Improved anti-aging compounds and uses thereof

Abstract

The present invention relates to improved compounds for use in treating a disease or condition that can benefit from removal of senescent cells, scar cells, and / or cancer cells, e.g., cancer. The present invention also relates to methods for treating an individual afflicted with, or suspected of being afflicted with, a disease or condition that can benefit from removal of senescent cells, scar cells, and / or cancer cells.

Description

Technical Field

[0001] This invention relates to improved compounds that can be used to treat diseases or conditions, such as cancer, from which the removal of senescent cells, scar cells, and / or cancer cells can be beneficial. The invention also relates to methods for treating individuals who have or are suspected of having diseases or conditions from which the removal of senescent cells, scar cells, and / or cancer cells can be beneficial. Background Technology

[0002] Apoptosis (programmed cell death) kills damaged cells. Dead cells undergoing the final stages of apoptosis exhibit phagocytic molecules, allowing them to be engulfed by cells with suitable receptors, such as macrophages.

[0003] Senescent cells are thought to disrupt tissue function, and their genetic clearance could delay aging characteristics. Senescent cells are permanently withdrawn from the cell cycle and often develop a persistent pro-inflammatory phenotype known as senescence-associated secretory phenotype (SASP). SASP influences the cellular microenvironment, which can be beneficial early in life or during the acute phase of wound healing. However, unlike permanently eliminated apoptotic cells, senescent cells can persist in large areas over extended periods and accumulate with age. Because of their lower but chronic SASP, persistent senescent cells are thought to accelerate aging and the development of age-related diseases.

[0004] It has been shown that senescent cells accumulate with age, particularly at age-related pathological sites. Furthermore, senescent cells can acquire mutations that allow them to re-enter a proliferative state. Thus, benign senescent lesions retain the ability to become malignant. Indeed, aging is associated with many age-related pathologies, and conversely, the genetic clearance of senescent cells can delay aging characteristics.

[0005] Indeed, it has recently been shown that the genetically significant clearance of senescent cells can significantly improve health and reduce aging parameters in mouse models of accelerated aging. These mice exhibited reduced signs of aging, such as those measured by kyphosis (excessive skeletal curvature), muscle strength, fat accumulation, and cataracts. This provides further evidence that cellular senescence and SASP are causally linked to age-related phenotypes and cancer (Bakeretal., 2011. Nature 479(7372): 232-6). This concept-validation evidence is obtained in a genetic sense, with poor therapeutic applicability.

[0006] Studies have consistently revealed that FOXO transcription factors are crucial determinants of aging and longevity. FOXO proteins represent a conserved subfamily of transcription factors from *C. elegans* to mammals, acting as key lifespan regulators downstream of the insulin and insulin-like growth factor pathways. Invertebrates have one FOXO gene, while mammals have four: FOXO1, FOXO3, FOXO4, and FOXO6. In mammals, this subfamily participates in a wide range of key cellular processes, regulating stress tolerance, metabolism, cell cycle arrest, and apoptosis. Their roles in lifespan determination are complex and require further comprehensive understanding.

[0007] Bourgeois and Madl (Regulation of cellular senescence via the FOXO4-p53 axis. FEBS Lett. 2018 Jun; 592(12): 2083-2097) published that FOXO and p53 have been identified as key regulators of senescence over the past decade, and their dysregulation is associated with a variety of diseases, including cancer. However, many fundamental molecular mechanisms remain unknown, including the regulation of senescence by FOXO and p53. Some activities appear to be shared between FOXO and p53, including their central role in the regulation of cellular senescence. They focused on recent advances in the association between FOXO and p53, with particular attention to the FOXO4-p53 axis and the role of FOXO4 / p53 in cellular senescence. Possible strategies for targeting the FOXO4-p53 interaction to regulate cellular senescence as a pharmacological target for the treatment of age-related diseases and pathologies are discussed.

[0008] WO2013 / 152038 relates to the use of agents that inhibit Jun kinase and / or FOXO4 in the treatment of cancer and / or removal of senescent cells in an individual. In some embodiments, the agent is a small molecule, such as SP600125. In some embodiments, the agent is a small molecule, such as AS601245. In some embodiments, the agent is a disclosed peptide.

[0009] WO2013 / 152041 relates to agents that inhibit FOXO4 function and their use in treating cancer and / or removing senescent cells in individuals. In some embodiments, the agent that inhibits FOXO4 is a peptide that inhibits FOXO4 function in cells, wherein the peptide comprises an amino acid sequence having at least 80% identity with the disclosed FOXO4 fragment.

[0010] WO2016 / 118014 relates to a peptide comprising the amino acid sequence LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRP (SEQ ID NO: 5), and a method of using the peptide in the treatment of age-related conditions, wherein the amino acids in the amino acid sequence are D-amino acid residues. This peptide exhibits apoptosis-inducing activity in senescent cells, or in cells with increased FOXO4 expression and expression of Ser15-phosphorylated p53 (pSer15-p53) relative to control cells.

[0011] WO2018 / 129007 relates to a conditionally active protein that targets senescent cells, and a method for generating the conditionally active protein.

[0012] From the above, it is clear that there is still a need in the field for compounds that can selectively induce apoptosis in senescent cells, because cellular senescence is associated with degenerative (loss of function) diseases and cancer (gain of function). Currently, there are no suitable therapeutically feasible compounds that can selectively induce apoptosis in senescent cells in vivo. Summary of the Invention

[0013] Therefore, the object of the present invention is to provide new improved compounds that therapeutically target senescent cells and can be effectively used to treat diseases related to cellular senescence.

[0014] In its first aspect, the present invention addresses this problem by providing a compound and a pharmaceutically acceptable salt thereof, the compound being selected from:

[0015] i) Contains amino acid sequences X3X2X4X5X7X5X4X4X6X 18 X8X3QNX9X8X 10 X 10 X 11 X 12 S*X 13 X 14 X 11 X 11 (SEQ ID NO: 1) A peptide with at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably more than 95% amino acid sequence identity.

[0016] in,

[0017] S* can be S, or it may not exist;

[0018] X2 does not exist, or is selected from K, E, R, and H;

[0019] X3 does not exist, or it is selected from A, J, and S;

[0020] X4 is selected from I, Z, and L, where Z is cyclohexyl-alanine;

[0021] X5 is selected from A, G, S, E, and D;

[0022] X6 is selected from E and D;

[0023] X7 is selected from J, G, Q, A, S, and P;

[0024] X8 is selected from B, W, Y, and F, where B is 2-methyl-tryptophan;

[0025] X9 does not exist, or it can be selected from A and G;

[0026] X 10 It does not exist, or it is selected from A and N;

[0027] X 11 Selected from R and K;

[0028] X 12 It does not exist, or it is selected from R;

[0029] X 13 It does not exist, or it is selected from G and S;

[0030] X 14 It does not exist, or is selected from A and C; and

[0031] X 18 Selected from A and E;

[0032] The rule states that there is no J, or that there are two Js forming a staple-like structure;

[0033] ii) The peptide according to i) comprising non-natural amino acids and / or D-amino acids, preferably the peptide according to i) comprising at least 80%, preferably at least 90%, more preferably at least 95% D-amino acids, or consisting of at least 80%, preferably at least 90%, more preferably at least 95% D-amino acids, and

[0034] iii) According to i) or ii), the inverted peptide, that is, the peptide whose amino acid sequence is flipped and placed as a D-not L-isomer when compared with the original protein sequence, and

[0035] This compound induces apoptosis in senescent cells, scar cells, and / or cancer cells.

[0036] Preferably, the compound according to this application is wherein the peptide comprises amino acid sequences X1X. 17 K*X2X 16 X3X3X2X4X5X7X5X4X4X6AX8X3QNX9X8X 10 X 10 X11 X 12 S*X 13 X 14 X 11 X 11 X 15 (SEQ ID NO: 2) An amino acid sequence with at least 70%, preferably at least 80%, more preferably at least 90% identity, wherein 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 defined above;

[0037] K* can be K, or it may not exist;

[0038] X1 is absent or represents the amino acid sequence LTL;

[0039] X 15 It does not exist or is selected from A and C;

[0040] X 16 Selected from A and P; and

[0041] X 17 There is no R and S; and

[0042] This compound induces apoptosis in senescent cells, scar cells, and / or cancer cells.

[0043] More preferably, the compounds according to the invention comprise an amino acid motif X3QNX9X8 (e.g., 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), wherein a "-" indicates a non-existent amino acid. Even more preferably, the compounds according to the invention comprise an amino acid motif X2X4X5X7X5 (e.g., 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).

[0044] More preferably, the compounds according to the invention comprise the amino acid motif X4X5X7X5X4X4X6AX8X3QNX9X8 (SEQ ID NO: 3, starting from position 9 of SEQ ID NO: 2), which is selected from the general formula IX5X7X5ILX6AFX3QNX9W (SEQ ID NO: 4).

[0045] in,

[0046] X3 does not exist or is selected from A, J, and S;

[0047] X5 is selected from A, G, S, E, and D;

[0048] X6 is selected from E and D;

[0049] X7 is selected from J, G, Q, A, S, and P; and X9 does not exist or is selected from A and G.

[0050] Preferably, the compounds according to this application are composed of D-amino acids. The amino acids are named according to standard single-letter codes.

[0051] Most preferably, the compound according to this application is a peptide comprising an amino acid sequence selected from the following, and a pharmaceutically acceptable salt thereof:

[0052] LTLRKEASSEIAQSILDAYSQNGWANRRSSCKRP (SEQ ID NO: 7),

[0053] LTLRKKASSKIAQSILDAFSQNGWANRRSSCKRP (SEQ ID NO: 8),

[0054] LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRP (SEQ ID NO: 9),

[0055] RKKASSKIAQSILDAFSQNGWANRRSSCKRP (SEQ ID NO: 10),

[0056] RKKASSKIAAAILDAFSQNGWANRRSSCKRP (SEQ ID NO: 11),

[0057] RKKASSKIAAAILDAFSQNAWANRRSSCKRP (SEQ ID NO: 12),

[0058] RKKASSKIAAAILDAFSQNWRRKR (SEQ ID NO: 13),

[0059] RKKASSKIEAAILDAFSQNWRRKR (SEQ ID NO: 14),

[0060] RKKASSKIAAEILDAFSQNWRRKR (SEQ ID NO: 15),

[0061] RKKASSKIEAEILDAFSQNWRRKR(SEQ ID NO:16)、

[0062] RKKSKIAAAILDAFSQNWRRKR(SEQ ID NO:17)、

[0063] RKKSKIEAEILDAFSQNWRRKR(SEQ ID NO:18)、

[0064] AKIAAAILDAFSQNWRRKR(SEQ ID NO: 19)ぁ

[0065] AKIEAILDAFSQNWRRKR(SEQ ID NO:20)、

[0066] LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRPPPRRRQRRKKRG(SEQ ID NO: 21)ぁ

[0067] RKKASSKIAAAILDAFSQNGWANRRSSCKRPPPRRRQRRKKRG(SEQ ID NO: 22)ぁ

[0068] RKKASSKIAAAILDAFSQNAWANRRSSCKRPPPPRRRQRRKKRA(SEQ ID NO:23)、

[0069] RKKASSKIAAAILDAFSQNWRRKRPPRRRQRRKKRG(SEQ ID NO:24)、

[0070] RKKASSKIEAAILDAFSQNWRRKRPPRRRQRRKKRG(SEQ ID NO:25)、

[0071] RKKASSKIAAEILDAFSQNWRRKRPPRRRQRRKKRG(SEQ ID NO:26)、

[0072] RKKASSKIEAEILDAFSQNWRRKRPPRRRQRRKKRG(SEQ ID NO:27)、

[0073] RKKSKIAAAILDAFSQNWRRKRRRRQRRKKRG(SEQ ID NO:28)、

[0074] RKKSKIEAEILDAFSQNWRRKRRRRQRRKKRG(SEQ ID NO:29)、

[0075] AKIAAAILDAFSQNWRRKRRRRQRRKKRG(SEQ ID NO:30)、

[0076] AKIEAAILDAFSQNWRRKRRRRQRRKKRG(SEQ ID NO:31)、

[0077] RKKASSKIEAEILDAFSQNWRRKRPPRRRQRRKKRG(SEQ ID NO:53)、

[0078] RKKSKIEAEILDAFSQNWRKRRRRQRRKKRG(SEQ ID NO:54)、

[0079] AKIEAAILDAFSQNWRKRRRRQRRKKRG(SEQ ID NO:55)、

[0080] AKIEAEILDAFSQNWRKRRRRQRRKKRG(SEQ ID NO:56)、

[0081] AKIEAAILDEFSQNWRKRRRRQRRKKRG(SEQ ID NO:57)、

[0082] RKKASJKIAJAILDAFSQNWRRKRPPRRRQRRKKRG(SEQ ID NO:58)、

[0083] RKKASSKIAAAZLDAFSQNAWANRRSSCKRPPPRRRQRRKKRA(SEQ ID NO:59)、

[0084] AKIEAAILDAFSQNBRKRRRRQRRKKRG(SEQ ID NO:60)、

[0085] AKIEAEILEAFSQNBRKRRRRQRRKKRG(SEQ ID NO:61)、

[0086] AKIEAAZLDAFSQNBRKRRRRQRRKKRG(SEQ ID NO:62)、

[0087] RKKASSKIEAEILDAFSQNBRRKRPPRRRQRRKKRG(SEQ ID NO:63)、

[0088] RKKASSKIEAEZLDAFSQNBRRKRPPRRRQRRKKRG (SEQ ID NO: 64),

[0089] RKKASSKIEEAIZDAFSQNBRRKRPPRRRQRRKKRG (SEQ ID NO: 65),

[0090] RKKSKIAAAILDAFSQNWRKRRRRQRRKKRG (SEQ ID NO: 67), and

[0091] RKKASSKIEAAILDAFSQNWRRKRPPRRRQRRKKRG (SEQ ID NO: 68),

[0092] Wherein J, B, and Z are as defined above, and peptides, particularly peptides comprising an amino acid sequence selected from any of SEQ ID NO: 1-4, 7-31, 53-65, 67, and 68, comprising at least 80%, preferably at least 90%, more preferably at least 95% identity with an amino acid sequence, and peptides composed of any of the amino acids according to SEQ ID NO: 1-4, 7-31, 53-65, 67, and 68. These peptides are particularly D-peptides.

[0093] In a second aspect, the present invention relates to a method for identifying an improved compound that binds to p53, or preferably binds to p53 and inhibits the interaction between FOXO4 and p53 in cells, comprising the steps of: a) providing at least one compound according to the present application; b) suitably modifying the compound of a); c) determining at least one of the following: the binding of the at least one compound of b) to p53 or a fragment thereof, the stability of the at least one compound, and the binding of FOXO4 or a fragment thereof to p53 or a fragment thereof in the presence of the at least one compound (compared to the binding of FOXO4 or a fragment thereof to p53 or a fragment thereof in the absence of the compound); and d) based on the determination in step c), identifying a compound that is 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).

[0094] Preferably, the method according to the invention involves binding of the at least one compound specifically to p53 or a fragment thereof. The preferred method further includes testing the activity of the identified compound in inducing apoptosis and / or killing senescent cells, scar cells, and / or tumor cells, preferably including the determination of caspase activity.

[0095] In a third aspect, the present invention relates to a screening tool for screening compounds that bind to p53, and preferably bind to p53 and inhibit the interaction between FOXO4 and p53 in cells, comprising isolated cells expressing FOXO4, and / or cells expressing their p53-binding fragments (wherein the cells optionally express p53), and / or cells expressing their FOXO4-binding fragments.

[0096] Preferably, the screening tool according to the invention is a peptide of the sequence PRKGGSRRNAWGNQSYAELISQAIESAPEKRLTLAQIYEWMVRTVPYFKDKGDSNSSAGWKNSIRHNLSLHSKFIKVHNEATGKSSWWMLN (complete forkhead domain) (SEQ ID NO: 32) or PRKGGSRRNAWGNQSYAELISQAIESAPEKRLTL (SEQ ID NO: 33), wherein the p53 fragment is a peptide of the sequence AMDDLMLSPDDIEQWFTEDPGP (SEQ ID NO: 34).

