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IMPROVED ANTI-SENESCENCE COMPOUNDS AND USES THEREOF

20230090099 · 2023-03-23

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention relates to improved compounds for use in the treatment of diseases or conditions where the removal of senescent cells, scarred cells, and/or cancerous cells is beneficial, for example cancer. The invention also relates to methods of treating an individual suffering, or suspected of suffering, from a disease or condition wherein the removal of senescent cells, scarred cells, and/or cancerous cells is beneficial.

    Claims

    1. A compound selected from i) a peptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence TABLE-US-00011 (SEQ ID NO: 1) X.sub.3X.sub.2X.sub.4X.sub.5X.sub.7X.sub.5X.sub.4X.sub.4X.sub.6X.sub.18X.sub.8X.sub.3QNX.sub.9X.sub.8X.sub.10X.sub.10X.sub.11X.sub.12S*X.sub.13X.sub.14X.sub.11X.sub.11, wherein S* can be S or is absent; X.sub.2 is absent or is selected from K, E, R, and H; X.sub.3 is absent or selected from A, J, and S; X.sub.4 is selected from I, Z, and L, wherein Z is Cyclo-Hexyl-Alanine; X.sub.5 is selected from A, G, S, E, and D; X.sub.6 is selected from E and D; X.sub.7 is selected from J, G, Q, A, S, and P; X.sub.8 is selected from B, W, Y, and F, wherein B is 2-Methyl-Tryptophan; X.sub.9 is absent or is selected from A and G; X.sub.10 is absent or is selected from A and N; X.sub.11 is selected from R and K; X.sub.12 is absent or is R; X.sub.13 is absent or is selected from G and S; X.sub.14 is absent or is selected from A and C; and X.sub.18 is selected from A and E; wherein J is Fmoc-L-2-(4′-pentenyl)alanine; with the provisio that either none or two Js are present that form a staple; ii) the peptide according to i) comprising at least 80% D-amino acids, and iii) a retro inverso peptide according to i) or ii), and pharmaceutically acceptable salts thereof; and wherein said compound induces apoptosis in senescent, scarred cells, and/or cancerous cells.

    2. The compound according to claim 1, wherein said peptide comprises an amino acid sequence that is at least 70% identical to the amino acid sequence TABLE-US-00012 (SEQ ID NO: 2) X.sub.1X.sub.17K*X.sub.2X.sub.16X.sub.3X.sub.3X.sub.2X.sub.4X.sub.5X.sub.7X.sub.5X.sub.4X.sub.4X.sub.6AX.sub.8X.sub.3QNX.sub.9X.sub.8X.sub.10X.sub.10X.sub.11X.sub.12S*X.sub.13 X.sub.14X.sub.11X.sub.11X.sub.15, wherein Z, B, J, S*, X.sub.2, X.sub.3, X.sub.4, X.sub.5, X.sub.6, X.sub.7, X.sub.8, X.sub.9, X.sub.10, X.sub.11, X.sub.12, X.sub.13, X.sub.14, and X.sub.is are as defined in claim 1; K* can be K or is absent; X.sub.1 is absent or indicates the amino acid sequence LTL; X.sub.15 is absent or is selected from A and C; X.sub.16 is selected from A and P; and X.sub.17 is absent or is selected from R and S; and wherein said compound induces apoptosis in senescent, scarred cells, and/or cancerous cells.

    3. The compound according to claim 1, wherein the amino acid motif X.sub.3QNX.sub.9X.sub.8 is selected from SQNAW (SEQ ID NO: 43), SQNGW (SEQ ID NO: 44) and SQN-W (SEQ ID NO: 45), wherein “-” indicates an absent amino acid.

    4. The compound according to claim 1, wherein the amino acid motif X.sub.2X.sub.4X.sub.5X.sub.7X.sub.5 is selected from KIAAA (SEQ ID NO: 46), KIEAA (SEQ ID NO: 47), KIAAE (SEQ ID NO: 48) and KIEAE (SEQ ID NO: 49).

    5. The compound according to claim 1, wherein the amino acid motif X.sub.4X.sub.5X.sub.7X.sub.5X.sub.4X.sub.4X.sub.6AX.sub.8X.sub.3QNX.sub.9X.sub.8 (SEQ ID NO: 3) is selected from the general formula
    IX.sub.5X.sub.7X.sub.5ILX.sub.6AFX.sub.3QNX.sub.9W (SEQ ID NO: 4), wherein X.sub.3 is absent or selected from A, J, and S; X.sub.5 is selected from A, G, 5, E, and D; X.sub.6 is selected from E and D; X.sub.7 is selected from J, G, Q, A, S, and P; and X.sub.9 is absent or is selected from A and G.

