSSCKRP (Sequence ID 8), LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRP (Sequence ID 9), RKKASSKIAQSILDAFSQNGWANRRSSCKRP (Sequence ID 10), RKKASSKIAAAILDAFSQNGWANRRSSCKRP (Sequence ID 11), RKKASSKIAAAILDAFSQNAWANRRSSCKRP (Sequence ID 12), RKKASSKIAAAILDAFSQNWRRKR (Sequence ID 13), RKKASSKIEAAILDAFSQNWRRKR (Sequence No. 14), RKKASSKIAAEILDAFSQNWRRKR (Sequence ID 15), RKKASSKIEAEILDAFSQNWRRKR (Sequence No. 16), RKKSKIAAAILDAFSQNWRRKR (Sequence ID 17), RKKSKIEAEILDAFSQNWRRKR (Sequence ID 18), AKIAAAILDAFSQNWRRKR (Sequence ID 19), AKIEAAILDAFSQNWRRKR (Sequence ID 20), LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRPPPRRRQRRKKRG (Sequence ID 21), RKKASSKIAAAILDAFSQNGWANRRSSCKRPPPRRRQRRKKRG (Sequence ID 22), RKKASSKIAAAILDAFSQNAWANRRSSCKRPPPRRRQRRKKRA (Sequence ID 23), RKKASSKIAAAILDAFSQNWRRKRPPRRRQRRKKRG (Sequence ID 24), RKKASSKIEAAILDAFSQNWRRKRPPRRRQRRKKRG (Sequence No. 25), RKKASSKIAAEILDAFSQNWRRKRPPRRRQRRKKRG (Sequence No. 26), RKKASSKIEAEILDAFSQNWRRKRPPRRRQRRKKRG (Sequence No. 27), RKKSKIAAAILDAFSQNWRRKRRRRQRRKKRG (Sequence ID 28), RKKSKIEAEILDAFSQNWRRKRRRRQRRKKRG (Sequence ID 29), AKIAAAILDAFSQNWRRKRRRRQRRKKRG (Sequence ID 30), AKIEAAILDAFSQNWRRKRRRRQRRKKRG (Sequence ID 31), RKKASSKIEAEILDAFSQNWRRKRPPRRRQRRKKRG (Sequence ID 53), RKKSKIEAEILDAFSQNWRKRRRRQRRKKRG (Sequence ID 54), AKIEAAILDAFSQNWRKRRRRQRRKKRG (Sequence ID 55), AKIEAEILDAFSQNWRKRRRRQRRKKRG (Sequence ID 56), AKIEAAILDEFSQNWRKRRRRQRRKKRG (Sequence ID 57), RKKASJKIAJAILDAFSQNWRRKRPPRRRQRRKKRG (Sequence ID 58), RKKASSKIAAAZLDAFSQNAWANRRSSCKRPPPRRRQRRKKRA (Sequence ID 59), AKIEAAILDAFSQNBRKRRRRQRRKKRG (Sequence ID 60), AKIEAEILEAFSQNBRKRRRRQRRKKRG (Sequence ID 61), AKIEAAZLDAFSQNBRKRRRRQRRKKRG (Sequence ID 62), RKKASSKIEAEILDAFSQNBRRKRPPRRRQRRKKRG (Sequence No. 63), RKKASSKIEAEZLDAFSQNBRRKRPPRRRQRRKKRG (Sequence ID 64), RKKASSKIEAEIZDAFSQNBRRKRPPRRRQRRKKRG (Sequence No. 65), (In the formula, J, B, and Z are defined as above) An amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to any one of sequence numbers 1-4, 7-31, 53-65, and 67 and 68, A peptide comprising the amino acid sequence described in any one of SEQ ID NOs: 1-4, 7-31, 53-65, and 67 and 68, or a peptide comprising the amino acid sequence described in any one of SEQ ID NOs: 55, 60, 63, 64, or 65, Selected from, A compound according to any one of claims 1 to 5, which is a peptide containing an amino acid sequence or a D-amino acid sequence.

7. A compound according to any one of claims 1 to 6, further comprising a sequence that confers cell permeability, organelle targeting properties, nuclear localization, mitochondrial localization, blood-brain barrier permeability, cell membrane localization, and / or peptidase cleavage, or the HIV TAT sequence (GRKKRRQRRRPP, SEQ ID NO: 41) or ARKKRRQRRRPPP (SEQ ID NO: 66).

8. The compound according to any one of claims 1 to 7, wherein the compound contains an amino acid that does not occur naturally or an amino acid that has the same or substantially the same properties as a desired natural amino acid at that position, and comprises a modified amino acid, or a linker amino acid, a staple, a cysteine ​​crosslink, a glycolation site, a ubiquitination and / or pegylation site.

9. The compound according to any one of claims 1 to 8, wherein the compound binds to p53, or binds to p53 and inhibits the interaction between FOXO4 and p53 in cells.

10. A method for identifying improved compounds that bind to p53, or that bind to p53 and inhibit the interaction between FOXO4 and p53 in cells, a) A step of preparing at least one compound according to any one of claims 1 to 9, b) A step of appropriately modifying the compound in a), A step of identifying at least one of the following: the binding of the at least one compound in c) and p53 or a fragment thereof, the stability of the at least one compound, and the binding of FOXO4 or a fragment thereof and p53 or a fragment thereof in the presence of the at least one compound compared to the binding of FOXO4 or a fragment thereof and p53 or a fragment thereof in the absence of the compound, d) A step of identifying an improved compound that, based on what was identified in step c), binds to p53 or binds to p53 and inhibits the interaction between FOXO4 and p53 in cells, compared to the compound provided in step a), Methods that include...