[0097] In its fourth aspect, the present invention relates to a method for preparing 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 invention into a suitable pharmaceutical composition or carrying out the method of the invention, and formulating an identified compound into a suitable pharmaceutical composition. The pharmaceutical composition according to the invention may contain pharmaceutically acceptable excipients, such as, but not limited to, stabilizers, swelling agents, buffers, carriers, diluents, solvents, solubilizers, and binders. Those skilled in the art will understand that the selection of a suitable excipient depends on the route of administration and dosage form, as well as the active ingredient and other factors. The pharmaceutical composition according to the invention is preferably suitable for parenteral administration.

[0098] In its fifth aspect, the present invention relates to pharmaceutical compositions obtained by the method of the present invention for treating or preventing senescent cells, scar cells, and / or cancer cells in subjects.

[0099] In its sixth aspect, the present invention relates to the use in medicine of at least one compound according to the invention, or a pharmaceutical composition according to the invention. Preferably, it is at least one compound or pharmaceutical composition according to the invention, wherein the disease or condition to be prevented and / or treated is selected from symptoms caused by senescent cells, scar cells, age-related diseases, kidney disease, osteoarthritis, COPD, skeletal muscle diseases, cognitive impairment, and cancer.

[0100] In its seventh aspect, the present invention relates to a method for treating or preventing diseases or conditions caused by senescent cells, scar cells, and / or cancer cells, age-related diseases, kidney disease, osteoarthritis, COPD, skeletal muscle diseases, cognitive impairment, or cancer in a subject in need, comprising administering to the subject an effective amount of at least one compound of the present invention, or a pharmaceutical composition according to the present invention.

[0101] Other aspects and benefits can be readily obtained by reading the following descriptions and non-limiting examples. Detailed Implementation

[0102] In the context of the experiments conducted in this invention, the inventors have surprisingly discovered that substantial functional improvements can still be achieved by introducing changes into the compound after careful further analysis of the interaction between FOXO4 and p53.

[0103] Therefore, a key aspect of the present invention relates to improved compounds and pharmaceutically acceptable salts thereof, the compounds being selected from...

[0104] i) Contains amino acid sequences X3X2X4X5X7X5X4X4X6X 18 X8X3QNX9X8X 10 X 10 X 11 X 12 S*X 13 X 14 X 11 X 11 (SEQ ID NO: 1) A peptide with at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably more than 95% amino acid sequence identity.

[0105] in,

[0106] S* can be S, or it may not exist;

[0107] X2 does not exist, or is selected from K, E, R, and H;

[0108] X3 does not exist, or it is selected from A, J, and S;

[0109] X4 is selected from I, Z, and L, where Z is cyclohexyl-alanine;

[0110] X5 is selected from A, G, S, E, and D;

[0111] X6 is selected from E and D;

[0112] X7 is selected from J, G, Q, A, S, and P;

[0113] X8 is selected from B, W, Y, and F, where B is 2-methyl-tryptophan;

[0114] X9 does not exist, or it can be selected from A and G;

[0115] X 10 It does not exist, or it is selected from A and N;

[0116] X 11 Selected from R and K;

[0117] X 12 It does not exist, or it is selected from R;

[0118] X 13 It does not exist, or it is selected from G and S;

[0119] X 14 It does not exist, or is selected from A and C; and

[0120] X 18 Selected from A and E;

[0121] The rule states that there is no J, or that there are two Js forming a staple-like structure;

[0122] ii) The peptide according to i) comprising non-natural amino acids and / or D-amino acids, preferably the peptide according to i) comprising at least 80%, preferably at least 90%, more preferably at least 95% D-amino acids, or consisting of at least 80%, preferably at least 90%, more preferably at least 95% D-amino acids, and

[0123] iii) According to i) or ii), the inverted peptide, that is, the peptide whose amino acid sequence is flipped and placed as a D-not L-isomer when compared with the original protein sequence, and

[0124] This compound induces apoptosis in senescent cells, scar cells, and / or cancer cells. Preferably, the compound is composed of D-amino acids.

[0125] Preferred compounds are those that bind to p53, or more preferably bind to p53 and inhibit the interaction between FOXO4 and p53 in cells, and can be detected by existing assays in the art and / or the assays disclosed herein. These assays include NMR, which determines the ability of the peptide to bind to (fragment of) TP53; pull-down assays, in which (biotinylated version) peptides are bound to (streptavidin) beads, which are poured into cell lysates containing TP53, and the TP53 moiety bound to the peptide is studied; and FRET / HTRF assays, in which the ability of the peptide to interfere with the interaction between (fragment of) TP53 and FOXO4 is evaluated.

[0126] The evolutionary differences between FOXO1 / 3 / 6 and FOXO4 suggest that some amino acids can still be modified to create improved compounds that exhibit the desired inhibitory activity.

[0127] The evolutionary differences between FOXO4 and its orthologs in FOXO1, 3, and 6, as well as in lower species, lie in certain unique residues. The inventors have never considered FOXO1 or FOXO3 as a focus, and there is no evidence that FOXO1 and 3 are important for the survival of senescent cells. Therefore, the analysis explored whether these amino acids at least partially explain the efficacy of the FOXO4-peptide in eliminating senescent cells. This was confirmed in D-trans-inverted peptides based on domains similar to FOXO4, while sequences based on FOXO1 or FOXO3 did not show selectivity in eliminating senescent cells.

[0128] Analysis of the FOXO4-FH fragment and the TP53-TAD2 domain revealed that the negative charge in TP53 and the hydrophobic Tyr mediated the binding to FOXO4. Therefore, mutations to positively charged amino acids, such as Glu->Lys and Tyr->Phe, are considered beneficial to the strength of the interaction. NMR also showed that the N-terminal amino acid LTL is not essential for the interaction. Finally, NMR revealed that the negatively charged amino acid in TP53 is responsible for interacting with FOXO4 around the SQ motif (site). Modification of the NG amino acid site can improve molecular stability. All these modifications present a novel strategy, where any one or a combination thereof leads to the rational design of improved molecules for use in this invention.

[0129] It has also been found that the active peptides in the context of this invention can be considerably shorter than those proposed in the prior art, while still retaining strong activity. However, longer and more active peptides are also possible; therefore, the preferred compounds according to the invention comprise the amino acid sequence X1X. 17 K*X2X 16 X3X3X2X4X5X7X5X4X4X6AX8X3QNX9X8X 10 X 10 X 11 X 12 S*X 13 X 14 X 11 X 11 X 15 (SEQ ID NO: 2) An amino acid sequence with at least 70%, preferably at least 80%, more preferably at least 90% identity, wherein 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 defined above;

[0130] K* can be K, or it may not exist;

[0131] X1 is absent or represents the amino acid sequence LTL;

[0132] X 15 It does not exist or is selected from A and C;

[0133] X 16 Selected from A and P; and

[0134] X 17 There is no R and S; and

[0135] This compound induces apoptosis in senescent cells, scar cells, and / or cancer cells.

[0136] It has also been found that the particularly active peptides according to the invention contain certain amino acid motifs, preferably the amino acid motif X3QNX9X8 selected from SQNAW (SEQ ID NO: 43), SQNGW (SEQ ID NO: 44), and SQN-W (SEQ ID NO: 45) (e.g., starting from position 18 of SEQ ID NO: 2), wherein “-” indicates an absent amino acid.

[0137] Surprisingly, it was found that when X9 is alanine (A), the peptide exhibits improved stability in solution, as exemplified in the cases of peptides SEQ ID NO: 12 and 23 (see Examples). Therefore, this transformation is preferred to provide additional stability to the compounds according to the invention.

[0138] Even more preferably, the compounds according to the invention comprise an amino acid motif of X2X4X5X7X5 selected from KIAAA (SEQ ID NO: 46), KIEAA (SEQ ID NO: 47), KIAAE (SEQ ID NO: 48), and KIEAE (SEQ ID NO: 49) (e.g., starting from position 8 of SEQ ID NO: 2).

[0139] More preferably, the compounds according to the invention comprise the amino acid motif X4X5X7X5X4X4X6AX8X3QNX9X8 (SEQ ID NO: 3, starting from position 9 of SEQ ID NO: 2), which is selected from the general formula IX5X7X5ILX6AFX3QNX9W (SEQ ID NO: 4).

[0140] in,

[0141] X3 does not exist or is selected from A, J, and S;

[0142] X5 is selected from A, G, S, E, and D;

[0143] X6 is selected from E and D;

[0144] X7 is selected from J, G, Q, A, S, and P; and

[0145] X9 does not exist or is selected from A and G.

[0146] In the context of this invention, it was found that the amino acid motif LTLRKEASSE (SEQ ID NO: 35) is non-essential for improving the basic function of the peptide and can be reduced to a single Ala, replaced by Ala, a "neutral" or positively charged amino acid. Instead, the "core motif" X4X5X7X5X4X4X6AX8X3QNX9X8 (SEQ ID NO: 3, starting from position 9 of SEQ ID NO: 2), which can be selected from the above general formula IX5X7X5ILX6AFX3QNX9W (SEQ ID NO: 4), is crucial. However, in this motif, the amino acid "SQNG" (SEQ ID NO: 50) appears to act as a structural linker, causing the "W" to contract into the hydrophobic core. Some residues can be deleted; a single or double "G" is sufficient, and the peptide remains functional when using AANG (SEQ ID NO: 51) or SQ-G or SQAG (SEQ ID NO: 52). The terminal W remains essential and is difficult to replace with native AA. The initial "I" is necessary and must be a hydrophobic amino acid, preferably I / L, but can also be non-natural. In the IX5X7X5IL (SEQ ID NO: 36, e.g., IAQSIL, SEQ ID NO: 37) portion, the interval between "I"s needs to be 2-3 residues. Some variations are tolerable as long as they do not disrupt the helix or hydrophobic core. Similarly, the terminal "IL" is necessary and must be a hydrophobic amino acid, preferably I / L, but can also be non-natural. Furthermore, the interval between the subsequent "L" and "Y" is preferably 2 residues, but a single amino insertion is tolerable. Again, this variation is tolerable as long as it does not disrupt the helix or hydrophobic core. "Y" is necessary and must be a hydrophobic amino acid, preferably F / Y, but can also be non-natural. Similar to the N-terminal portion described above, the motif ANRRSSCKRP (SEQ ID NO: 38) is not essential for improving the basic function of the peptide and can be reduced to at least 3 positively charged residues; although fewer, the peptide remains active.

[0147] More preferably, the compound according to the invention is a peptide comprising an amino acid sequence selected from the following, and a pharmaceutically acceptable salt thereof:

[0148] LTLRKEASSEIAQSILDAYSQNGWANRRSSCKRP (SEQ ID NO: 7),

[0149] LTLRKKASSKIAQSILDAFSQNGWANRRSSCKRP(SEQ ID NO: 8)ぁ

[0150] LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRP(SEQ ID NO:9)、

[0151] RKKASSKIAQSILDAFSQNGWANRRSSCKRP(SEQ ID NO: 10)ぁ

[0152] RKKASSKIAAAILDAFSQNGWANRRSSCKRP(SEQ ID NO:11)、

[0153] RKKASSKIAAAILDAFSQNAWANRRSSCKRP(SEQ ID NO:12)、

[0154] RKKASSKIAAAILDAFSQNWRRKR(SEQ ID NO: 13)ぁ

[0155] RKKASSKIEAAILDAFSQNWRRKR(SEQ ID NO:14)、

[0156] RKKASSKIAAEILDAFSQNWRRKR(SEQ ID NO: 15)ぁ

[0157] RKKASSKIEAEILDAFSQNWRRKR(SEQ ID NO:16)、

[0158] RKKSKIAAAILDAFSQNWRRKR(SEQ ID NO:17)、

[0159] RKKSKIEAEILDAFSQNWRRKR(SEQ ID NO:18)、

[0160] AKIAAAILDAFSQNWRRKR(SEQ ID NO: 19)ぁ

[0161] AKIEAILDAFSQNWRRKR(SEQ ID NO:20)、

[0162] LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRPPPRRRQRRKKRG(SEQ ID NO: 21)ぁ

[0163] RKKASSKIAAAILDAFSQNGWANRRSSCKRPPPRRRQRRKKRG(SEQ ID NO:22)、

[0164] RKKASSKIAAAILDAFSQNAWANRRSSCKRPPPRRRQRRKKRA(SEQ ID NO:23)、

[0165] RKKASSKIAAAILDAFSQNWRRKRPPRRRQRRKKRG(SEQ ID NO:24)、

[0166] RKKASSKIEAAILDAFSQNWRRKRPPRRRQRRKKRG(SEQ ID NO:25)、

[0167] RKKASSKIAAEILDAFSQNWRRKRPPRRRQRRKKRG(SEQ ID NO:26)、

[0168] RKKASSKIEAEILDAFSQNWRRKRPPRRRQRRKKRG(SEQ ID NO:27)、

[0169] RKKSKIAAAILDAFSQNWRRKRRRRQRRKKRG(SEQ ID NO:28)、

[0170] RKKSKIEAEILDAFSQNWRRKRRRRQRRKKRG(SEQ ID NO:29)、

[0171] AKIAAAILDAFSQNWRRKRRRRQRRKKRG(SEQ ID NO:30)、

[0172] AKIEAAILDAFSQNWRRKRRRRQRRKKRG(SEQ ID NO:31)、

[0173] RKKASSKIEAEILDAFSQNWRRKRPPRRRQRRKKRG(SEQ ID NO:53)、

[0174] RKKSKIEAEILDAFSQNWRKRRRRQRRKKRG(SEQ ID NO:54)、

[0175] AKIEAAILDAFSQNWRKRRRRQRRKKRG(SEQ ID NO:55)、

[0176] AKIEAEILDAFSQNWRKRRRRQRRKKRG (SEQ ID NO: 56),

[0177] AKIEAAILDEFSQNWRKRRRRQRRKKRG (SEQ ID NO: 57),

[0178] RKKASJKIAJAILDAFSQNWRRKRPPRRRQRRKKRG (SEQ ID NO: 58),

[0179] RKKASSKIAAAZLDAFSQNAWANRRSSCKRPPPRRRQRRKKRA (SEQ ID NO: 59),

[0180] AKIEAAILDAFSQNBRKRRRRQRRKKRG (SEQ ID NO: 60),

[0181] AKIEAEILEAFSQNBRKRRRRQRRKKRG (SEQ ID NO: 61),

[0182] AKIEAAZLDAFSQNBRKRRRRQRRKKRG (SEQ ID NO: 62),

[0183] RKKASSKIEAEILDAFSQNBRRKRPPRRRQRRKKRG (SEQ ID NO: 63),

[0184] RKKASSKIEAEZLDAFSQNBRRKRPPRRRQRRKKRG (SEQ ID NO: 64),

[0185] RKKASSKIEAEIZDAFSQNBRRKRPPRRRQRRKKRG (SEQ ID NO: 65),

[0186] RKKSKIAAAILDAFSQNWRKRRRRQRRKKRG (SEQ ID NO: 67), and

[0187] RKKASSKIEAAILDAFSQNWRRKRPPRRRQRRKKRG (SEQ ID NO: 68), wherein J, B, and Z are as defined above, and a peptide comprising an amino acid sequence selected from any of SEQ ID NO: 1-4, 7-31, 53-65, 67, and 68, wherein at least 80%, preferably at least 90%, more preferably at least 95%, of the same amino acid sequence, and a peptide consisting of any of the amino acids according to SEQ ID NO: 1-4, 7-31, 53-65, 67, and 68, preferably a peptide consisting of any of the amino acid sequences of SEQ ID NO: 55, 60, 63, 64, or 65.