    6. The compound according to claim 1, wherein said compound is a peptide comprising an amino acid sequence selected from TABLE-US-00013 (SEQ ID NO: 7) LTLRKEASSEIAQSILDAYSQNGWANRRSSCKRP, (SEQ ID NO: 8) LTLRKKASSKIAQSILDAFSQNGWANRRSSCKRP, (SEQ ID NO: 9) LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRP, (SEQ ID NO: 10) RKKASSKIAQSILDAFSQNGWANRRSSCKRP, (SEQ ID NO: 11) RKKASSKIAAAILDAFSQNGWANRRSSCKRP, (SEQ ID NO: 12) RKKASSKIAAAILDAFSQNAWANRRSSCKRP, (SEQ ID NO: 13) RKKASSKIAAAILDAFSQNWRRKR, (SEQ ID NO: 14) RKKASSKIEAAILDAFSQNWRRKR, (SEQ ID NO: 15) RKKASSKIAAEILDAFSQNWRRKR, (SEQ ID NO: 16) RKKASSKIEAEILDAFSQNWRRKR, (SEQ ID NO: 17) RKKSKIAAAILDAFSQNWRRKR, (SEQ ID NO: 18) RKKSKIEAEILDAFSQNWRRKR, (SEQ ID NO: 19) AKIAAAILDAFSQNWRRKR, (SEQ ID NO: 20) AKIEAAILDAFSQNWRRKR, (SEQ ID NO: 21) LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRPPPRRRQRRKKRG, (SEQ ID NO: 22) RKKASSKIAAAILDAFSQNGWANRRSSCKRPPPRRRQRRKKRG, (SEQ ID NO: 23) RKKASSKIAAAILDAFSQNAWANRRSSCKRPPPRRRQRRKKRA, (SEQ ID NO: 24) RKKASSKIAAAILDAFSQNWRRKRPPRRRQRRKKRG, (SEQ ID NO: 25) RKKASSKIEAAILDAFSQNWRRKRPPRRRQRRKKRG, (SEQ ID NO: 26) RKKASSKIAAEILDAFSQNWRRKRPPRRRQRRKKRG, (SEQ ID NO: 27) RKKASSKIEAEILDAFSQNWRRKRPPRRRQRRKKRG, (SEQ ID NO: 28) RKKSKIAAAILDAFSQNWRRKRRRRQRRKKRG, (SEQ ID NO: 29) RKKSKIEAEILDAFSQNWRRKRRRRQRRKKRG, (SEQ ID NO: 30) AKIAAAILDAFSQNWRRKRRRRQRRKKRG, (SEQ ID NO: 31) AKIEAAILDAFSQNWRRKRRRRQRRKKRG, (SEQ ID NO: 53) RKKASSKIEAEILDAFSQNWRRKRPPRRRQRRKKRG, (SEQ ID NO: 54) RKKSKIEAEILDAFSQNWRKRRRRQRRKKRG, (SEQ ID NO: 55) AKIEAAILDAFSQNWRKRRRRQRRKKRG, (SEQ ID NO: 56) AKIEAEILDAFSQNWRKRRRRQRRKKRG, (SEQ ID NO: 57) AKIEAAILDEFSQNWRKRRRRQRRKKRG, (SEQ ID NO: 58) RKKASJKIAJAILDAFSQNWRRKRPPRRRQRRKKRG, (SEQ ID NO: 59) RKKASSKIAAAZLDAFSQNAWANRRSSCKRPPPRRRQRRKKRA, (SEQ ID NO: 60) AKIEAAILDAFSQNBRKRRRRQRRKKRG, (SEQ ID NO: 61) AKIEAEILEAFSQNBRKRRRRQRRKKRG, (SEQ ID NO: 62) AKIEAAZLDAFSQNBRKRRRRQRRKKRG, (SEQ ID NO: 63) RKKASSKIEAEILDAFSQNBRRKRPPRRRQRRKKRG, (SEQ ID NO: 64) RKKASSKIEAEZLDAFSQNBRRKRPPRRRQRRKKRG,  and (SEQ ID NO: 65) RKKASSKIEAEIZDAFSQNBRRKRPPRRRQRRKKRG, wherein J, B, and Z are as defined in claim 1; an amino acid sequence that is at least 80% identical to any one of SEQ ID NO: 1 to 4, 7 to 31, 53 to 65, 67 and 68; and a peptide consisting of the amino acid sequence according to any one of SEQ ID NO: 1 to 4; 7 to 31, 53 to 65, 67 and 68.

    7. The compound according to claim 1, further comprising a sequence conferring cell-penetrating properties, organelle targeting properties, nuclear localization, mitochondrial localization, blood brain barrier permeability, cell membrane localization, and/or peptidase cleavage.

    8. The compound according to claim 1, comprising non-naturally occurring amino acids, modified amino acids, linker amino acids, staples, cysteine bridges, glycolysation sites, ubiquitination and/or pegylation sites.

    9. The compound according to claim 1, wherein said compound binds to p53 and inhibits the interaction of FOXO4 with p53 in a cell.

    10. A method for identifying an improved compound that binds to p53 comprising the steps of a) providing at least one compound according to claim 1; b) modifying said compound of a); c) determining at least one of binding of said at least one compound of b) to p53 or a fragment thereof, the stability of said at least one compound, and binding of FOXO4 or said fragment thereof to p53 or said fragment thereof in the presence of said at least one compound, compared to binding of FOXO4 or said fragment thereof to p53 or said fragment thereof in the absence of said compound; and d) identifying an improved compound that binds to p53 in a cell based on said determining in step c) when compared to a compound as provided in step a).