11. The method according to claim 10, wherein the bond of the at least one compound is specific to p53 or the fragment thereof.

12. The method according to claim 10 or 11, further comprising testing the compound as identified for activity to induce apoptosis in senescent cells, scar cells, and / or tumor cells and / or to kill said cells, or comprising identifying a decrease in viability, which is an increase in caspase 3 / 7 activity, a decrease in mitochondrial cytochrome C, TUNEL positivity, extracellular annexin V positivity, and / or cell uptake of propidium iodide, and / or a decrease in calcein AM uptake, or comprising an MTS viability assay.

13. The method according to any one of claims 10 to 12, wherein the FOXO4 or the fragment thereof, and / or p53 or the fragment thereof, are expressed by recombination in cells.

14. The method according to any one of claims 10 to 13, wherein the cells are selected from the group consisting of cancer cells, scar cells, senescent cells, human non-embryonic stem cells, yeast cells, bacterial cells, and recombinant host cells expressing FOXO4 or its p53 binding fragment, and the recombinant host cells optionally express p53 or its FOXO4 binding fragment.

15. The method according to any one of claims 10 to 14, wherein the FOXO4 or its p53-binding fragment, p53 or its FOXO4-binding fragment, and / or the compound are detectably labeled by fluorescent labeling, mass labeling, and / or stable isotope labeling.

16. A screening tool for screening compounds that bind to p53 or bind to p53 and inhibit the interaction between FOXO4 and p53 in cells, comprising isolated cells expressing FOXO4 and / or cells expressing its p53-binding fragment, wherein the cells optionally include cells expressing p53 and / or its FOXO4-binding fragment.

17. The screening tool according to claim 16, wherein FOXO4 or a fragment thereof, and / or p53 or a fragment thereof, are expressed by recombination in the cells.

18. The screening tool according to claim 16 or 17, wherein the cells are selected from the group consisting of cancer cells, senescent cells, scar cells, human non-embryonic stem cells, yeast cells, bacterial cells, and recombinant host cells expressing FOXO4 or its p53 binding fragment, and the recombinant host cells optionally express p53 or its FOXO4 binding fragment.

19. The screening tool according to any one of claims 16 to 18, wherein the FOXO4 or its p53-binding fragment, p53 or its FOXO4-binding fragment, and / or the compound are detectably labeled by fluorescent labeling and / or mass labeling.

20. The aforementioned fragment of FOXO4 is arranged The peptide is described in PRKGGSRRNAWGNQSYAELISQAIESAPEKRLTLAQIYEWMVRTVPYFKDKGDSNSSAGWKNSIRHNLSLHSKFIKVHNEATGKSSWWMLN (SEQ ID NO: 32) or PRKGGSRRNAWGNQSYAELISQAIESAPEKRLTL (SEQ ID NO: 33), and the aforementioned fragment of p53 is sequence A screening tool according to any one of claims 16 to 19, wherein the peptide is AMDDLMLSPDDIEQWFTEDPGP (SEQ ID NO: 34).

21. A method for producing a pharmaceutical composition for treating or preventing senescent cells, scar cells, and / or cancer cells in a subject, comprising the steps of: formulating a compound according to any one of claims 1 to 9 to make a suitable pharmaceutical composition, or carrying out the method according to any one of claims 10 to 15; and formulating the compound as identified to make a suitable pharmaceutical composition.

22. A pharmaceutical composition obtained by the method described in claim 21, for treating or preventing senescent cells in a subject.

23. The pharmaceutical composition according to claim 22, comprising a mixture of compounds, and / or a mixture of at least one of the compounds and an additional pharmaceutically active ingredient.

24. A pharmaceutical composition for use in pharmaceuticals, comprising at least one compound according to any one of claims 1 to 9, or according to claim 22 or 23.

25. At least one compound or pharmaceutical composition for use according to claim 24, wherein the disease or pathological condition to be prevented and / or treated is selected from diseases or pathological conditions caused by senescent cells, scar cells and / or cancer cells, age-related diseases, kidney diseases, non-alcoholic steatohepatitis (NASH) / non-alcoholic fatty liver disease (NAFLD), hepatic fibrosis, idiopathic pulmonary fibrosis (IPF), amyotrophic lateral sclerosis (ALS), osteoarthritis, COPD, musculoskeletal diseases, cognitive impairment, and cancer.

26. The treatment is a combination therapy with an anticancer chemotherapy agent or other standard therapeutic agents for the respective disease or condition, or the treatment is applied to senescent cells, scar cells, and / or cancer cells that have been pretreated with an anticancer chemotherapy agent or other standard therapeutic agents for the respective disease or condition, and have survived the treatment, wherein the treatment is at least one compound or pharmaceutical composition for use according to claim 24 or 25.

27. A method for treating or preventing a disease or symptom caused by senescent cells, scar cells and / or cancer cells, age-related diseases, kidney diseases, non-alcoholic steatohepatitis (NASH) / non-alcoholic fatty liver disease (NAFLD), hepatic fibrosis, idiopathic pulmonary fibrosis (IPF), amyotrophic lateral sclerosis (ALS), osteoarthritis, COPD, musculoskeletal diseases, cognitive decline, or cancer, comprising administering to the subject in an effective amount at least one compound described in any one of claims 1 to 9, or a pharmaceutical composition described in claim 22 or 23.

28. The method according to claim 27, wherein the treatment is a combination therapy with an anticancer chemotherapy agent or other standard therapeutic agents for the respective disease or condition, or the treatment is administered to senescent cells, scar cells, and / or cancer cells that have survived the treatment, after being pretreated with an anticancer chemotherapy agent or other standard therapeutic agents for the respective disease or condition.

Images