[0188] In the compounds of this invention, according to the single-letter coding of amino acids, X1 is absent or represents the amino acid LTL, because the N-terminal amino acid LTL is not essential for the interaction. X2 is selected from positively charged amino acids, preferably K, E, R, and H. X3 is selected from small nonpolar amino acids, preferably A and S, and can be used to form staples as defined herein. X4 is selected from hydrophobic amino acids, preferably I and L, and modified amino acid Z, wherein Z is cyclohexyl-alanine. X5 is selected from small positively or negatively charged amino acids, preferably A, G, S, E, and D. X6 is selected from negatively charged amino acids, preferably E and D. X7 is selected from small or polar amino acids, preferably G, Q, A, S, and P, and can be used to form staples as defined herein. X8 is selected from aromatic amino acids, preferably W, Y, and F, and B, wherein B is 2-Me-tryptophan. X9 is selected from small polar or nonpolar amino acids, preferably A and G. 10 Selected from A and N, as well as electrically neutral polar amino acids, with Q being preferred. X 11 and X 12 Selected from positively charged amino acids, preferably R and K. X 13 Selected from G and S, X 14 and / or X 15 Selected from A and C, X 16 Selected from A and P, X 17 Selected from R and S, X 18 Selected from A and E. Preferably, the compound may contain a non-naturally occurring amino acid having the same or substantially the same desired properties as the natural amino acid indicated herein, and at the relevant position in the compound represented by the formula herein. Modifying amino acids may also be introduced, such as linker amino acids, staple needles, cysteine ​​bridges, glycosylation sites, ubiquitination and / or polyethylene glycol sites, if desired.

[0189] A stapled peptide is a short peptide, typically in an α-helical configuration, bound by a synthetic support (“staple”). This staple is formed by covalent bonds between two amino acid side chains, creating a macrocyclic peptide. Therefore, a staple typically refers to the covalent connection of two previously independent groups. In other applications, peptide binding is primarily used to enhance the pharmacological properties of the peptide.

[0190] The peptides of the present invention can be of any length, as long as the compound has the function of binding to p53 and / or binding to p53 and inhibiting or substantially inhibiting the interaction between FOXO4 and p53 in cells. Preferably, compounds have a length of 100-19, more preferably 75-19, and even more preferably 50-19 amino acids. Specifically preferred are peptides of the present invention having a length selected from one of SEQ ID NO: 1-4, 7-31, 53-65, and 67 and 68.

[0191] The compounds according to the invention are preferably inverted peptides comprising an amino acid sequence that is at least 80%, preferably at least 90%, more preferably at least 95% identical to any one of SEQ ID NO: 1-4, 7-31, 53-65, 67 and 68.

[0192] As used herein, the term "peptide" preferably refers to a synthetically synthesized peptide, more preferably a peptide analogue, and more preferably a D-peptide. The term "peptide" includes peptide analogues that may have modifications, for example, to make the peptide more stable in vivo or more permeable into cells. Such modifications include, but are not limited to, N-terminal modifications, C-terminal modifications, peptide bond modifications (including but 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), and skeletal modifications. Methods for preparing peptide analogues are well known in the art and are described, for example, in Quantitative Drug Design, CARAMSden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992).

[0193] Further preferred modifications involve the amino acid itself and include the introduction of a non-naturally occurring amino acid (preferably having the same or substantially the same desired characteristics as the natural amino acid), preferably at the same or substantially the same position in the compound. Preferred examples are 2-Me-tryptophan or cyclohexyl-alanine. Other modifications may include modified amino acids, such as linker amino acids, staples, cysteine ​​bridges, glycosylation sites, ubiquitination and / or polyethylene glycolation sites. Staples can be achieved by introducing two α-pentenyl-glycine residues, or α,a'-pentenyl-alanine residues, for crosslinking the i and i+4 or i and i+7 residues within the peptide. These residues can be used to create all-hydrocarbon, olefin-containing, demethylated, or staple-like structures. Preferred options are Fmoc-L-2-(4′-pentenyl)alanine and ring-closing metathesis (RCM) (see, for example, Kim YW, Kutukian PS, Verdine GL. Introduction of all-hydrocarboni,i+3staples 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 for crosslinking or binding peptides include lactam bridges introduced by “click” chemistry, hydrogen bond substitution, light-controlled switching, thioethers, and triazoles.

[0194] In a preferred embodiment, the compound according to the invention further comprises a peptide sequence that imparts cell permeability, organelle targeting, nuclear localization, mitochondrial localization, blood-brain barrier permeability, cell membrane localization, and / or peptidase cleavage. Cell permeation sequences are, for example, the HIV TAT-sequence (GRKKRRQRRRPP, SEQ ID NO: 41) or ARKKRRQRRRPP (SEQ ID NO: 66). Other suitable sequences are known from the literature and may be derived, for example, from 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 peptide sequence can preferably be fused to the N- and / or C-terminal portion of the peptide.

[0195] The term "D-isomer," as used herein, refers to an amino acid sequence in which at least a portion of the amino acid residues have a molecular spatial configuration called "D" (Latin dexter; right). The compounds of the present invention comprise at least 80%, more preferably at least 90%, more preferably at least 95%, most preferably 99% or 100% (i.e., "all-D") D-amino acids. The peptides of the present invention preferably comprise 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. Furthermore, the peptides of the present invention preferably comprise 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.

[0196] As used herein, the abbreviation "DRI" refers to the D-trans-inverted isomer, in which the amino acid sequence is reversed and placed in a D- rather than L-isomer configuration, specifically with reference to the sequence of the human FOXO4 protein or a peptide fragment thereof. Those skilled in the art will recognize that the DRI peptide according to the invention may comprise a combination of L-amino acid residues and D-amino acid residues, or may be composed entirely of D-amino acid (all-D) residues, as disclosed above.

[0197] The term "% sequence identity" or "% identity sequence" is defined herein as the percentage of nucleotides in a nucleic acid sequence or amino acids in an amino acid sequence that are identical to those in a target nucleic acid or amino acid sequence, after alignment and optionally the introduction of necessary spaces to achieve the maximum percentage sequence identity. Methods and computer programs used for alignment are well known in the art. Sequence identity is calculated over almost the entire length, preferably the full (complete) length, of the target amino acid sequence. Those skilled in the art will understand that consecutive amino acid residues in one amino acid sequence are compared to consecutive amino acid residues in another amino acid sequence. Preferably, the conformation of the amino acid residues, such as D or L, is irrelevant to determining amino acid sequence identity. For example, in the context of this invention, D-Ala (DA) exhibits sequence identity with L-Ala (LA).

[0198] Those skilled in the art also know that there are in vitro standard assays for determining the degree of apoptosis in cells and / or cell cultures, such as assays for assessing the levels of cytochrome C (an apoptosis marker) and TUNEL (an apoptosis marker). Using these standard assays, those skilled in the art can readily assess and compare the apoptosis-inducing activity of different compounds on different cell types or cells at different developmental stages (e.g., senescent cells vs. non-senescent cells). Other standard apoptosis assays are annexin V assays and staining for caspase-3 cleavage. To detect cell viability, which is essentially the opposite of apoptosis, MTT assays (colorimetric assays for assessing cell viability), ATP assays, real-time cell density assays (e.g., xCELLigence), or community formation assays can be used. Further information on the assays to be used can be obtained from the literature, such as Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007; 35(4): 495-516. Preferred detection methods include the determination of elevated caspase-3 / 7 activity, loss of mitochondrial cytochrome C, TUNEL positivity, extracellular connectin V positivity, and / or elevated cell death markers (e.g., inclusion of propidium iodide), and / or loss of vitality (e.g., incorporation of calcein AM or MTS activity assay).

[0199] Typically and preferably, senescent cells can be identified by the presence / reduction of markers LMNB1 and / or HMGB1, and p21. Cip1 and / or p16 INK4aCells exhibiting elevated levels and / or presence of DNA-SCARS markers yH2AX and / or 53BP1 and / or PML, and / or presence of SASP markers such as IL1 and / or IL6 and / or MMP1. Cells do not need to show a complete absence of TP53 or its mutants. Typically and preferably, cancer cells can be identified as p21. Cip1 Cells exhibiting elevated and / or present levels of DNA-SCARS markers yH2AX and / or 53BP1 and / or PM, and / or present SASP markers such as IL1 and / or IL6 and / or MMP1. Cells do not need to show a complete absence of TP53 or its mutants. Typically and preferably, scar cells can be identified as cells with elevated levels of DNA-SCARS (larger clusters of 53BP1 and / or yH2AX, and / or PML / FOXO4 nucleoplasms).

[0200] In another aspect, the present invention provides a nucleic acid (to the extent that it is biologically probable) encoding the peptide of the present invention, optionally contained in a vector (e.g., an expression vector). The nucleic acid may be DNA, RNA, cDNA, PNA, or a combination thereof. In another aspect, the present invention provides a host cell comprising the nucleic acid or vector of the present invention. The host cell may be any suitable prokaryotic or eukaryotic cell of any kind, such as those selected from 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 recombinant host cells expressing p53 and / or its FOXO4-binding fragment.

[0201] As described above, this invention relates to improved inhibitory compounds designed because the evolutionary differences between FOXO1 / 3 / 6 and FOXO4 indicate that some amino acids can still be modified or altered to create improved inhibitory compounds. All these modifications present novel strategies, in which any one or a combination of these ultimately leads to the “rational design” of improved molecules for the purposes of this invention. The invention also includes strategies for further improving compounds that have only undergone partial “directed evolution” or “directed mutation.”

[0202] Therefore, another important aspect of the present invention is a method for identifying improved compounds that bind to p53, or preferably bind to p53 and inhibit the interaction between FOXO4 and p53 in cells, comprising the following steps: a) providing at least one compound according to the present invention; b) suitably modifying the compound of a); c) determining at least one of the following: the binding of the at least one compound of b) to p53 or a fragment thereof, the stability of the at least one compound, and the binding of FOXO4 or a fragment thereof to p53 or a fragment thereof in the presence of the at least one compound (compared to the binding of FOXO4 or a fragment thereof to p53 or a fragment thereof in the absence of the compound); and d) based on the determination in step c), identifying improved compounds that bind to p53, or preferably bind to p53 and inhibit the interaction between FOXO4 and p53 in cells, compared to the compounds provided in step a).

[0203] Preferably, the method according to the invention is wherein the binding of the at least one compound is specific to, and / or determined / detected to be specific to p53 or a fragment thereof.

[0204] Another preferred embodiment of the method according to the invention further includes testing the activity of the identified compound in inducing apoptosis and / or killing senescent cells, scar cells, or cancer or tumor cells, preferably including the determination of caspase activity.

[0205] In the process of “rationally designing” improved molecules for the purposes of this invention, and in order to further improve compounds that have only undergone “directed evolution” or “directed mutation” in part, the compounds may be subjected to the above-described methods in successive rounds.

[0206] After a compound (e.g., a peptide) is provided, such as after its appropriate chemical synthesis, the compound can be modified. In general, many methods for modifying the compounds of the present invention are known to those skilled in the art and are described in the literature. Modification of the compound will generally fall into several categories, such as a) amino acid mutation / change to different amino acids, b) chemical modification of amino acids, such as by adding additional chemical groups, c) modification of amino acid structure (e.g., L-to-D-type) or linkage (e.g., introduction of anti-inverted bonds), d) change of compound length, and e) attachment of additional groups to the molecule (including labeling groups, tags, linkers, or carriers, such as chelating agents).

[0207] Evolutionary differences between FOXO1 / 3 / 6 and FOXO4 indicate that some amino acids can still be modified to create improved compounds exhibiting the desired binding and—fundamentally—inhibitory activities. Analysis of the FOXO4-FH fragment and the TP53-TAD2 domain showed that the negative charge of TP53 and its hydrophobic Tyr mediate binding to FOXO4. Therefore, the amino acids in the peptides of this invention can be mutated to positively charged amino acids, which is beneficial to the strength of the interaction. Examples are lysine (K), arginine (R), and histidine (H). NMR experiments also showed that the negatively charged amino acid in TP53 is responsible for interacting with FOXO4 around the SQ motif (site). This site can be phosphorylated to prevent this, and the site can be mutated to small amino acids, such as alanine (A), glycine (G), or serine (S). Methods for introducing these mutations are known to those skilled in the art and include genetic engineering methods such as introducing modifications during the chemical synthesis of peptides, or corresponding alterations to the nucleotide sequence encoding the peptide, for example, through oligonucleotide mutations, PCR-involved mutations, etc. Random mutations are also feasible. Further guidance on promising amino acid modifications can be found above regarding X1-X. 12 This can be found within the context of the definition, and it also conveniently applies to the remaining X. 13 -X 18 Definition.

[0208] In addition, amino acids can also be altered through chemical modification, for example by adding additional chemical groups during synthesis or through post-translational or post-synthetic modification. Methods for altering amino acids are well known in 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.

[0209] Furthermore, alterations can be made to the amino acid structure (e.g., L-to-D-type) or the bonds connecting amino acids (e.g., introducing trans-inverted bonds during synthesis). NMR also shows that the three N-terminal amino acid LTLs are unnecessary for the interactions. Therefore, the length of the compound can be altered, for example, by removing these three amino acids.

[0210] Finally, additional chemical and / or functional groups for the molecule, such as labeling groups, tags, linker amino acids, staples, cysteine ​​bridges, glycosylation sites, ubiquitination and / or polyethylene glycolation sites, linkers, or carriers, such as chelating agents, may be included. This also includes the introduction or addition of amino acids (staples) that allow the preparation of dimers or polymers of the FOXO4-derived DRI peptide. Furthermore, stabilization modifications (e.g., so-called end-capping) may be included to enhance the stability of the FOXO4-derived DRI peptide and / or eliminate the potential for senescent cells. Stabilization of the FOXO4 peptide can also be achieved by mutagenesis at sites where the peptide frequently breaks (e.g., NG-sites). Staples can be achieved by introducing two α-pentenyl-glycine residues, or α,a'-pentenyl-glycine residues, for crosslinking the i and i+4 or i and i+7 residues within the peptide. These residues are used to create all-hydrocarbon, olefinic, demethylated, or stapled confinements. Other strategies that can be used to crosslink or bind peptides include lactam bridges introduced via "click" chemistry, hydrogen bond substitution, light-controlled switching, thioethers, and triazoles.

[0211] In the next step, the modified compound is tested for binding to at least one of the at least one compound with p53 or a fragment thereof. As discussed herein, the property of a compound to bind to p53 or a binding fragment thereof (like TP53) is essential for all uses of the compound, whether therapeutic or diagnostic. In the context of this invention, “improved” binding includes two scenarios where the modified compound binds to the same extent as the unmodified (i.e., the starting) compound, even though the compound has been modified (e.g., by dimerization, or by adding a label or other group). Preferably, the modified compound exhibits a stronger binding to the target, i.e., p53 or a binding fragment thereof. Also preferred are compounds that exhibit a longer binding to the target, i.e., p53 or a binding fragment thereof, for example, due to improved in vitro or in vivo stability of the modified compound. Additionally, compounds exhibiting reduced phosphorylation of the modified compound are also preferred. Combinations of at least two of the above properties are also included.

[0212] Assays for detecting the binding of a compound to a target (i.e., p53 and / or its binding fragments) are well known to those skilled in the art, and preferably include mass spectrometry, NMR detection, pull-down detection, etc.

[0213] In addition, competitive detection is included, which involves detecting the properties of the modified compound in the presence of at least one compound, compared with the binding of FOXO4 or a fragment thereof with p53 or a fragment thereof in the absence of the compound.

[0214] Another preferred method according to the invention is one in which the binding of the at least one compound is specific to, and / or confirmed / detected to be specific to p53 or a fragment thereof. As mentioned above, the property of the compound to bind to p53 or a binding fragment thereof (e.g., TP53) is essential for all uses of the compound, therapeutic or diagnostic. Ideally, the binding is also specific to, or at least substantially or essentially specific to, the intended target, i.e., p53 or a binding fragment thereof. This will reduce or avoid unwanted side effects of the compound in medical use, and reduce or avoid any background or false positive results in diagnostic use.