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

    12. The method according to claim 10, further comprising testing said compound as identified for its activity to induce apoptosis and/or to kill senescent, scarred and/or tumor cells, comprising determination of elevated caspase-3/7 activity, loss of mitochondrial cytochrome C, TUNEL positivity, extracellular annexin-V positivity, an elevation in cell death markers, and/or loss of viability.

    13. The method according to claim 10, wherein said FOXO4 or said fragment thereof, and/or p53 or said fragment thereof, is recombinantly expressed in a cell.

    14. The method according to claim 10, wherein said cell is selected from cancer cells, scarred cells, senescent cells, human non-embryonic stem cells, yeast cells, bacterial cells, and recombinant host cells expressing FOXO4 or the p53 binding fragment thereof, wherein said recombinant host cells optionally express p53 or the FOXO4 binding fragment thereof.

    15. The method according to claim 10, wherein said FOXO4 or the p53 binding fragment thereof, p53 or the FOXO4 binding fragment thereof, and/or the compound are detectably labeled.

    16. A screening tool for screening for a compound that binds to p53 comprising an isolated cell expressing FOXO4, and/or a cell expressing a p53 binding fragment thereof, wherein said cell optionally expresses p53, and/or a FOXO4 binding fragment thereof.

    17. The screening tool according to claim 16, wherein said FOXO4 or said fragment thereof, and/or to p53 or said fragment thereof is recombinantly expressed in said cell.

    18. The screening tool according to claim 16, wherein said cell is selected from cancer cells, senescent cells, scarred cells, human non-embryonic stem cells, yeast cells, bacterial cells, and recombinant host cells expressing FOXO4 or the p53 binding fragment thereof, wherein said recombinant host cells optionally express p53 or the FOXO4 binding fragment thereof.

    19. The screening tool according to claim 16, wherein said FOXO4 or the p53 binding fragment thereof, p53 or the FOXO4 binding fragment thereof, and/or the compound are detectably labeled.

    20. The screening tool according to claim 16, wherein said fragment of FOXO4 is a peptide according to the sequence PRKGGSRRNAWGNQSYAELISQAIESAPEKRLTLAQIYEWMVRTVPYFKDKGDSNSS AGWKNSIRHNLSLHSKFIKVHNEATGKSSWWMLN (SEQ ID NO: 32) or PRKGGSRRNAWGNQSYAELISQAIESAPEKRLTL (SEQ ID NO: 33), and wherein said fragment of p53 is a peptide according to the sequence AMDDLMLSPDDIEQWFTEDPGP (SEQ ID NO: 34).

    21. A method for manufacturing a pharmaceutical composition for treating or preventing senescent cells, scarred cell and/or cancer cells in a subject, comprising the steps of formulating A) a compound selected from i) a peptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence X.sub.3X.sub.2X.sub.4X.sub.5X.sub.7X.sub.5X.sub.4X.sub.4X.sub.6X.sub.18X.sub.8X.sub.3QNX.sub.9X.sub.8X.sub.10X.sub.10X.sub.11X.sub.12S*X.sub.13X.sub.14X.sub.11X.sub.11 (SEQ ID NO: 1), wherein S* can be S or is absent; X.sub.2 is absent or is selected from K, E, R, and H; X.sub.3 is absent or selected from A, J, and S; X.sub.4 is selected from I, Z, and L, wherein Z is Cyclo-Hexyl-Alanine; X.sub.5 is selected from A, G, S, E, and D; X.sub.6 is selected from E and D; X.sub.7 is selected from J, G, Q, A, S, and P; X.sub.8 is selected from B, W, Y, and F, wherein B is 2-Methyl-Tryptophan; X.sub.9 is absent or is selected from A and G; X.sub.10 is absent or is selected from A and N; X.sub.11 is selected from R and K; X.sub.12 is absent or is R; X.sub.13 is absent or is selected from G and S; X.sub.14 is absent or is selected from A and C; and X.sub.18 is selected from A and E; wherein J is Fmoc-L-2-(4′-pentenyl)alanine; with the provisio that either none or two Js are present that form a staple; ii) the peptide according to i) comprising at least 80% D-amino acids, and iii) a retro inverso peptide according to i) or ii), and pharmaceutically acceptable salts thereof; and wherein said compound induces apoptosis in senescent, scarred cells, and/or cancerous cells into a suitable pharmaceutical composition, or B) performing a method according to claim 10, and formulating said compound as identified into a suitable pharmaceutical composition.

    22. A pharmaceutical composition for treating or preventing senescent cells in a subject, wherein said composition comprises: A) a compound selected from i) a peptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence X.sub.3X.sub.2X.sub.4X.sub.5X.sub.7X.sub.5X.sub.4X.sub.4X.sub.6X.sub.18X.sub.8X.sub.3QNX.sub.9X.sub.8X.sub.10X.sub.10X.sub.11X.sub.12S*X.sub.13X.sub.14X.sub.11X.sub.11 (SEQ ID NO: 1), wherein S* can be S or is absent; X.sub.2 is absent or is selected from K, E, R, and H; X.sub.3 is absent or selected from A, J, and S; X.sub.4 is selected from I, Z, and L, wherein Z is Cyclo-Hexyl-Alanine; X.sub.5 is selected from A, G, S, E, and D; X.sub.6 is selected from E and D; X.sub.7 is selected from J, G, Q, A, S, and P; X.sub.8 is selected from B, W, Y, and F, wherein B is 2-Methyl-Tryptophan; X.sub.9 is absent or is selected from A and G; X.sub.10 is absent or is selected from A and N; X.sub.11 is selected from R and K; X.sub.12 is absent or is R; X.sub.13 is absent or is selected from G and X.sub.14 is absent or is selected from A and C; and X.sub.18 is selected from A and E; wherein J is Fmoc-L-2-(4′-pentenyl)alanine; with the provisio that either none or two Js are present that form a staple; ii) the peptide according to i) comprising at least 80% D-amino acids, and iii) a retro inverso peptide according to i) or ii), and pharmaceutically acceptable salts thereof; and wherein said compound induces apoptosis in senescent, scarred cells, and/or cancerous cells; or B) a compound obtained by a method according to claim 21.

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

    24-26. (canceled)

    27. A method for treating or preventing a disease or condition caused by senescent cells, scarred cells, and/or cancer cells; age-related diseases; kidney disease; non-alcoholic steohepatitis (NASH)/non-alcoholic fatty liver diseases (NAFLD); liver fibrosis; idopathic pulmonary fibrosis (IPF); amyotrofic lateral sclerosis (ALS); osteoarthritis; COPD; musculoskeletal diseases; reductions of cognitive functions; or cancer, in a subject in need thereof, comprising administering to said subject, an effective amount of the pharmaceutical composition according to claim 22.

    28. The method according to claim 27, wherein said therapy is a combination therapy with anti-cancer chemotherapeutic agents or other standard of care drugs for the respective disease or condition or wherein said therapy is applied in senescent cells, scarred cells, and/or cancer cells that have been pretreated with anti-cancer chemotherapeutic agents or other standard of care drugs for the respective disease or condition, and have survived said treatment.

    Description

    [0151] The invention shall now be further described in the following examples with reference to the accompanying Figures, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties.

    [0152] FIG. 1 shows the sequence of human FOXO4 (Homo sapiens) with the sequence of the TP53 interaction domain underlined.

    [0153] FIG. 2 shows that exemplary peptide CL04009 (SEQ ID NO: 11) is potent in binding recombinant TP53-TAD2 and full-length TP53, isolated from cells and superior to a reference compound (FOXO4-DRI). A) shows the relative NMR chemical shift perturbation of 15N-labeled TP53-TAD2 alone (bottom), in the presence of CL04009 (SEQ ID NO: 11) (top), or a reference compound (middle). The NMR chemical shift perturbation induced by the reference compound has been set to 100%. B) shows a pull-down with streptavidin beads for endogenous TP53 from lysates ofHEK293T cells, either in the absence of any peptide, biotinylated CL04009 (SEQ ID NO: 11), or a biotinylated reference peptide (FOXO4-DRI).

    [0154] FIG. 3 shows that peptide CL04009 (SEQ ID NO: 11) is potent and selective against senescent, but not healthy control, IMR90 lung fibroblasts. This figure shows that CL04009 (SEQ ID NO: 11), in which modifications were made to improve the binding to TP53, has superior selectivity to induce apoptosis by Caspase-3/7 activation in senescent, but not healthy control IMR90 lung fibroblasts. Furthermore, it is effective at a lower concentration, indicating improved potency. Top graph shows Caspase activity, bottom graph viability of cells. FOXO4-DRI was used as a control.

    [0155] FIG. 4 shows that CL04022 (SEQ ID NO: 12) is more potent in binding TP53-TAD than the reference compound, FOXO4-DRI. Fluorescence polarization measurements of FITC-labeled TP53-TAD2 (250 nM) in the presence of increasing concentrations of peptides are shown.

    [0156] FIG. 5 shows peptide CL04022 (SEQ ID NO: 12) to be more stable than CL04009 (SEQ ID NO: 11) over a prolonged period of multiple days, based on the alanine exchange in the peptide. Both peptides were dissolved in H.sub.2O and kept at room temperature. The peptide concentration was determined for the indicated time points.

    [0157] FIG. 6 shows that peptide CL04022 (SEQ ID NO: 12) starts inducing Caspase-3/7 activation, a measure of canonical apoptosis, at 24 h after administration. Full caspase-3/7 activation is complete after 60 h, top row is control.

    [0158] FIG. 7 shows that changes in peptide CL04009 (SEQ ID NO: 11) to improve the selective TP53 binding, also result in a very potent and selective capacity to induce apoptosis in senescent (top blue line), but not healthy control (bottom black line) IMR90 cells. The bottom figure shows peptide CL04088 (SEQ ID NO: 13) to be superior to peptide CL04022 (SEQ ID NO: 12), which is still better than the control.

    [0159] FIG. 8 shows that modifications of peptide CL04009 ((SEQ ID NO: 11) here indicated as CL05055) can further improve the potency to eliminate senescent cells of different origins. RPE=Retinal pigment epithelial cells CL03001 is the same as FOXO4-DRI that was used as a control.