[0215] In the final step of the method, based on the determination in step c), compounds that are improved compared to the compounds provided in step a) – in a preferred aspect of the invention – bind to p53, or preferably bind to p53 and inhibit the interaction between FOXO4 and p53 in cells are identified. Although this function is considered necessary for the therapeutic function of the compounds of the invention via the elimination of senescent cells, such a property / function may not be mandatory for the improved diagnostic molecules according to the invention.

[0216] The identification of these improved compounds may include detecting the efficacy of these compounds, such as the FOXO4 peptide, in binding to TP53 and eliminating senescent cells, optionally, for the identification of structural requirements that determine this property; and / or detecting the selectivity of these compounds, such as the FOXO4 peptide, in eliminating senescent cells, again optionally, for the identification of structural requirements that determine this property. FOXO4 differs evolutionarily from FOXO1 / 3 and 6, and from FOXO orthologs in lower species, in some unique residues. The inventors have never found FOXO1 or FOXO3 to be the focus, nor is there evidence that FOXO1 and 3 are important for the survival of senescent cells. Therefore, it was analyzed whether these amino acids at least partially explain the efficacy of the FOXO4-peptide in eliminating senescent cells. This was confirmed in D-trans-inverted peptides based on domains similar to FOXO4, while sequences based on FOXO1 or FOXO3 did not show selectivity in eliminating senescent cells.

[0217] Also preferred is the method according to the invention, wherein the method further includes testing the activity of the identified compound in inducing apoptosis and / or killing senescent cells, scar cells, cancer or tumor cells, preferably including the determination of caspase activity. As mentioned above, those skilled in the art also know that there are in vitro standard assays for determining the degree of apoptosis in cell cultures, such as assays assessing the levels of cytochrome C (a marker of apoptosis) and TUNEL (a marker of apoptosis). Using these standard assays, those skilled in the art can readily assess and compare the apoptosis-inducing activity of different compounds on different cell types or cells at different developmental stages (e.g., senescent cells vs. non-senescent cells). Other standard apoptosis assays are annexin V detection and staining for caspase-3 cleavage. Preferred assays include the determination of elevated caspase-3 / 7 activity, loss of mitochondrial cytochrome C, TUNEL positivity, extracellular annexin V positivity, and / or elevated cell death markers (e.g., inclusion of propidium iodide), and / or loss of viability (e.g., incorporation of calcein AM, or MTS viability assay). To detect cell viability, which is essentially the opposite of apoptosis, MTT assay (a colorimetric assay for assessing cell viability), ATP assay, real-time cell density (e.g., xCELLigence) assay, or community formation assay can be used. Further information on the assays to be used can be obtained from the literature, for example, Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007; 35(4): 495-516. Preferably, the peptide or modified peptide according to the invention is considered to exhibit apoptosis-inducing activity in senescent cells if it kills, eliminates, or removes at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, or 80% of cells (or reduces their viability) in senescent cell cultures. Preferably, the peptide according to the invention selectively exhibits apoptosis-inducing activity in senescent cells, i.e., not in non-senescent cells. The peptides according to the invention favor apoptosis in senescent cells, and even more so in non-senescent cells, by a factor of 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.

[0218] The peptides according to the invention are preferably isolated. The peptides according to the invention are preferably prepared using appropriate peptide synthesis.

[0219] In one aspect of the method according to the invention, FOXO4 or its p53-binding fragment, p53 or its FOXO4-binding fragment, and / or the compound (peptide) are tagged, for example, with fluorescent, detectable, stable isotopic, and / or mass tags. These tags are known in the literature and include FITC, Alexa, Dylight, FAM, and others. Detectable tags can be recognized by antibodies, streptavidin / avidin, or based on an enzymatic reaction that induces a detectable product. Mass tags can be implemented by attaching isotopes or other suitable mass markers. In principle, any tag suitable for experimental and therapeutic / imaging purposes can also be used.

[0220] Furthermore, the method according to the invention is preferred, wherein FOXO4 or a fragment thereof, and / or p53 or a fragment thereof, is recombinantly expressed in cells. Methods for recombinantly expressing peptides and for isolating and purifying the recombinant preparation products are well known in the art and are described in various documents. Generally, any suitable cell can be used for the recombinant preparation of FOXO4 or a fragment thereof, and / or p53 or a fragment thereof, preferably cells selected from cancer cells, senescent cells, human non-embryonic stem cells, yeast cells, bacterial cells, and recombinant host cells expressing FOXO4 and / or its p53-binding fragment (wherein the recombinant host cell optionally expresses p53 or its FOXO4-binding fragment, i.e., using two or more constructs).

[0221] Another aspect of the invention relates to a screening tool for screening compounds of the present invention that bind to p53, and preferably bind to p53, and inhibit the interaction between FOXO4 and p53 in cells, comprising isolated cells expressing FOXO4, and / or cells expressing their p53-binding fragments (wherein the cells optionally express p53), and / or cells expressing FOXO4-binding fragments.

[0222] The screening tool of this invention is an important part of carrying out the method of this invention and can also be used as a first step in verifying the in vivo drug activity of the compound. Preferably, FOXO4 or a fragment thereof, and / or p53 or a fragment thereof, are recombinantly expressed in cells. Methods for recombinant expression of peptides and for isolating and purifying the recombinant preparation products are well known in the art and are described in various literatures. Generally, any suitable cells can be used for the recombinant preparation of FOXO4 or a fragment thereof, and / or p53 or a fragment thereof, preferably cells selected from cancer cells, scar cells, senescent cells, human non-embryonic stem cells, yeast cells, bacterial cells, and recombinant host cells expressing FOXO4 and / or its p53-binding fragment (wherein the recombinant host cells optionally express p53 or its FOXO4-binding fragment, i.e., using two or more constructs). In one aspect of the tool according to the invention, FOXO4 or its p53-binding fragment, p53 or its FOXO4-binding fragment, and / or the compound (peptide) are tagged, for example, with fluorescent, detectable, and / or quality tags. As mentioned above, such tags are known in the literature and include FITC, Alexa, Dylight, FAM, and others. Detectable tags can be recognized by antibodies, streptavidin / avidin, or based on enzymatic reactions that induce detectable products. Quality labeling can be achieved by attaching isotopes or other suitable quality markers.

[0223] Preferred is the screening tool according to the invention, comprising fragments of FOXO4, such as peptides (preferably all-D-peptides) according to the sequence RKK PRKGGSRRNAWGNQSYAELISQAIESAPEKRLTLAQIYEWMVRTVPYFKDKGDSNSSAGWKNSIRHNLSLHSKFIKVHNEATGKSSWWMLN (SEQ ID NO: 32) or PRKGGSRRNAWGNQSYAELISQAIESAPEKRLTL (SEQ IFD NO: 33), and fragments of p53, such as peptides according to the sequence AMDDLMLSPDDIEQWFTEDPGP (SEQ ID NO: 34).

[0224] Another important aspect of this invention relates to the use of the compounds of the invention in diagnostics, for example, as diagnostic compounds. Clearly, recognizing / using the compounds of the invention to bind to p53 or p53 fragments preferably also inhibits the interaction between FOXO4 and p53 in cells. These properties are also used to diagnose background. More preferably, the binding of at least one compound is specific to, and / or determined / detected to be specific to p53 or p53 fragments or fragments thereof. This will reduce or avoid any background or false positive results in diagnostic use. The compounds (peptides) according to the invention are preferably tagged, for example, with fluorescent, detectable, stable isotope, and / or mass tags. These tags are known in the literature and include FITC, Alexa, Dylight, FAM, and others. Detectable tags can be recognized by antibodies, streptavidin / avidin, or based on an enzymatic reaction that induces a detectable product. Mass tags can be implemented by attaching isotopes or other suitable mass markers. However, diagnostic methods can also be implemented without tags, and the binding can be detected directly or indirectly using, for example, NMR.

[0225] One specific diagnostic use or method could be a method for detecting the expression of p53 or fragments thereof in cells, including using the compounds of the present invention to detect p53 or fragments thereof, wherein increased binding compared to control cells that do not express p53 or fragments thereof indicates the expression of p53 or fragments thereof in those cells. Due to the high specificity of the compounds of the present invention, the compounds provide improved molecules for diagnostic purposes. Diagnosis could also include detecting p53-related diseases or symptoms, such as cancers or other conditions caused by senescent cells, such as age-related diseases, kidney disease, non-alcoholic steatohepatitis (NASH) / non-alcoholic fatty liver disease (NAFLD), liver fibrosis, idiopathic pulmonary fibrosis (IPF), amyotrophic lateral sclerosis (ALS), osteoarthritis, COPD, skeletal muscle diseases, and cognitive decline.

[0226] In the context of this invention, the term "about" means including a given amount of + / - 20%, preferably + / - 10%, unless otherwise indicated.

[0227] Preferably, the method according to the invention is applicable to automation, and preferably implemented in an automated and / or high-throughput manner. Generally, this involves the use of chips and respective mechanical devices such as robotic arms. Automation is particularly preferred in the identification and / or screening of improved compounds. Simultaneously, the use of the screening tool of the invention can also be included in the automation.

[0228] Another important aspect of the invention relates to a method for preparing a pharmaceutical composition for treating or preventing senescent cells in a subject, comprising formulating a compound according to the invention into a suitable pharmaceutical composition, or carrying out the method of the invention for identifying an improved compound that preferably inhibits the interaction between FOXO4 and p53 in cells, and the step of formulating the identified compound into a suitable pharmaceutical composition. The invention also relates to pharmaceutical compositions for treating or preventing senescent cells in a subject, obtained by the method of the invention.

[0229] As used herein, the term "pharmaceutical composition" refers to a composition prepared under conditions suitable for administration to mammals (preferably humans), such as under GMP conditions. Pharmaceutical compositions according to the invention may contain pharmaceutically acceptable excipients, such as, but not limited to, stabilizers, swelling agents, buffers, carriers, diluents, solvents, solubilizers, and binders. Those skilled in the art will understand that the selection of suitable excipients depends on the route of administration and dosage form, as well as the active ingredient and other factors. Pharmaceutical compositions according to the invention are preferably suitable for parenteral administration.

[0230] Preferably, the pharmaceutical composition according to the invention comprises a mixture of compounds, and / or a mixture of at least one compound with other pharmaceutically active ingredients. As used herein, the term "pharmaceutical active ingredient" refers to a compound that inhibits or prevents cell viability and / or function, and / or causes cell destruction (cell death), and / or exerts antitumor / antiproliferative effects, such as directly or indirectly preventing the development, maturation, or spread of tumor cells. The term also includes agents that cause growth inhibition effects only, and not merely cytotoxic effects. The term also includes alkylating agents such as platinum-based drugs (e.g., cisplatin, carboplatin, and oxaliplatin), antimetabolites such as 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (Xeloda), cladribine, chlorofarapine, cytarabine (Ara-C), deoxyfluorouridine, fludarabine, gemcitabine (Gemzar), hydroxyurea, and methotrexate, antitumor antibiotics, preferably doxorubicin, topoisomerase inhibitors, mitotic inhibitors, corticosteroids, antiangiogenic agents, tyrosine kinase inhibitors, protein kinase A inhibitors, cytokine family members, and radioisotopes. Chemotherapeutic agents as used in this invention also include paclitaxel, such as Paclitaxel or Doxetaxel.

[0231] In another aspect, the present invention provides the use of at least one peptide, pharmaceutical composition, or nucleic acid according to the invention in medicine, or as a medicine, or for the treatment of symptoms or diseases or conditions.

[0232] As 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 mammalian (preferably human) subject in which the presence of senescent cells or the presence of cellular senescence is associated with the disease or condition in that subject.

[0233] It is believed that normal and pathological degenerative aging phenotypes (loss of function) and cancer (gain of function) are causally related to cellular senescence. Senescent fibroblasts are associated with reduced milk production in the mammary glands, senescent pulmonary artery smooth muscle cells are associated with pulmonary hypertension, senescent skin cells are associated with thinning of the dermis and reduced collagen content, senescent astrocytes are associated with Alzheimer's disease and Parkinson's disease, and senescent chondrocytes are associated with osteoarthritis.

[0234] Symptoms and diseases confirmed to be associated with cellular senescence include: atherosclerosis, emphysema, diabetic ulcers, kidney disease (see, e.g., Valentijn FA, et al. Cellular senescence in the aging and disordered kidney. J Cell Commun Signal. 2018 Mar; 12(1): 69-82), kyphosis, osteoporosis, macular degeneration, COPD and insulin resistance, diabetes, obesity, laminopathies (e.g., progeria, hernia, sarcopenia, and cachexia), arthritis, scoliosis, and cancer. Therefore, the clearance of senescent cells at the genetic level significantly improves health and reduces aging parameters, as shown in accelerated aging mouse models (Baker et al., 2011. Nature 479(7372): 232-6). This concept-validation evidence is obtained in a genetic sense, with poor therapeutic applicability. This invention provides novel and improved compounds, as well as methods for identifying them, which therapeutically target senescent cells and can be used in the treatment of diseases related to cellular senescence.

[0235] Another aspect relates to the use of at least one compound or pharmaceutical composition in the prevention or treatment of symptoms, wherein the symptoms to be prevented and / or treated are selected from symptoms caused by senescent cells, age-related diseases, kidney disease, non-alcoholic steatohepatitis (NASH) / non-alcoholic fatty liver disease (NAFLD), liver fibrosis, idiopathic pulmonary fibrosis (IPF), amyotrophic lateral sclerosis (ALS), arthritis (such as osteoarthritis), COPD, skeletal muscle diseases, cognitive impairment, atherosclerosis, emphysema, diabetic ulcers, kyphosis, osteoporosis, macular degeneration, COPD and insulin resistance, diabetes, obesity, laminar diseases (such as progeria, hernia, sarcopenia, and cachexia), scoliosis, and cancer.

[0236] Preferably, at least one compound or pharmaceutical composition is used in the present invention, wherein the therapy is a combination therapy with an anticancer chemotherapeutic agent or other standard care medicine for each disease or condition, or wherein the therapy is applied to senescent cells, scar cells, and / or cancer cells that have been pretreated with an anticancer chemotherapeutic agent or a standard care medicine for each disease or condition and have survived the treatment.

[0237] As a result, another aspect relates to a method for treating or preventing diseases or conditions caused by senescent cells, scar cells, age-related diseases, kidney disease, non-alcoholic steatohepatitis (NASH) / non-alcoholic fatty liver disease (NAFLD), liver fibrosis, idiopathic pulmonary fibrosis (IPF), amyotrophic lateral sclerosis (ALS), arthritis (such as osteoarthritis), COPD, skeletal muscle diseases, cognitive decline, atherosclerosis, emphysema, diabetic ulcers, kyphosis, osteoporosis, macular degeneration, COPD and insulin resistance, diabetes, obesity, laminar diseases (such as progeria, hernia, sarcopenia, and cachexia), scoliosis, and cancer in a subject of need, comprising administering to the subject an effective amount of at least one compound of the present invention, or a pharmaceutical composition of the present invention.

[0238] Preferably, the method according to the invention is a combination therapy with an anticancer chemotherapeutic agent or other standard care medicine for each disease or condition, or the therapy is applied to senescent cells, scar cells, and / or cancer cells that have been pretreated with an anticancer chemotherapeutic agent or standard care medicine for each disease or condition and have survived the treatment.

[0239] "Treatment" refers to the reduction and / or alleviation of disease symptoms. Effective treatment achieves, for example, the removal of senescent cells, a reduction in tumor quality and the number of cancer cells. Treatment can also prevent (avoid) and reduce the number of senescent cells, reduce cancer spread, for example, by influencing its metastasis and / or formation. Treatment can be initial treatment (before the initiation of treatment for any other disease) or treatment after the first round of treatment (e.g., after surgery, or after recurrence). Treatment can also be combined therapy, involving, for example, chemotherapy, surgery, and / or radiation therapy. It can be used to treat subjects, especially humans, who have or are suspected of having a disease or condition that can benefit from the removal of senescent cells.