    [0160] FIG. 9 shows that CL04022 (SEQ ID NO: 12) is superior to FOXO4-DRI in inducing apoptotic cell death in glioblastoma cells. A) CL04022 (SEQ ID NO: 12) is more potent than FOXO4-DRI in inducing Caspase3/-7 activation (apoptosis) and b) loss of LDH (cell death) in GBM8 glioblastoma cells. C) shows CL04022 (SEQ ID NO: 12) to induce apoptosis in GBM8 glioblastoma cells, but not in healthy Wi38 cells. CL04022 (SEQ ID NO: 12) is also selective for inducing apoptosis in GBM8 glioblastoma vs. healthy WI38 cells.

    [0161] FIG. 10 shows that CL04022 (SEQ ID NO: 12) is effective against colon carcinoma. A) Peptide CL04022 (SEQ ID NO: 12) is effective against two microsatellite-unstable (MSI) colon carcinoma 3D organoid cultures. The peptide is ineffective against one Microsatellite stable (MSS) line. B) shows that CL04022 (SEQ ID NO: 12) is selective against patient-derived colorectal cancers vs. healthy control samples. CL04022 (SEQ ID NO: 12) shows less potency against normal wildtype colon organoids (compared to A) above). The peptide is effective when such organoids are depleted for APC.

    [0162] FIG. 11 shows show superior stability of CL04183 (SEQ ID NO: 55) and CL05114 (SEQID NO: 60) vs. FOXO4-DRI (CL03001) in solution. In the table, CL02001 designates the “L” form of the peptide CL03001, CL02015 designates the “L” form of peptide CL04183 (SEQ ID NO: 55).

    [0163] FIG. 12 shows that A) peptide CL04022 (SEQ ID NO: 12) is more effective in colon cancer 3D organoids when they survived chemotherapy (5′FluoroUracil or Oxaliplatin), B) shows that CL04022 (SEQ ID NO: 12) induces cell death, not merely causes a cell cycle arrest, in contrast to standard of care, SFU.

    [0164] FIG. 13 shows that there is no more tumor outgrowth of 3D colon organoids 14 d after exposure to CL04009 (SEQ ID NO: 11). This indicates CL04009 (SEQ ID NO: 11) is cytotoxic on colon organoids and not just cytostatic, as here shown for the example of colon carcinoma.

    [0165] FIG. 14 shows that within one experiment peptide CL04183 (B) (SEQ ID NO: 55) shows superior selectivity and potency vs. the original FOXO4-DRI (CL03001) (A) in senescent vs. ctrl human RPE1 cells.

    [0166] FIG. 15 shows that colorectal cancer associated mutations in colon organoids sensitizes to FOXO4-based peptides, dependent on p53. WT colon and distinct tumor progression (TPO) organoids were treated with increasing concentrations of CL04124 (SEQ ID NO: 68) or CL04183 (SEQ ID NO: 55). Cell viability was measured 5 days later.

    [0167] FIG. 16 shows 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 either ′5-Fluorouracil (5-FU) or CL04183 (SEQ ID NO: 55). 3 days later the cells were treated with Calcein-AM and Propidium Iodide, colouring alive and dead cells respectively. The organoids were imaged with the EVOS imaging system. B) CRC29 organoids were incubated with Calcein-AM and a Caspase-3/7 red dye for apoptosis before treatment with CL04183 (SEQ ID NO: 55). Live imaging was started immediately using a LSM880 microscope and images were taken every hour. C) CRC29 were treated with increasing concentrations of CL04183 (SEQ ID NO: 55). Cell viability was measured 5 days later.

    [0168] FIG. 17 shows that peptide CL04009 (SEQ ID NO: 11) is more potent in inducing apoptosis in senescent human breast epithelial cells than the reference compound, FOXO4-DRI/CL03001. MCF10 cells were treated with increasing concentrations of CL04009 (SEQ ID NO: 11) and FOXO4-DR and a caspase assay was performed 2 days later to determine apoptosis induction.

    [0169] FIG. 18 shows that CL04183 (SEQ ID NO: 55) reduces grey hair in naturally aged mice. 24-month old C57BL/6J mice were photographed before receiving 3 doses of either PBS or CL04183 (SEQ ID NO: 55) through i.v. injection. 4 weeks after the first treatment the mice were photographed again.

    [0170] FIG. 19 shows that peptide CL04183 (SEQ ID NO: 55) reduces luciferase-expressing colorectal cancer cells in vivo. A) The colorectal cancer organoid line CRC29 containing firefly luciferase was transplanted in the caecum of immunodeficient mice. Two weeks after transplantation bioluminescence levels were determined and the mice were treated with 3 doses of CL04183 (SEQ ID NO: 55) or PBS. All animals were imaged again two days after the last dose and luminescence values were compared to baseline. B) The mice were injected with luciferin before sacrifice. Individual organs were imaged and the luciferase signal quantified using the M3 vision software.

    [0171] FIG. 20 shows that peptide CL04183 (SEQ ID NO: 55) reduces colorectal cancer metastasis in liver and lung. The colorectal cancer organoid line CRC29 was transplanted in the caecum of immunodeficient mice. Two weeks after transplantation the animals were treated with 3 doses of CL04183 (SEQ ID NO: 55) or PBS. A) The lung and liver were stained for human nucleoli to detect human cancer cells in the mouse organs. B) The amount of metastatic cells in lung was quantified using CellProfiler software. Six tile scans were analysed per mouse. All counts were compared to the average of the PBS treated group. The number of liver metastases per stained section was counted visually.

    [0172] FIG. 21 shows that CL04183 (SEQ ID NO: 55) induces apoptosis in metastatic cancer cells in vivo. A TUNEL assay was performed on CRC29 lung metastases treated with PBS or CL04183 (SEQ ID NO: 55) to determine apoptosis induction. Three mice per group were included and the percentage of TUNEL positive cancer cells was quantified using FIJI.

    [0173] FIG. 22 shows that triple negative breast cancer lines exhibit an open conformation of p53, correlating with CL04183 (SEQ ID NO: 55) sensitivity. The indicated human breast cancer cell lines were treated with increasing concentrations of CL04183 (SEQ ID NO: 55) and an MTS assay was performed 2 days later to determine cell viability. The EC50 values were calculated using GraphPad Prism.

    [0174] FIG. 23 shows CL04183 (SEQ ID NO: 55) efficacy against NRAS mutated and BRAF mutated melanoma. See also FIG. 14.

    [0175] FIG. 24 shows NMR peptide binding data. Zoom of the .sup.1H,.sup.15N cross-peak of the p53-TAD2 T55 extracted from the .sup.1H,.sup.15N HSQC NMR spectra obtained with p53-TAD2 alone (lower circle, red), upon addition of the reference peptide FOXO4-DRI/CL03001 (black) or upon addition of the tested peptides (upper circle, blue). A) peptide SEQ ID NO: 22, B) peptide SEQ ID NO: 23, C) peptide SEQ ID NO: 24, D) peptide SEQ ID NO: 25, E) peptide SEQ ID NO: 26, and F) peptide SEQ ID NO: 27.

    [0176] FIG. 25 shows NMR peptide binding data. Zoom of the .sup.1H,.sup.15N cross-peak of the p53-TAD2 T55 extracted from the .sup.1H,.sup.15N HSQC NMR spectra obtained with p53-TAD2 alone (lower circle, red), upon addition of the reference peptide FOXO4-DRI/CL03001 (black) or upon addition of the tested peptides (upper circle, blue). A) peptide CL04022, SEQ ID NO: 12, B) peptide CL04121, SEQ ID NO: 67, C) peptide CL04183, SEQ ID NO: 55, D) peptide CL04180, SEQ ID NO: 54, E) peptide CL04230, SEQ ID NO: 56, and F) peptide CL04231, SEQ ID NO: 57.

    [0177] FIG. 26 shows NMR peptide binding data. Zoom of the .sup.1H,.sup.15N cross-peak of the p53-TAD2 T55 extracted from the .sup.1H,.sup.15N HSQC NMR spectra obtained with p53-TAD2 alone (lower circle, red), upon addition of the reference peptide FOXO4-DRI/CL03001 (black) or upon addition of the tested peptides (upper circle, blue). A) peptide CL04235, SEQ ID NO: 61, B) peptide CL05114, SEQ ID NO: 60.

    [0178] SEQ ID NOs: 1 to 4, and 7 to 31, and 43 to 66 show peptides of the invention, or parts thereof.

    [0179] SEQ ID NOs: 5, 6 and 42 show control peptides, e.g. FOXO4-DRI/CL03001.

    [0180] SEQ ID NOs: 39 and 40 show the amino acid sequences of human FOXO4 and p53, respectively.

    EXAMPLES

    [0181] In the context of the present invention, the terms FOXO4-DRI or CL03001 when relating to a peptide and/or control peptide refer to the peptide with the D-amino acid sequence LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRPPPRRRQRRKKRG (SEQ ID NO: 6) or L-amino acid sequence PRKGGSRRNAWGNQSYAELISQAIESAPEKRLTL (SEQ ID NO 42).

    Cell Culture:

    [0182] Human IMR90, WI38 and RPE cells were grown in Dulbecco's Modified Eagle's Medium (DMEM; Lonza) containing 10% FCS and 1% pen/strep at 37° C. with 3.0% 02 and 5.0% CO.sub.2. To induce senescence, cells were irradiated with 10Gy ionizing radiation (Gammacell 1000) and were left for a minimum of 10 days to become senescent.

    [0183] GBM8 cells were cultured in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 (DMEM F-12), containing high glucose, L-Glutamine, no phenol red, supplemented with 1% (10 mM) HEPES, 1% Penicillin/Streptomycin, 2% B-27 (50×), 5 μg/ml Heparin sodium salt from porcine intestinal mucosa Grade I-A, 20 ng/ml recombinant human Fibroblast Growth Factor (rhFGF) and 20 ng/ml recombinant human Epidermal Growth Factor (rhEGF). These cells were cultured in plastic ware coated with Matrigel Matrix (20× diluted in DMEM F-12) at 37° C., 5% CO.sub.2 and 3% O.sub.2 in a humified incubator.