[0240] In the context of this invention, it has been surprisingly discovered that the compounds according to the invention can be particularly beneficial for treating resistant cancers, and are most effective, as exemplified in the present embodiments and figures. In addition to their effectiveness, it has been shown that the compounds of the present invention are not merely proliferation inhibitors, but are indeed cytotoxic to cancer cells. Peptides are particularly effective in cells that have been pretreated with disruptive agents such as chemotherapy / radiotherapy and survived the treatment. Therefore, treatment using peptides can be administered after a primary cell-disrupting therapy, such as chemotherapy / radiotherapy, dietary stress, exogenous stress, oxidative stress, etc., or as described above, as a combination therapy, i.e., applied simultaneously or acting concurrently, rather than sequentially. This is particularly effective when using, for example, small molecule inhibitors such as BRAF inhibitors.

[0241] The present invention also relates to a kit comprising a first container and a second container, the first container containing the compound of the present invention and the second container containing a chemotherapeutic agent. The kit may suitably include instructions for administration in mammalian (preferably human) subjects. Preferably human subjects have or are suspected of having diseases or conditions caused by senescent cells, scar cells, age-related diseases, kidney disease, non-alcoholic steatohepatitis (NASH) / non-alcoholic fatty liver disease (NAFLD), liver fibrosis, idiopathic pulmonary fibrosis (IPF), amyotrophic lateral sclerosis (ALS), arthritis (such as osteoarthritis), COPD, skeletal muscle diseases, cognitive decline, atherosclerosis, emphysema, diabetic ulcers, kyphosis, osteoporosis, macular degeneration, COPD and insulin resistance, diabetes, obesity, laminar diseases (e.g., progeria, hernia, sarcopenia, and cachexia), scoliosis, and cancer. Although the compounds and chemotherapeutic agents of the present invention can be formulated in a single dosage form, such as a pharmaceutical composition, they are preferably formulated in multiple dosage forms, wherein the compounds of the present invention are in one container and the chemotherapeutic agents are in another container. In the kits used in the present invention, the compounds of the present invention are preferably administered together with the chemotherapeutic agents. Preferably, the peptide is administered after the administration of the chemotherapeutic agent. Preferably, the compounds of the present invention are administered as adjuvants, thereby enabling them to clear, kill, or reduce the viability of cells that have become senescent due to treatment with the chemotherapeutic agent. Unexpectedly, the compounds of the present invention can reduce the off-target effects of current chemotherapeutic agents. Preferably, the kits according to the present invention include instructions for dosing regimens to obtain the optimized combined effect of the compounds of the present invention and the chemotherapeutic agents.

[0242] In another aspect of the invention, the invention also relates to a diagnostic kit comprising materials for carrying out the methods of the invention, as described herein, in one or separate containers, optionally together with excipients and / or instructions for carrying out the methods of the invention. The kit may contain compounds described herein and identified compounds. Additionally, it may contain dyes, antibodies, and other components for the aforementioned detection assays. The kit may contain compounds described herein and / or identified compounds in labeled form as described above.

[0243] The present invention will be further described in the following embodiments with reference to the accompanying drawings; however, the invention is not limited thereto. For the purposes of this invention, all references cited herein are incorporated herein by reference in their entirety. Attached Figure Description

[0244] Figure 1 The sequence of human FOXO4 (Homo sapiens) is shown, with the underlined part representing the sequence of the TP53 interaction domain.

[0245] Figure 2 The exemplary peptide CL04009 (SEQ ID NO: 11) is shown to be more potent and superior to the reference compound (FOXO4-DRI) in binding to recombinant TP53-TAD2 and full-length TP53 isolated from cells. A) Shows the relative NMR chemical shift perturbations of 15N-labeled TP53-TAD2 alone (bottom), in the presence of CL04009 (SEQ ID NO: 11) (top), or in the presence of the reference compound (middle). The NMR chemical shift perturbation caused by the reference compound has been set to 100%. B) Shows the pull-down of endogenous TP53 in HEK293T cell lysates using streptavidin beads in the absence of any peptide, in the presence of biotinylated CL04009 (SEQ ID NO: 11) or biotinylated reference peptide (FOXO4-DRI).

[0246] Figure 3 The figure shows that peptide CL04009 (SEQ ID NO: 11) is potent and selective in inducing apoptosis via caspase-3 / 7 activation in senescent, non-healthy control IMR90 lung fibroblasts. The modified CL04009 (SEQ ID NO: 11) with improved TP53 binding exhibits good selectivity in inducing apoptosis via caspase-3 / 7 activation in senescent, non-healthy control IMR lung fibroblasts. Furthermore, it is effective even at lower concentrations, indicating its improved efficacy. The top figure shows caspase activity, and the bottom figure shows cell viability. FOXO4-DRI was used as a control.

[0247] Figure 4The results show that CL04022 (SEQ ID NO: 12) is more potent than the reference compound FOXO4-DRI in binding TP53-TAD. The fluorescence polarity measurement of FITC-labeled TP53-TAD2 (250 nM) in the presence of increasing peptide concentrations is shown.

[0248] Figure 5 As shown, based on the alanine substitution in the peptide, peptide CL04022 (SEQ ID NO: 12) is more stable than CL04009 (SEQ ID NO: 11) over a longer period of several days. Both peptides are soluble in H2O and stored at room temperature. Peptide concentrations were determined at specified time points.

[0249] Figure 6 The image shows that peptide CL04022 (SEQ ID NO: 12) began to induce caspase-3 / 7 activation 24 h after administration, a typical process of apoptosis. Complete caspase-3 / 7 activation was completed after 60 h; the top row shows the control.

[0250] Figure 7 The diagram shows the alterations made by peptide CL04009 (SEQ ID NO: 11) to improve TP53 selective binding, resulting in a highly effective and selective ability to induce apoptosis in senescent (upper blue line) and non-healthy controls (lower black line) IMR90 cells. The graph below shows that peptide CL04088 (SEQ ID NO: 13) is superior to peptide CL04022 (SEQ ID NO: 12) and still outperforms the control.

[0251] Figure 8 The modification of peptide CL04009 (SEQ ID NO: 11) (represented herein as CL05055) shows that it can further improve the efficacy of eliminating senescent cells from different sources. RPE = retinal pigment epithelial cells, and CL03001 is the same as FOXO4-DRI used as a control.

[0252] Figure 9 This study demonstrates that CL04022 (SEQ ID NO: 12) is superior to FOXO4-DRI in inducing programmed cell death 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 loss (cell death) in GBM8 glioblastoma. C) This study demonstrates that CL04022 (SEQ ID NO: 12) induces apoptosis in GBM8 glioblastoma cells but not in healthy Wi38 cells. CL04022 (SEQ ID NO: 12) also exhibits selectivity in inducing apoptosis in GBM8 glioblastoma cells relative to healthy Wi38 cells.

[0253] Figure 10 The efficacy of CL04022 (SEQ ID NO: 12) against colorectal cancer is shown. A) The peptide CL04022 (SEQ ID NO: 12) is effective against two microsatellite instability (MSI) colorectal cancer 3D organoid cultures. The peptide is ineffective against one microsatellite stable (MSS) lineage. B) CL04022 (SEQ ID NO: 12) is shown to be selective for colorectal cancer derived from patients compared to healthy control samples. CL04022 (SEQ ID NO: 12) shows lower potency against normal wild-type colon organoids (compared to A above). The peptide is effective when the organoids lack APCs.

[0254] Figure 11 The results show that CL04183 (SEQ ID NO: 55) and CL05114 (SEQ ID NO: 60) have better stability in solution compared to FOXO4-DRI (CL03001). In the table, CL02001 represents the "L" form of peptide CL03001, and CL02015 represents the "L" form of peptide CL04183 (SEQ ID NO: 55).

[0255] Figure 12 A) Peptide CL04022 (SEQ ID NO: 12) is shown to be more effective in 3D organoids of colon cancer that survive chemotherapy (5'-fluorouracil or oxaliplatin), and B) CL04022 (SEQ ID NO: 12) induces cell death rather than cell cycle arrest, which is the opposite of standard care 5FU.

[0256] Figure 13 The results show that 14 days after exposure to CL04009 (SEQ ID NO: 11), the 3D colon organoids no longer exhibited tumor vegetations. This indicates that CL04009 (SEQ ID NO: 11) is cytotoxic to colon organoids, and not merely inhibits cell proliferation, as illustrated in the colon cancer example.

[0257] Figure 14 In one experiment, peptide CL04183(B) (SEQ ID NO: 55) showed better selectivity and potency against senescent human RPE1 cells compared to control human RPE1 cells, while pristine FOXO4-DRI (CL03001) (A).

[0258] Figure 15This study demonstrates colorectal cancer-related mutations in colonic organoids, based on p53, and sensitive to FOXO4-like peptides. WT colon and organoids with different tumor progression (TPO) stages were treated with escalating concentrations of CL04124 (SEQ ID NO: 68) or CL04183 (SEQ ID NO: 55). Cell viability was measured after 5 days.

[0259] Figure 16 This study demonstrates that CL04183 (SEQ ID NO: 55) potently induces apoptosis in patient-derived colorectal cancer organoids. A) Patient-derived colorectal cancer organoid line CRC29 was treated with 5'-fluorouracil (5-FU) or CL04183 (SEQ ID NO: 55). After 3 days, cells were treated with calcein-AM and propidium iodide, and both live and dead cells were stained. Organoids were imaged using an EVOS imaging system. B) Before treatment with CL04183 (SEQ ID NO: 55), CRC29 organoids were co-incubated with calcein-AM and caspase-3 / 7 red dye for apoptosis assay. Real-time imaging was performed immediately using an LSM880 microscope, with images taken hourly. C) CRC29 was treated with increasing concentrations of CL04183 (SEQ ID NO: 55). Cell viability was measured after 5 days.

[0260] Figure 17 The study showed that peptide CL04009 (SEQ ID NO: 11) induced apoptosis in senescent human mammary epithelial cells more effectively than the reference compound FOXO4-DRI / CL03001. MCF10 cells were treated with increasing concentrations of CL04009 (SEQ ID NO: 11) and FOXO4-DR, and caspase assays were performed after 2 days to determine the induction of apoptosis.

[0261] Figure 18 The image shows that CL04183 (SEQ ID NO: 55) reduced gray fur in naturally aging mice. 24-month-old C57BL / 6J mice were photographed before receiving three doses of PBS or CL04183 (SEQ ID NO: 55) via intravenous injection. Mice were photographed again four weeks after the first treatment.

[0262] Figure 19This study demonstrates that peptide CL04183 (SEQ ID NO: 55) reduces luciferase expression in colorectal cancer cells in vivo. A) The CRC29 colorectal cancer organoid line, containing firefly luciferase, was transplanted into the cecum of immunodeficient mice. Two weeks after transplantation, bioluminescence levels were determined by treating mice with three doses of CL04183 (SEQ ID NO: 55) or PBS. All animals were photographed two days after the last dose, and the luminescence values ​​were compared with baseline values. B) Mice were injected with luciferin prior to sacrifice. Organ imaging was performed, and luciferase signals were quantified using M3 visualization software.

[0263] Figure 20 The peptide CL04183 (SEQ ID NO: 55) was shown to reduce colorectal cancer metastasis to the liver and lungs. The colorectal cancer organoid line 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) Human nucleolar staining was performed on the lungs and liver to detect human cancer cells in the mouse organs. B) The amount of metastatic cancer cells in the lungs was quantified using CellProfiler software. Six tiled scans were analyzed for each mouse. All counts were compared to the mean of the PBS-treated group. The number of liver metastases in each stained section was visualized and counted.

[0264] Figure 21 This study demonstrates that CL04183 (SEQ ID NO: 55) induces apoptosis in metastatic cancer cells in vivo. TUNEL assays were performed on CRC29 lung metastases treated with PBS or CL04183 (SEQ ID NO: 55) to determine apoptosis induction. Each group consisted of 3 mice, and the percentage of TUNEL-positive cancer cells was quantified using FIJI.

[0265] Figure 22 The study showed that triple-negative breast cancer lines exhibited an open p53 configuration, which correlated with the sensitivity of CL04183 (SEQ ID NO: 55). This indicates that human breast cancer cell lines treated with escalating concentrations of CL04183 (SEQ ID NO: 55) were subjected to MTS assays after 2 days to determine cell viability. EC50 was calculated using GraphPad Prism. 50 value.

[0266] Figure 23 This demonstrates the efficacy of CL04183 (SEQ ID NO: 55) against NRAS-mutant and BRAF-mutant melanomas. See also... Figure 14 .

[0267] Figure 24NMR peptide binding data are shown. Data were obtained using 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). 1 H, 15 p53-TAD2 T55 extracted from NHSQC NMR spectra 1 H, 15 Enlarged view of the N cross peak. 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.

[0268] Figure 25 NMR peptide binding data are shown. Data were obtained using 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). 1 H, 15 p53-TAD2 T55 extracted from N HSQC NMR spectra 1 H, 15 Enlarged view of the N cross peaks. 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.

[0269] Figure 26 NMR peptide binding data are shown. Data were obtained using 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). 1 H, 15 p53-TAD2 T55 extracted from N HSQC NMR spectra 1 H, 15 Magnified view of the N cross peak. A) Peptide CL04235, SEQ ID NO: 61, B) Peptide CL05114, SEQ ID NO: 60.

[0270] SEQ ID NOs: 1-4, 7-31, and 43-66 illustrate peptides or portions thereof of the present invention.

[0271] SEQ ID NOs: 5, 6 and 42 show control peptides, such as FOXO4-DRI / CL03001.

[0272] SEQ ID NOs: 39 and 40 show the amino acid sequences of human FOXO4 and p53, respectively.

[0273] Example

[0274] In the context of this invention, the terms FOXO4-DRI or CL03001, when associated with 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).

[0275] Cell culture

[0276] Human IMR90, WI38, and RPE cells were grown in Dulbecco modified Eagle medium (DMEM; Lonza) containing 10% FCS and 1% penicillin / streptomycin at 37°C, 3.0% O2, and 5.0% CO2. To induce senescence, cells were irradiated with 10 Gy of ionizing radiation (Gammacell 1000) and left to stand for at least 10 days to become senescent.

[0277] GBM8 cells were cultured in a Dulbecco modified Eagle medium / nutrient mixture F-12 (DMEMF-12) containing high glucose, L-glutamine, phenol red-free, supplemented with 1% (10 mM) HEPES, 1% penicillin / streptomycin, 2% B-27 (50X), 5 μg / ml heparin sodium (derived from porcine small intestinal mucosa, grade IA), 20 ng / ml recombinant human fibroblast growth factor (rhFGF), and 20 ng / ml recombinant human epidermal growth factor (rhEGF). These cells were then incubated in artificial incubators at 37°C, 5% CO2, and 3% O2 ​​in plastic dishes coated with matrix gel (20x diluted in DMEMF-12).

[0278] To perform viability and apoptosis assays, cells were divided into 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.

[0279] organoid culture

[0280] Organoids were grown in an artificial incubator at 37°C and 5% CO2. All organoids were cultured in modified DMEM / F12 (Lonza) Corning droplets supplemented with 1% Glutamax, 1% penicillin / streptomycin, 1% (10mM) HEPES, 10% Noggin, 2% B-27 (50X; Thermo / Life Technologies), N-acetylcysteine ​​(Sigma-Aldrich, 1.25mM), A83-01 (Tocris, 500nM), and SB203580 (Invitrogen / Life Technologies, 3μM). For viability and apoptosis assays, organoids were resuspended in ice-cold medium and then centrifuged at 4°C in 15ml tubes. The resulting clumps were treated with trypsin at 37°C for 5 min to obtain single cells, followed by washing twice with modified DMEM / F12 medium. These cells were then resuspended in Matrigel and seeded in 96-well plates at 5 μl droplets. After 15 minutes, 100 μl of fresh culture medium was added to the wells. Two days after seeding, peptide and chemotherapy treatments were added to the organoids.