    [0184] To perform viability and apoptosis assays, cells were split into 96-well plates and treated with peptides 2 days later. Non-senescent and cancer cells were plated 1500 cells/well, while senescent cells were plated at 8.000 cells/well.

    Organoid Culture:

    [0185] Organoids were grown at 37° C. and 5% CO.sub.2 in a humidified incubator. All organoids were cultured in matrigel (Corning) droplets in advanced DMEM/F12 (Lonza) supplemented with 1% glutamax, 1% Penicillin/Streptomycin, 1% (10 mM) HEPES, 10% Noggin conditioned medium, 2% B-27 (50×; (Thermo/Life Technologies), N-acetylcysteine ((Sigma-Aldrich, 1.25 mM)), A83-01 (Tocris, 500 nM) and SB203580 (Invitrogen/Life Technologies, 3 μM). To perform viability and apoptosis assays, the organoids were passaged through resuspension in ice-cold medium, followed by centrifugation in 15 ml tubes at 4° C. The resulting pellet was trypsinized for 5 minutes at 37° C. to obtain single cells and subsequently washed twice with advanced DMEM/F12 media. These cells were then resuspended in Matrigel and plated in 96-well plates in 5 ul droplets. 100 ul fresh medium was added to the wells 15 minutes later. Peptide and chemotherapy treatment was added to the organoids 2 days after plating.

    Caspase Assay:

    [0186] For a caspase assay, Caspase-Glo 3/7 assay reagent (Promega) was added to the medium 2 days after treatment. The plates were then incubated in aluminium foil at room temperature on a shaker for 1 hour and subsequently measured with a Luminescence Plate Reader.

    MTS Assay:

    [0187] Six days after treatment, cells were incubated with 10 ul CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega) for 1 hour at 37° C. to perform an MTS assay. Subsequently, absorbance was measured at 490 nm using a Spectramax M5e.

    Live/Dead Assay on Organoids:

    [0188] A Live/dead assay was performed on both larger organoids 3 days after peptide and/or chemotherapy treatment, or on organoids that were treated as single cells and allowed to grow out for 2 weeks before the assay was performed. To perform the assay, Calcein-AM (1:1000) and Propidium Iodide (PI; 1:100) were added to the medium of the organoids to detect living and dead cells respectively. The organoids were then imaged on a Zeiss Cell Observer microscope and the signal was quantified using FIJI.

    Live Imaging:

    [0189] Organoids and IMR90 cells were grown in 96-well cell culture plates and either the 488 IncuCyte Caspase 3/7 Reagent For Apoptosis (Sartorius) or the IncuCyte Caspase 3/7 red apoptosis assay reagent (Essen Bioscience) was added to the wells. Peptides were added directly before the plate was transferred to a Zeiss Cell Observer microscope that contains a Heat and CO2-controlled compartment. Subsequently, live imaging was started where a picture was recorded every 2 hours for 60 hours. Images were processed using Zen imaging software (Zeiss).

    [0190] Alternatively, live imaging was performed on a LSM880 confocal microscope. For this experiment organoids were plated on a glass bottom cell culture dish and incubated with Calcein-AM (Sigma-Aldrich) to measure cell viability and the IncuCyte Caspase 3/7 red apoptosis assay reagent (Essen Bioscience). Peptides were added directly before imaging was started.

    Mice

    [0191] Mouse experiments were performed after approval from the Dutch animal ethics committee. For these experiments male NOD.Cg-Prkdc.sup.scid Il2rg.sup.tmlWjl/SzJ/J mice (Charles River) were employed. Human CRC29 colorectal cancer organoids containing firefly luciferase were implanted into the caecum, resulting in the formation of a primary tumor at this site. The implantation was performed under ketamine and dexmedetomidine anaesthesia. In addition, the animals received carpofen 30 min before surgery and the day after. 14 days after transplantation, the mice were sedated using isoflurane and imaged under the BioSpace Imager. Subsequently, The animals were treated with either PBS or with ′5-Fluorouracil (5-FU) at a dose of 50 mg/kg via i.p. injection. The first dose of CL04183 (2.5 mg/kg) was administered via i.v. injection 1 week after 5-FU treatment and this treatment was repeated 2 and 4 days later. 4 weeks after transplantation, the mice were imaged again to determine treatment effect and subsequently sacrificed. All organs were harvested and imaged under the BioSpace Imager again and the luciferase signal was quantified using the M3 vision software.

    Immunohistochemistry

    [0192] Liver and lung paraffin sections were rehydrated in decreasing concentrations of ethanol before being washed in TBS and boiled for 20 minutes in 10 mM sodium citrate buffer (PH6) for antigen unmasking. After the slides were left to cool for 30 minutes, the tissue was permeabilized with 0.2% TX-100 in TBS for 5 minutes at room temperature. Subsequently, the sections were washed with TBS and incubated for 1 hour in blocking buffer containing 2% w/v secondary antibody-appropriate sera (e.g. donkey or goat) and 0.1% fish gelatin in 1% BSA. The sections were then encircled with a water-repellent pen and incubated with the primary antibody diluted in TB S/1% BSA overnight at 4° C. The next day, the tissues were washed 3 times with TBS before an hour incubation with fluorescent labelled secondary antibody diluted in blocking buffer (containing nuclear staining with Hoechst 33342). Subsequently, the slides were washed twice in TBS, incubated in Sudan black solution for 20 min to reduce background and washed in demineralized water. The sections were then mounted with Vectashield and imaged using a LSM880 Zeiss confocal microscope.