[0281] Caspase detection

[0282] For caspase detection, caspase-Glo 3 / 7 assay reagent (Promega) was added to the culture medium 2 days after treatment. The plates were placed in aluminum foil and incubated on a shaker at room temperature for 1 hour, followed by measurement using a luminescent microplate reader.

[0283] MTS testing

[0284] After 6 days of treatment, the cells were treated with 10 μl CellTiter. Cell proliferation assays were performed using Aqueous One solution (Promega) at 37°C for 1 hour to conduct MTS assays. Afterwards, absorbance at 490 nm was measured using a Spectramax M5e.

[0285] Live / dead cell detection on organoids

[0286] Live / dead cell assays were performed on larger organoids three days after peptide and / or chemotherapy treatment, or on organoids treated as single cells and grown for two weeks prior to assay. For the assay, calcein-AM (1:1000) and propidium iodide (PI; 1:100) were added to the organoid culture medium to determine live and dead cells, respectively. Organoids were imaged using a Zeiss cell observation microscope, and signals were quantified using FIJI.

[0287] Real-time imaging

[0288] Organoids and IMR90 cells were grown in 96-well cell culture plates. 488 IncuCyte apoptosis assay with caspase 3 / 7 (Sartorius) or IncuCyte caspase 3 / 7 red blood cell apoptosis assay (EssenBioscience) were added to the wells. Peptides were added before transferring the plates to a Zeiss cell observation microscope containing heat and CO2 control components. Real-time imaging was then initiated, with images recorded every 2 hours for 60 hours. Images were processed using Zen imaging software (Zeiss).

[0289] Alternatively, real-time imaging can be performed using an LSM880 confocal microscope. For this experiment, organoids were plated on glass-bottomed cell culture dishes and incubated with calcein-AM (Sigma-Aldrich) for measuring cell viability and IncuCyte caspase 3 / 7 red apoptosis assay kit (Essen Bioscience). Peptides were added before imaging began.

[0290] mice

[0291] After obtaining approval from the Dutch Animal Ethics Committee, mouse experiments were conducted. For these experiments, male NOD. Cg-Prkdc mice were used. scid Il2rg tm1Wj1 / SzJ / J mice (Charles River). Human CRC29 colorectal cancer organoids containing firefly luciferase were transplanted into the cecum to form a primary tumor. Transplantation was performed under ketamine and dexmedetomidine anesthesia. Additionally, animals received carbofen for 30 min before and one day after surgery. Fourteen days post-transplantation, mice were sedated with isoflurane and imaged using a BioSpace imaging system. Next, animals were treated with PBS or 5'-fluorouracil (5-FU) via intraperitoneal injection at a dose of 50 mg / kg. One week after 5-FU treatment, the first dose of CL04183 (2.5 mg / kg) was administered intravenously, and this treatment was repeated at 2 and 4 days. Four weeks post-transplantation, mice were imaged again to determine treatment effectiveness, after which they were sacrificed. All organs were harvested and imaged again using a BioSpace imaging system, and luciferase signals were quantified using M3 visualization software.

[0292] Immunohistochemistry

[0293] Before washing with TBS, paraffin sections of liver and lungs were rehydrated in decreasing concentrations of ethanol and boiled in 10 nM sodium citrate buffer (pH 6) for 20 minutes for antigen retrieval. After cooling for 30 minutes, the tissues were permeated with 0.2% TX-100 in TBS solution at room temperature for 5 minutes. Next, the sections were washed with TBS and incubated for 1 hour in blocking buffer containing 2% w / v secondary antibody, appropriate serum (e.g., donkey or goat), and 0.1% fish glue in 1% BSA solution. The sections were then wrapped with a waterproof pen and incubated overnight at 4°C with primary antibody diluted with TBS / 1% BSA. The next day, the tissues were washed three times with TBS and incubated for 1 hour with fluorescently labeled secondary antibody diluted with blocking buffer (containing Hoechst 33342 nuclear stain). Afterward, the sections were washed twice with TBS, incubated in Sudan Black solution for 20 minutes to reduce background, and washed in demineralized water. After sectioning, the slides were mounted using a Vectashield mount and imaged using an LSM880 Zeiss confocal microscope.

[0294] Apoptosis staining

[0295] TUNEL assays were performed on lung metastases to determine the induction of apoptosis after treatment. Rehydrated sections were treated with PBS solution containing 20 μg / ml ProtK for 15 min and permeabilized in 0.1% Triton X-100 and 0.1% sodium citrate solution. The tissues were then labeled with a 10% TUNELase-containing labeling solution (ROCHE) at 37°C for 1 h. Before mounting with Vectashield soft mounting medium, the nuclei were labeled with Hoechst 33342 (ThermoFisher). Images were acquired using an LSM880 confocal microscope (Zeiss), and the percentage of TUNEL-positive cells was analyzed using FIJI.

[0296] Protein expression and purification

[0297] Expression constructs for human p53 fragments 1-312 (p53-TADBD), 94-312 (p53-DBD), 1-94 (p53-TAD), and 37-57 (p53-TAD2) were generated by synthesizing corresponding optimized p53 cDNA constructs and inserting them into the pETM11-ZZ-His6 vector (Genscript) via NcoI / BamHI restriction enzyme sites. For the human FOXO4 amino acid 86-208 fragment (FOXO4-FH), the inventors also generated optimized cDNA expression constructs (Genscript) in the pETM11-ZZ-His6 vector. The inventors used three different chemically competent Escherichia coli strains (E. coli BL21(DE3) and E. coli BL21-(DE3 Star) for protein expression, and E. coli TOP10 for plasmid DNA amplification).

[0298] Protein expression and purification were performed using publicly available methodologies known in the art.

[0299] Fluorescence polarization

[0300] Prepare a 116 μl solution of the specific peptide (1-100 μM). Then add 4 μl of FITC-labeled p53 peptide (stock concentration 15 μM, final concentration 500 nM). Transfer 35 μl to each well of a 384-well plate. Perform measurements in triplicate.

[0301] At ClarioStar + Data was acquired using a microplate reader. An endpoint measurement of 200 flashes was performed on each well. Gain and focus were adjusted for each measurement using an excitation filter at 482 nm and an emission filter at 530 nm. Fluorescence intensity, parallel fluorescence polarization, and vertical fluorescence polarization were recorded.

[0302] Data analysis was performed using MARS version (BMG), Microsoft Excel, and GraphPad Prism version 8.

[0303] NMR chemical shift perturbation

[0304] Prepare a 5 mm NMR tube containing 500 μl of 100 μM p53-TAD2 and record... 1 H, 15 N HSQC NMR spectra. Then, incremental amounts of peptide were added, with measurements taken after each step. 1 H, 15N HSQC spectra. NMR spectra were obtained using a 600 MHz Bruker Avance NOE NMR spectrometer equipped with a TXI 600S3 probe tip. Data acquisition and processing were performed using Topspin 3.5 and Topspin 4.0 (Bruker). Data analysis, peak extraction, and assignment were performed using ccpNMR.

[0305]

[0306] sequence list <110> Kelier Biotechnology Private Limited <120> Improved anti-aging compounds and their uses <130> C32322WO <140> PCT / EP2021 / 054338 <141> 2021-02-22 <150> US62 / 979,819 <151> 2020-02-21 <160> 68 <170> PatentIn version 3.5 <210> 1 <211> 25 <212> PRT <213> Homo sapiens <220> <221> Other features <222> (1)..(1) <223> Xaa can be Ala, Ser, or fluorenemethoxycarbonyl-L-2-(4'-pentenyl)alanine (staple) <220> <221> Other features <222> (2)..(2) <223> Xaa can be Lys, Glu, Arg, or His. <220> <221> Other features <222> (3)..(3) <223> Xaa can be Ile, Leu, or cyclohexyl-alanine. <220> <221> Other features <222> (4)..(4) <223> Xaa can be Ala, Gly, Ser, Glu, or Asp. <220> <221> Other features <222> (5)..(5) <223> Xaa can be Gly, Gln, Ala, Ser, or Pro, or fluorenemethoxycarbonyl-L-2-(4'-pentenyl)alanine (stapple) <220> <221> Other features <222> (6)..(6) <223> Xaa can be Ala, Gly, Ser, Glu, or Asp. <220> <221> Other features <222> (7)..(8) <223> Xaa can be Ile, Leu, or cyclohexyl-alanine. <220> <221> Other features <222> (9)..(9) <223> Xaa can be Glu or Asp. <220> <221> Other features <222> (10)..(10) <223> Xaa can be Ala or Glu <220> <221> Other features <222> (11)..(11) <223> Xaa can be Trp, Tyr, or Phe or 2-methyl-tryptophan. <220> <221> Other features <222> (12)..(12) <223> Xaa can be Ala, Ser, or fluorenemethoxycarbonyl-L-2-(4'-pentenyl)alanine (staple) <220> <221> Other features <222> (15)..(15) <223> Xaa can be Ala or Gly <220> <221> Other features <222> (16)..(16) <223> Xaa can be Trp, Tyr, or Phe or 2-methyl-tryptophan. <220> <221> Other features <222> (17) (18) <223> Xaa can be Ala <220> <221> Other features <222> (19)..(19) <223> Xaa can be Arg or Lys <220> <221> Other features <222> (20)..(20) <223> Xaa can be Arg <220> <221> Other features <222> (22)..(22) <223> Xaa can be Gly or Ser <220> <221> Other features <222> (23)..(23) <223> Xaa can be Ala or Cys <220> <221> Other features <222> (24) (25) <223> Xaa can be Arg or Lys <400> 1 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gln Asn Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Ser Xaa Xaa Xaa Xaa 20 25 <210> 2 <211> 32 <212> PRT <213> Homo sapiens <220> <221> Other features <222> (1)..(1) <223> Xaa can be Leu, Thr, or Leu. <220> <221> Other features <222> (2)..(2) <223> Xaa can be Arg or Ser <220> <221> Other features <222> (4)..(4) <223> Xaa can be Lys, Glu, Arg, and His. <220> <221> Other features <222> (5)..(5) <223> Xaa can be Ala or Pro <220> <221> Other features <222> (6)..(6) <223> Xaa can be Ala or Ser <220> <221> Other features <222> (7)..(7) <223> Xaa can be Ala, Ser, or fluorenemethoxycarbonyl-L-2-(4'-pentenyl)alanine (staple) <220> <221> Other features <222> (8)..(8) <223> Xaa can be Lys, Glu, Arg, or His. <220> <221> Other features <222> (9)..(9) <223> Xaa can be Ile, Leu, or cyclohexyl-alanine. <220> <221> Other features <222> (10)..(10) <223> Xaa can be Ala, Gly, Ser, Glu, or Asp. <220> <221> Other features <222> (11)..(11) <223> Xaa can be Gly, Gln, Ala, Ser, or Pro, or fluorenemethoxycarbonyl-L-2-(4'-pentenyl)alanine (stapple) <220> <221> Other features <222> (12)..(12) <223> Xaa can be Ala, Gly, Ser, Glu, or Asp. <220> <221> Other features <222> (13) (14) <223> Xaa can be Ile, Leu, or cyclohexyl-alanine. <220> <221> Other features <222> (15)..(15) <223> Xaa can be Glu or Asp. <220> <221> Other features <222> (17)..(17) <223> Xaa can be Trp, Tyr, and Phe <220> <221> Other features <222> (18)..(18) <223> Xaa can be Ala, Ser, or fluorenemethoxycarbonyl-L-2-(4'-pentenyl)alanine (staple) <220> <221> Other features <222> (21)..(21) <223> Xaa can be Ala or Gly <220> <221> Other features <222> (22)..(22) <223> Xaa can be Trp, Tyr, and Phe <220> <221> Other features <222> (23)..(24) <223> Xaa can be Ala <220> <221> Other features <222> (25)..(25) <223> Xaa can be Arg or Lys <220> <221> Other features <222> (26)..(26) <223> Xaa can be Arg <220> <221> Other features <222> (28)..(28) <223> Xaa can be Gly or Ser <220> <221> Other features <222> (29)..(29) <223> Xaa can be Ala or Cys <220> <221> Other features <222> (30) (31) <223> Xaa can be Arg or Lys <220> <221> Other features <222> (32)..(32) <223> Xaa can be Ala or Cys <400> 2 Xaa Xaa Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala 1 5 10 15 Xaa Xaa Gln Asn Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa Xaa Xaa Xaa Xaa 20 25 30 <210> 3 <211> 14 <212> PRT <213> Homo sapiens <220> <221> Other features <222> (1)..(1) <223> Xaa can be Ile or Leu <220> <221> Other features <222> (2)..(2) <223> Xaa can be Ala, Gly, Ser, Glu, or Asp. <220> <221> Other features <222> (3)..(3) <223> Xaa can be Gly, Gln, Ala, Ser, or Pro, or fluorenemethoxycarbonyl-L-2-(4'-pentenyl)alanine (stapple) <220> <221> Other features <222> (4)..(4) <223> Xaa can be Ala, Gly, Ser, Glu, or Asp. <220> <221> Other features <222> (5)..(6) <223> Xaa can be Ile or Leu <220> <221> Other features <222> (7)..(7) <223> Xaa can be Glu or Asp. <220> <221> Other features <222> (9)..(9) <223> Xaa can be Trp, Tyr, and Phe <220> <221> Other features <222> (10)..(10) <223> Xaa can be Ala, Ser, or fluorenemethoxycarbonyl-L-2-(4'-pentenyl)alanine (staple) <220> <221> Other features <222> (13)..(13) <223> Xaa can be Ala or Gly <220> <221> Other features <222> (14)..(14) <223> Xaa can be Trp, Tyr, and Phe <400> 3 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Xaa Gln Asn Xaa Xaa 1 5 10 <210> 4 <211> 14 <212> PRT <213> Homo sapiens <220> <221> Other features <222> (2)..(2) <223> Xaa can be Ala, Gly, Ser, Glu, or Asp. <220> <221> Other features <222> (3)..(3) <223> Xaa can be Gly, Gln, Ala, Ser, or Pro. <220> <221> Other features <222> (4)..(4) <223> Xaa can be Ala, Gly, Ser, Glu, or Asp. <220> <221> Other features <222> (7)..(7) <223> Xaa can be Glu or Asp. <220> <221> Other features <222> (10)..(10) <223> Xaa can be Ala or Ser <220> <221> Other features <222> (13)..(13) <223> Xaa can be Ala or Gly <400> 4 Ile Xaa Xaa Xaa Ile Leu Xaa Ala Phe Xaa Gln Asn Xaa Trp 1 5 10 <210> 5 <211> 34 <212> PRT <213> Homo sapiens <400> 5 Leu Thr Leu Arg Lys Glu Pro Ala Ser Glu Ile Ala Gln Ser Ile Leu 1 5 10 15 Glu Ala Tyr Ser Gln Asn Gly Trp Ala Asn Arg Arg Ser Gly Gly Lys 20 25 30 Arg Pro <210> 6 <211> 46 <212> PRT <213> Homo sapiens <400> 6 Leu Thr Leu Arg Lys Glu Pro Ala Ser Glu Ile Ala Gln Ser Ile Leu 1 5 10 15 Glu Ala Tyr Ser Gln Asn Gly Trp Ala Asn Arg Arg Ser Gly Gly Lys 20 25 30 Arg Pro Pro Pro Arg Arg Arg Gln Arg Arg Lys Lys Arg Gly 35 40 45 <210> 7 <211> 34[[ID=3,8]] <212> PRT <213> Homo sapiens <400> 7 Leu Thr Leu Arg Lys Glu Ala Ser Ser Glu Ile Ala Gln Ser Ile Leu 1 5 10 15 Asp Ala Tyr Ser Gln Asn Gly Trp Ala Asn Arg Arg Ser Ser Cys Lys 20 25 30 Arg Pro <210> 8 <211> 34 <212> PRT <213> Homo sapiens <400> 8 Leu Thr Leu Arg Lys Lys Ala Ser Ser Lys Ile Ala Gln Ser Ile Leu 1 5 10 15 Asp Ala Phe Ser Gln Asn Gly Trp Ala Asn Arg Arg Ser Ser Cys Lys 20 25 30 Arg Pro <210> 9 <211> 34 <212> PRT <213> Homo sapiens <400> 9 Leu Thr Leu Arg Lys Glu Pro Ala Ser Glu Ile Ala Gln Ser Ile Leu 1 5 10 15 Glu Ala Tyr Ser Gln Asn Gly Trp Ala Asn Arg Arg Ser Gly Gly Lys 20 25 30 Arg Pro <210> 10 <211> 31 <212> PRT <213> Homo sapiens <400> 10 Arg Lys Lys Ala Ser Ser Lys Ile Ala Gln Ser Ile Leu Asp Ala Phe 1 5 10 15 Ser Gln Asn Gly Trp Ala Asn Arg Arg Ser Ser Cys Lys Arg Pro 20 25 30 <210> 11 <211> 31 <212> PRT <213> Homo sapiens <400> 11 Arg Lys Lys Ala Ser Ser Lys Ile Ala Ala Ala Ile Leu Asp Ala Phe 1 5 10 15 Ser Gln Asn Gly Trp Ala Asn Arg Arg Ser Ser Cys Lys Arg Pro 20 25 30 <210> 12 <211> 31 <212> PRT <213> Homo sapiens <400> 12 Arg Lys Lys Ala Ser Ser Lys Ile Ala Ala Ala Ile Leu Asp Ala Phe 1 5 10 15 Ser Gln Asn Ala Trp Ala Asn Arg Arg Ser Ser Cys Lys Arg Pro 20 25 30 <210> 13 <211> 24 <212> PRT <213> Homo sapiens <400> 13 Arg Lys Lys Ala Ser Ser Lys Ile Ala Ala Ala Ile Leu Asp Ala Phe [[ID=三十八]]1 5 10 15 Ser Gln Asn Trp Arg Arg Lys Arg 20 <210> 14 <211> 24 <212> PRT<00011三十三><213> Homo sapiens <400> 14 Arg Lys Lys Ala Ser Ser Lys Ile Glu Ala Ala Ile Leu Asp Ala Phe 1 5 10 15 Ser Gln Asn Trp Arg Arg Lys Arg 20 <210> 15 <211> 24 <212> PRT <213> Homo sapiens <400> 15 Arg Lys Lys Ala Ser Ser Lys Ile Ala Ala Glu Ile Leu Asp Ala Phe 1 5 10 15 Ser Gln Asn Trp Arg Arg Lys Arg 20 <210> 16 <211> 24 <212> PRT <213> Homo sapiens <400> 16 Arg Lys Lys Ala Ser Ser Lys Ile Glu Ala Glu Ile Leu Asp Ala Phe 1 5 10 15 Ser Gln Asn Trp Arg Arg Lys Arg 20 <210> 17 <211> 22 <212> PRT <213> Homo sapiens <e400> 17 Arg Lys Lys Ser Lys Ile Ala Ala Ala Ile Leu Asp Ala Phe Ser Gln 1 5 10 15 Asn Trp Arg Arg Lys Arg 20 <210> 18 <211> 22 <212> PRT <213> Homo sapiens [[ID=৬3]]<400> 18[[ID=৬4]] [[ID=৬5]]Arg Lys Lys Ser Lys Ile Glu Ala Glu Ile Leu Asp Ala Phe Ser Gln[[ID=৬6]] [[ID=৬7]]1 5 10 15 [[ID=৬8]] Asn Trp Arg Arg Lys Arg 20 <210> 19 <211> 19 <212> PRT <213> Homo sapiens <400> 19 Ala Lys Ile Ala Ala Ala Ile Leu Asp Ala Phe Ser Gln Asn Trp Arg 1 5 10 15 Arg Lys Arg <210> 20 <211> 19 <212> PRT <213> Homo sapiens <400> 20 Ala Lys Ile Glu Ala Ala Ile Leu Asp Ala Phe Ser Gln Asn Trp Arg 1 5 10 15 Arg Lys Arg <210> 21 <211> 46 <212> PRT <213> Homo sapiens <400> 21 Leu Thr Leu Arg Lys Glu Pro Ala Ser Glu Ile Ala Gln Ser Ile Leu 1 5 10 15 Glu Ala Tyr Ser Gln Asn Gly Trp Ala Asn Arg Arg Ser Gly Gly Lys 20 25 30 Arg Pro Pro Pro Arg Arg Arg Gln Arg Arg Lys Lys Arg Gly 35 40 45 <210> 22 <211> 43 <212> PRT <213> Homo sapiens <400> 22 Arg Lys Lys Ala Ser Ser Lys Ile Ala Ala Ala Ile Leu Asp Ala Phe 1 5 10 15 Ser Gln Asn Gly Trp Ala Asn Arg Arg Ser Ser Cys Lys Arg Pro Pro 20 25 30 Pro Arg Arg Arg Gln Arg Arg Lys Lys Arg Gly 35 40 <210> 23 <211> 43 <212> PRT <213> Homo sapiens <400> 23 Arg Lys Lys Ala Ser Ser Lys Ile Ala Ala Ala Ile Leu Asp Ala Phe 1 5 10 15 Ser Gln Asn Ala Trp Ala Asn Arg Arg Ser Ser Cys Lys Arg Pro Pro 20 25 30 Pro Arg Arg Arg Gln Arg Arg Lys Lys Arg Ala 35 40 <210> 24 <211> 36 <212> PRT <213> Homo sapiens <400> 24 Arg Lys Lys Ala Ser Ser Lys Ile Ala Ala Ala Ile Leu Asp Ala Phe 1 5 10 15 Ser Gln Asn Trp Arg Arg Lys Arg Pro Pro Arg Arg Arg Gln Arg Arg 20 25 30 Lys Lys Arg Gly 35 <210> 25 <211> 36 <212> PRT <213> Homo sapiens <400> 25 Arg Lys Lys Ala Ser Ser Lys Ile Glu Ala Ala Ile Leu Asp Ala Phe 1 5 10 15 Ser Gln Asn Trp Arg Arg Lys Arg Pro Pro Arg Arg Arg Gln Arg Arg 20 25 30 Lys Lys Arg Gly 35 <210> 26 <211> 36 <212> PRT <213> Homo sapiens <400> 26 Arg Lys Lys Ala Ser Ser Lys Ile Ala Ala Glu Ile Leu Asp Ala Phe 1 5 10 15 Ser Gln Asn Trp Arg Arg Lys Arg Pro Pro Arg Arg Arg Gln Arg Arg 20 25 30 Lys Lys Arg Gly 35 <210> 27 <211> 36 <212> PRT <213> Homo sapiens <400> 27 Arg Lys Lys Ala Ser Ser Lys Ile Glu Ala Glu Ile Leu Asp Ala Phe 1 5 10 15 Ser Gln Asn Trp Arg Arg Lys Arg Pro Pro Arg Arg Arg Gln Arg Arg 20 25 30 Lys Lys Arg Gly 35 <210> 28 <211> 32 <212> PRT <213> Homo sapiens <400> 28 Arg Lys Lys Ser Lys Ile Ala Ala Ala Ile Leu Asp Ala Phe Ser Gln 1 5 10 15 Asn Trp Arg Arg Lys Arg Arg Arg Arg Gln Arg Arg Lys Lys Arg Gly 20 25 30 <210> 29 <211> 32 <212> PRT <213> Homo sapiens <400> 29 Arg Lys Lys Ser Lys Ile Glu Ala Glu Ile Leu Asp Ala Phe Ser Gln 1 5 10 15 Asn Trp Arg Arg Lys Arg Arg Arg Arg Gln Arg Arg Lys Lys Arg Gly 20 25 30 <210> 30 <211> 29 <212> PRT <213> Homo sapiens <400> 30 Ala Lys Ile Ala Ala Ala Ile Leu Asp Ala Phe Ser Gln Asn Trp Arg 1 5 10 15 Arg Lys Arg Arg Arg Arg Gln Arg Arg Lys Lys Arg Gly 20 25 <210> 31 <211> 29 <212> PRT <213> Homo sapiens <400> 31 Ala Lys Ile Glu Ala Ala Ile Leu Asp Ala Phe Ser Gln Asn Trp Arg 1 5 10 15 Arg Lys Arg Arg Arg Arg Gln Arg Arg Lys Lys Arg Gly 20 25 <210> 32 <211> 91 <212> PRT <213> Homo sapiens <400> 32 Pro Arg Lys Gly Gly Ser Arg Arg Asn Ala Trp Gly Asn Gln Ser Tyr 1 5 10 15 Ala Glu Leu Ile Ser Gln Ala Ile Glu Ser Ala Pro Glu Lys Arg Leu 20 25 30 Thr Leu Ala Gln Ile Tyr Glu Trp Met Val Arg Thr Val Pro Tyr Phe 35 40 45 Lys Asp Lys Gly Asp Ser Asn Ser Ser Ala Gly Trp Lys Asn Ser Ile 50 55 60 Arg His Asn Leu Ser Leu His Ser Lys Phe Ile Lys Val His Asn Glu 65 70 75 80 Ala Thr Gly Lys Ser Ser Trp Trp Met Leu Asn 85 90 <210> 33 <211> 34 <212> PRT <213> Homo sapiens <400> 33 Pro Arg Lys Gly Gly Ser Arg Arg Asn Ala Trp Gly Asn Gln Ser Tyr 1 5 10 15 Ala Glu Leu Ile Ser Gln Ala Ile Glu Ser Ala Pro Glu Lys Arg Leu 20 25 30 Thr Leu <210> 34 <211> twenty two <212> PRT <213> Homo sapiens <400> 34 Ala Met Asp Asp Leu Met Leu Ser Pro Asp Asp Ile Glu Gln Trp Phe 1 5 10 15 Thr Glu Asp Pro Gly Pro 20 <210> 35 <211> 10 <212> PRT <213> Homo sapiens <400> 35 Leu Thr Leu Arg Lys Glu Ala Ser Ser Glu 1 5 10 <210> 36 <211> 6 <212> PRT <213> Homo sapiens <220> <221> Other features <222> (2)..(2) <223> Xaa can be Ala, Gly, Ser, Glu, or Asp. <220> <221> Other features <222> (3)..(3) <223> Xaa can be Gly, Gln, Ala, Ser, or Pro. <220> <221> Other features <222> (4)..(4) <223> Xaa can be Ala, Gly, Ser, Glu, or Asp. <400> 36 Ile Xaa Xaa Xaa Ile Leu 1 5 <210> 37 <211> 6 <212> PRT <213> Homo sapiens <400> 37 Ile Ala Gln Ser Ile Leu 1 5 <210> 38 <211> 10 <212> PRT <213> Homo sapiens <400> 38 Ala Asn Arg Arg Ser Ser Cys Lys Arg Pro 1 5 10 <210> 39 <211> 505 <212> PRT <213> Homo sapiens <400> 39 Met Asp Pro Gly Asn Glu Asn Ser Ala Thr Glu Ala Ala Ala Ile Ile 1 5 10 15 Asp Leu Asp Pro Asp Phe Glu Pro Gln Ser Arg Pro Arg Ser Cys Thr 20 25 30 Trp Pro Leu Pro Arg Pro Glu Ile Ala Asn Gln Pro Ser Glu Pro Pro 35 40 45 Glu Val Glu Pro Asp Leu Gly Glu Lys Val His Thr Glu Gly Arg Ser 50 55 60 Glu Pro Ile Leu Leu Pro Ser Arg Leu Pro Glu Pro Ala Gly Gly Pro 65 70 75 80 Gln Pro Gly Ile Leu Gly Ala Val Thr Gly Pro Arg Lys Gly Gly Ser 85 90 95 Arg Arg Asn Ala Trp Gly Asn Gln Ser Tyr Ala Glu Leu Ile Ser Gln 100 105 110 Ala Ile Glu Ser Ala Pro Glu Lys Arg Leu Thr Leu Ala Gln Ile Tyr 115 120 125 Glu Trp Met Val Arg Thr Val Pro Tyr Phe Lys Asp Lys Gly Asp Ser 130 135 140 Asn Ser Ser Ala Gly Trp Lys Asn Ser Ile Arg His Asn Leu Ser Leu 145 150 155 160 His Ser Lys Phe Ile Lys Val His Asn Glu Ala Thr Gly Lys Ser Ser 165 170 175 Trp Trp Met Leu Asn Pro Glu Gly Gly Lys Ser Gly Lys Ala Pro Arg 180 185 190 Arg Arg Ala Ala Ser Met Asp Ser Ser Ser Lys Leu Leu Arg Gly Arg 195 200 205 Ser Lys Ala Pro Lys Lys Lys Pro Ser Val Leu Pro Ala Pro Pro Glu 210 215 220 Gly Ala Thr Pro Thr Ser Pro Val Gly His Phe Ala Lys Trp Ser Gly 225 230 235 240 Ser Pro Cys Ser Arg Asn Arg Glu Glu Ala Asp Met Trp Thr Thr Phe 245 250 255 Arg Pro Arg Ser Ser Ser Asn Ala Ser Ser Val Ser Thr Arg Leu Ser 260 265 270 Pro Leu Arg Pro Glu Ser Glu Val Leu Ala Glu Glu Ile Pro Ala Ser 275 280 285 Val Ser Ser Tyr Ala Gly Gly Val Pro Pro Thr Leu Asn Glu Gly Leu 290 295 300 Glu Leu Leu Asp Gly Leu Asn Leu Thr Ser Ser His Ser Leu Leu Ser 305 310 315 320 Arg Ser Gly Leu Ser Gly Phe Ser Leu Gln His Pro Gly Val Thr Gly 325 330 335 Pro Leu His Thr Tyr Ser Ser Ser Leu Phe Ser Pro Ala Glu Gly Pro 340 345 350 Leu Ser Ala Gly Glu Gly Cys Phe Ser Ser Ser Gln Ala Leu Glu Ala 355 360 365 Leu Leu Thr Ser Asp Thr Pro Pro Pro Pro Ala Asp Val Leu Met Thr 370 375 380 Gln Val Asp Pro Ile Leu Ser Gln Ala Pro Thr Leu Leu Leu Leu Gly 385 390 395 400 Gly Leu Pro Ser Ser Ser Lys Leu Ala Thr Gly Val Gly Leu Cys Pro 405 410 415 Lys Pro Leu Glu Ala Pro Gly Pro Ser Ser Leu Val Pro Thr Leu Ser 420 425 430 Met Ile Ala Pro Ser Ala Pro Ile Pro Lys Ala 435 440 445 Leu Gly Thr Pro Val Leu Thr Pro Pro Thr Glu Ala Ala Ser Gln Asp 450 455 460 Arg Met Pro Gln Asp Leu Asp Leu Asp Met Tyr Met Glu Asn Leu Glu 465 470 475 480 Cys Asp Met Asp Asn Ile Ile Ser Asp Leu Met Asp Glu Gly Glu Gly 485 490 495 Leu Asp Phe Asn Phe Glu Pro Asp Pro 500 505 <210> 40 <211> 393 <212> PRT <213> Homo sapiens <400> 40 Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln 1 5 10 15 Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu 20 25 30 Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp 35 40 45 Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro 50 55 60 Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro Ala Pro Ala Ala Pro 65 70 75 80 Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser 85 90 95 Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly 100 105 110 Phe Leu His Ser Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro 115 120 125 Ala Leu Asn Lys Met Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln 130 135 140 Leu Trp Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met 145 150 155 160 Ala Ile Tyr Lys Gln Ser Gln His Met Thr Glu Val Val Arg Arg Cys 165 170 175 Pro His His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln 180 185 190 His Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp 195 200 205 Arg Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu 210 215 220 Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser 225 230 235 240 Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr 245 250 255 Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu Val 260 265 270 Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn 275 280 285 Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro Pro Gly Ser Thr 290 295 300 Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys 305 310 315 320 Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu 325 330 335 Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp 340 345 350 Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His 355 360 365 Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met 370 375 380 Phe Lys Thr Glu Gly Pro Asp Ser Asp 385 390 <210> 41 <211> 12 <212> PRT <213> Homo sapiens <400> 41 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro 1 5 10 <210> 42 <211> 34 <212> PRT <213> Homo sapiens <400> 42 Pro Arg Lys Gly Gly Ser Arg Arg Asn Ala Trp Gly Asn Gln Ser Tyr 1 5 10 15 Ala Glu Leu Ile Ser Gln Ala Ile Glu Ser Ala Pro Glu Lys Arg Leu 20 25 30 Thr Leu <210> 43 <211> 5 <212> PRT <213> Homo sapiens <400> 43 Ser Gln Asn Ala Trp 1 5 <210> 44 <211> 5 <212> PRT <213> Homo sapiens <400> 44 Ser Gln AsN Gly Trp 1 5 <210> 45 <211> 4 <212> PRT <213> Homo sapiens <400> 45 Ser Gln Asn Trp 1 <210> 46 <211> 5 <212> PRT <213> Homo sapiens <400> 46 Lys Ile Ala Ala Ala 1 5 <210> 47 <211> 5 <212> PRT <213> Homo sapiens <400> 47 Lys Ile Glu Ala Ala 1 5 <210> 48 <211> 5 <212> PRT <213> Homo sapiens <400> 48 Lys Ile Ala Ala Glu 1 5 <210> 49 <211> 5 <212> PRT <213> Homo sapiens <400> 49 Lys Ile Glu Ala Glu 1 5 <210> 50 <211> 4 <212> PRT <213> Homo sapiens <400> 50 Ser Gln Asn Gly 1 <210> 51 <211> 4 <212> PRT <213> Homo sapiens <400> 51 Ala Ala Asn Gly 1 <210> 52 <211> 4 <212> PRT <213> Homo sapiens <400> 52 Ser Gln Ala Gly 1 <210> 53 <211> 36 <212> PRT <213> Homo sapiens <400> 53 Arg Lys Lys Ala Ser Ser Lys Ile Glu Ala Glu Ile Leu Asp Ala Phe 1 5 10 15 Ser Gln Asn Trp Arg Arg Lys Arg Pro Pro Arg Arg Arg Gln Arg Arg 20 25 30 Lys Lys Arg Gly 35 <210> 54 <211> 31 <212> PRT <213> Homo sapiens <400> 54 Arg Lys Lys Ser Lys Ile Glu Ala Glu Ile Leu Asp Ala Phe Ser Gln 1 5 10 15 Asn Trp Arg Lys Arg Arg Arg Arg Gln Arg Arg Lys Lys Arg Gly 20 25 30 <210> 55 <211> 28 <212> PRT <213> Homo sapiens <400> 55 Ala Lys Ile Glu Ala Ala Ile Leu Asp Ala Phe Ser Gln Asn Trp Arg 1 5 10 15 Lys Arg Arg Arg Arg Gln Arg Arg Lys Lys Arg Gly 20 25 <210> 56 <211> 28 <212> PRT <213> Homo sapiens <400> 56 Ala Lys Ile Glu Ala Glu Ile Leu Asp Ala Phe Ser Gln Asn Trp Arg 1 5 10 15 Lys Arg Arg Arg Arg Gln Arg Arg Lys Lys Arg Gly 20 25 <210> 57 <211> 28 <212> PRT <213> Homo sapiens <400> 57 Ala Lys Ile Glu Ala Ala Ile Leu Asp Glu Phe Ser Gln Asn Trp Arg 1 5 10 15 Lys Arg Arg Arg Arg Gln Arg Arg Lys Lys Arg Gly 20 25 <210> 58 <211> 36 <212> PRT <213> Homo sapiens <220> <221> Other features <222> (6)..(6) <223> fluorenemethyloxycarbonyl-L-2-(4'-pentenyl)alanine <220> <221> Other features <222> (10)..(10) <223> fluorenemethyloxycarbonyl-L-2-(4'-pentenyl)alanine <400> 58 Arg Lys Lys Ala Ser Xaa Lys Ile Ala Xaa Ala Ile Leu Asp Ala Phe 1 5 10 15 Ser Gln Asn Trp Arg Arg Lys Arg Pro Pro Arg Arg Arg Gln Arg Arg 20 25 30 Lys Lys Arg Gly 35 <210> 59 <211> 43 <212> PRT <213> Homo sapiens <220> <221> Other features <222> (12)..(12) <223> Cyclohexyl-alanine <400> 59 Arg Lys Lys Ala Ser Ser Lys Ile Ala Ala Ala Xaa Leu Asp Ala Phe 1 5 10 15 Ser Gln Asn Ala Trp Ala Asn Arg Arg Ser Ser Cys Lys Arg Pro Pro 20 25 30 Pro Arg Arg Arg Gln Arg Arg Lys Lys Arg Ala 35 40 <210> 60 <211> 28 <212> PRT <213> Homo sapiens <220> <221> Other features <222> (15)..(15) <223> 2-Methyl-Tryptophan <400> 60 Ala Lys Ile Glu Ala Ala Ile Leu Asp Ala Phe Ser Gln Asn Xaa Arg 1 5 10 15 Lys Arg Arg Arg Arg Gln Arg Arg Lys Lys Arg Gly 20 25 <210> 61 <211> 28 <212> PRT <213> Homo sapiens <220> <221> Other features <222> (15)..(15) <223> 2-Methyl-Tryptophan <400> 61 Ala Lys Ile Glu Ala Glu Ile Leu Glu Ala Phe Ser Gln Asn Xaa Arg 1 5 10 15 Lys Arg Arg Arg Arg Gln Arg Arg Lys Lys Arg Gly 20 25 <210> 62 <211> 28 <212> PRT <213> Homo sapiens <220> <221> Other features <222> (7)..(7) <223> Cyclohexyl-alanine <220> <221> Other features <222> (15)..(15) <223> 2-Methyl-Tryptophan <400> 62 Ala Lys Ile Glu Ala Ala Xaa Leu Asp Ala Phe Ser Gln Asn Xaa Arg 1 5 10 15 Lys Arg Arg Arg Arg Gln Arg Arg Lys Lys Arg Gly 20 25 <210> 63 <211> 36 <212> PRT <213> Homo sapiens <220> <221> Other features <222> (20)..(20) <223> 2-Methyl-Tryptophan <400> 63 Arg Lys Lys Ala Ser Ser Lys Ile Glu Ala Glu Ile Leu Asp Ala Phe 1 5 10 15 Ser Gln Asn Xaa Arg Arg Lys Arg Pro Pro Arg Arg Arg Gln Arg Arg 20 25 30 Lys Lys Arg Gly 35 <210> 64 <211> 36 <212> PRT <213> Homo sapiens <220> <221> Other features <222> (12)..(12) <223> Cyclohexyl-alanine <220> <221> Other features <222> (20)..(20) <223> 2-Methyl-Tryptophan <400> 64 Arg Lys Lys Ala Ser Ser Lys Ile Glu Ala Glu Xaa Leu Asp Ala Phe 1 5 10 15 Ser Gln Asn Xaa Arg Arg Lys Arg Pro Pro Arg Arg Arg Gln Arg Arg 20 25 30 Lys Lys Arg Gly 35 <210> 65 <211> 36 <212> PRT <213> Homo sapiens <220> <221> Other features <222> (13)..(13) <223> Cyclohexyl-alanine <220> <221> Other features <222> (20)..(20) <223> 2-Methyl-Tryptophan <400> 65 Arg Lys Lys Ala Ser Ser Lys Ile Glu Ala Glu Ile Xaa Asp Ala Phe 1 5 10 15 Ser Gln Asn Xaa Arg Arg Lys Arg Pro Pro Arg Arg Arg Gln Arg Arg 20 25 30 Lys Lys Arg Gly 35 <210> 66 <211> 13 <212> PRT <213> Homo sapiens <400> 66 Ala Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Pro 1 5 10 <210> 67 <211> 31 <212> PRT <213> Homo sapiens <400> 67 Arg Lys Lys Ser Lys Ile Ala Ala Ala Ile Leu Asp Ala Phe Ser Gln 1 5 10 15 Asn Trp Arg Lys Arg Arg Arg Arg Gln Arg Arg Lys Lys Arg Gly 20 25 30 <210> 68 <211> 36 <212> PRT <213> Homo sapiens <400> 68 Arg Lys Lys Ala Ser Ser Lys Ile Glu Ala Ala Ile Leu Asp Ala Phe 1 5 10 15 Ser Gln Asn Trp Arg Arg Lys Arg Pro Pro Arg Arg Arg Gln Arg Arg 20 25 30 Lys Lys Arg Gly 35

Claims

1. A peptide or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of the peptide is as follows: RKKASSKIAAAILDAFSQNGWANRRSSCKRP (SEQ ID NO: 11), RKKASSKIAAAILDAFSQNAWANRRSSCKRP (SEQ ID NO: 12), RKKASSKIAAAILDAFSQNWRRKR (SEQ ID NO: 13), RKKASSKIAAAILDAFSQNGWANRRSSCKRPPPRRRQRRKKRG (SEQ ID NO: 22), RKKASSKIAAAILDAFSQNAWANRRSSCKRPPPRRRQRRKKRA (SEQ ID NO: 23), RKKASSKIAAAILDAFSQNWRRKRPPRRRQRRKKRG (SEQ ID NO: 24), RKKASSKIEAAILDAFSQNWRRKRPPRRRQRRKKRG (SEQ ID NO: 25), RKKASSKIAAEILDAFSQNWRRKRPPRRRQRRKKRG (SEQ ID NO: 26), RKKASSKIEAEILDAFSQNWRRKRPPRRRQRRKKRG (SEQ ID NO: 27), RKKSKIEAEILDAFSQNWRKRRRRQRRKKRG (SEQ ID NO: 54), AKIEAAILDAFSQNWRKRRRRQRRKKRG (SEQ ID NO: 55), AKIEAEILDAFSQNWRKRRRRQRRKKRG (SEQ ID NO: 56), AKIEAAILDEFSQNWRKRRRRQRRKKRG (SEQ ID NO: 57), AKIEAAILDAFSQNBRKRRRRQRRKKRG (SEQ ID NO: 60), AKIEAEILEAFSQNBRKRRRRQRRKKRG (SEQ ID NO: 61), RKKSKIAAAILDAFSQNWRKRRRRQRRKKRG (SEQ ID NO: 67), or RKKASSKIEAAILDAFSQNWRRKRPPRRRQRRKKRG (SEQ ID NO: 68) is shown, In SEQ ID NO: 60 and 61, B is 2-methyl-tryptophan.

2. The peptide of claim 1 or a pharmaceutically acceptable salt thereof, wherein the peptide comprises at least 80% D-amino acids.

3. The peptide of claim 2 or a pharmaceutically acceptable salt thereof, wherein the peptide comprises at least 90% D-amino acids.

4. The peptide of claim 3 or a pharmaceutically acceptable salt thereof, wherein the peptide comprises at least 95% D-amino acids.

5. The peptide of claim 1 or a pharmaceutically acceptable salt thereof, wherein the peptide is capable of binding to p53 in cells.

6. The peptide of claim 5 or a pharmaceutically acceptable salt thereof, wherein the peptide is capable of binding to p53 in cells and inhibiting the interaction between FOXO4 and p53.

7. A compound or a pharmaceutically acceptable salt thereof, comprising the peptide or a pharmaceutically acceptable salt thereof as described in any one of claims 1-6, and a sequence conferring cell permeability, organelle targeting, nuclear localization, mitochondrial localization, blood-brain barrier permeability, cell membrane localization, and / or peptidase cleavage.

8. The compound of claim 7 or a pharmaceutically acceptable salt thereof, wherein the sequence conferring cell permeability comprises the TAT-sequence of HIV.

9. The compound of claim 7 or a pharmaceutically acceptable salt thereof, wherein the sequence conferring cell permeability comprises the amino acid sequence GRKKRRQRRRPP (SEQ ID NO: 41) or ARKKRRQRRRPPP (SEQ ID NO: 66).

10. A method for identifying an improved peptide or a pharmaceutically acceptable salt thereof capable of binding to p53 in cells, or capable of binding to p53 in cells and inhibiting the interaction between FOXO4 and p53, comprising the steps of: a) Provide the peptide or a pharmaceutically acceptable salt thereof as described in any one of claims 1-6; b) Appropriately modify the peptide in step a); c) Determine the binding of the peptide obtained in step b) to p53 or a fragment thereof, its stability, and the binding of at least one of FOXO4 or a fragment thereof to p53 or a fragment thereof in its presence or absence, and d) Based on the determination in step c), identify peptides that are improved relative to the peptides provided in step a) and are capable of binding to p53 or binding to p53 and inhibiting the interaction between FOXO4 and p53.

11. The method of claim 10, further comprising testing the activity of the identified peptide or a pharmaceutically acceptable salt thereof in inducing apoptosis and / or killing senescent cells, scar cells, and / or tumor cells.

12. The method of claim 11, comprising the determination of increased caspase-3 / 7 activity, loss of mitochondrial cytochrome C, TUNEL positivity, extracellular connectin V positivity, increased cell death markers, and / or loss of viability.

13. The method of claim 12, wherein the elevation of cell death markers is manifested as the presence of propidium iodide in the cells.

14. The method of claim 12, wherein the loss of activity is the loss of calcein AM incorporation or the loss of activity in MTS activity assay.

15. The method of claim 10, wherein the FOXO4 or a fragment thereof, and / or the p53 or a fragment thereof, is recombinantly expressed in the cells.

16. The method of claim 10, wherein the FOXO4 or a fragment thereof, the p53 or a fragment thereof, the peptide provided in step a) or a pharmaceutically acceptable salt thereof, and / or the peptide obtained in step b) or a pharmaceutically acceptable salt thereof contains a detectable label.

17. The method of claim 16, wherein the detectable label is a fluorescent, mass, and / or stable isotope tag.

18. A method for preparing a pharmaceutical composition, comprising the step of formulating the peptide or a pharmaceutically acceptable salt thereof, as described in any one of claims 1-6, into a suitable pharmaceutical composition.

19. A pharmaceutical composition obtained by the method of claim 18.

20. A method for preparing a pharmaceutical composition, comprising the step of formulating the compound of claim 7 or a pharmaceutically acceptable salt thereof into a suitable pharmaceutical composition.

21. A pharmaceutical composition obtained by the method of claim 20.

22. A method for preparing a pharmaceutical composition, comprising the step of formulating the compound of claim 9 or a pharmaceutically acceptable salt thereof into a suitable pharmaceutical composition.

23. A pharmaceutical composition obtained by the method of claim 22.

24. A method for preparing a pharmaceutical composition, comprising carrying out the method of claim 10, and the step of formulating the identified peptide or a pharmaceutically acceptable salt thereof into a suitable pharmaceutical composition.

25. A pharmaceutical composition obtained by the method of claim 24.

26. The use of the peptide or a pharmaceutically acceptable salt thereof of any one of claims 1-6, or the pharmaceutical composition of claims 19, 21, or 23, in the preparation of a medicament for treating diseases comprising senescent cells, scar cells, and / or cancer cells. The amino acid sequence of this peptide is shown as RKKASSKIAAAILDAFSQNAWANRRSSCKRP (SEQ ID NO: 12), and the disease is glioblastoma or colon cancer. The amino acid sequence of this peptide is shown as RKKASSKIAAAILDAFSQNGWANRRSSCKRP (SEQ ID NO: 11), and the disease in question is colon cancer. The amino acid sequence of the peptide is shown in AKIEAAILDAFSQNWRKRRRRQRRKKRG (SEQ ID NO: 55), where the disease is colorectal cancer, breast cancer, or melanoma, or The amino acid sequence of the peptide is shown as RKKASSKIEAAILDAFSQNWRRKRPPRRRQRRKKRG (SEQ ID NO: 68), and the disease is colon cancer.

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