    Apoptosis Staining

    [0193] A TUNEL assay was performed on lung metastases to determine apoptosis induction after treatment. Rehydrated sections were treated with 20 ug/ml ProtK solution in PBS for 15 minutes and permeabilized in 0.1% Triton X-100 in 0.1% sodium citrate. Subsequently, the tissue was labelled for 1 hour with 10% TUNEL enzyme in labelling solution (ROCHE) at 37° C. Nuclei were labelled with Hoechst 33342 (ThermoFisher) before the slides were mounted with soft set mounting medium (Vectashield). Images were acquired using a LSM880 confocal microscope (Zeiss) and the percentage of TUNEL-positive cells was analysed using FIJI.

    Protein Expression and Purification:

    [0194] Expression constructs for the fragments of human p53 from 1 to 312 (p53-TADBD), 94 to 312 (p53-DBD), amino acid 1 to 94 (p53-TAD), and amino acid 37 to 57 (p53-TAD2) were generated by synthesis of the corresponding optimized p53 cDNA constructs and inserted into pETM11-ZZ-His.sub.6 vector using NcoI/BamHI restriction sites (Genscript). The inventors also generated optimized cDNA expression constructs (Genscript) in pETM11-ZZ-His.sub.6 vector for fragment of human FOXO4 from amino acids 86 to 208 (FOXO4-FH). The inventors employed three different chemically competent E. coli strains (E. coli BL21(DE3) and E. coli BL21-(DE3 Star) for protein expression and E. coli TOP10 for amplification of plasmid DNA).

    [0195] Protein expression and purification were carried out using protocols as published and known to the person of skill.

    Fluorescence Polarization:

    [0196] 116 μl of a solution of the specific peptides (ranging from 1 to 100 μM) were prepared. Subsequently 4 μl of FITC-labelled p53 peptide (stock concentration 15 resulting in a final concentration of 500 nM) were added. 35 μl were then transferred in each well of the 384-well plate. Measurements were performed in triplicates.

    [0197] Data was acquired on a ClarioStar Plus platereader. Endpoint measurement with 200 flashes per well were performed. An excitation filter with 482 nm wavelength and emission filter with 530 nm were used and gain adjustment as well as focal height adjustment were performed for each measurement. Fluorescence intensity, parallel fluorescence polarization and perpendicular fluorescence polarization were recorded.

    [0198] Data analysis was performed using MARS Version 3.4 (BMG), Microsoft Excel and GraphPad Prism Version 8.

    NMR Chemical Shift Mapping:

    [0199] A 5 mm NMR tube containing 500 μl 100 μM p53-TAD2 was prepared and a .sup.1H,.sup.15N HSQC NMR spectrum was recorded. Then increasing amounts of peptide were added stepwise, followed by measurement of another .sup.1H,.sup.15N HSQC spectrum after each step. NMR spectra were acquired on a 600 Mhz Bruker Avance NOE NMR spectrometer equipped with a TXI 600S3 probehead. Data acquisition and processing was performed with Topspin3.5 and Topspin4.0 (Bruker). Data analysis, peak picking and assignments were performed using ccpNMR.

    TABLE-US-00010 TABLE 1 Selection of peptides according to the present invention SEQ Intern. ID No. NO: CL7 7 LTL R K E A S S E I A Q S I L D A Y S Q N G W A N R R S S C K R P CL8 8 LTL R K K A S S K I A Q S I L D A F S Q N G W A N R R S S C K R P CL6 6 LTL R K E P A S E I A Q S I L E A Y S Q N G W A N R R S G G K R P CLIO 10     R K K A S S K I A Q S I L D A F S Q N G W A N R R S S C K R P CL4009 11     R K K A S S K I A A A I L D A F S Q N G W A N R R S S C K R P CL4022 12     R K K A S S K I A A A I L D A F S Q N A W A N R R S S C K R P CL4088 13     R K K A S S K I A A A I L D A F S Q N   W     R R       K R CL14 14     R K K A S S K I E A A I L D A F S Q N   W     R R       K R CL15 15     R K K A S S K I A A E I L D A F S Q N   W     R R       K R CL16 16     R K K A S S K I E A E I L D A F S Q N   W     R R       K R CL17 17     R K K     S K I A A A I L D A F S Q N   W     R R       K R CL18 18     R K K     S K I E A E I L D A F S Q N   W     R R       K R CL19 19     A K           I A A A I L D A F S Q N   W     R R       K R CL20 20     A K           I E A A I L D A F S Q N   W     R R       K R CL21 21 LTL R K E P A S E I A Q S I L E A Y S Q N G W A N R R S G G K R P - P P R R R Q CL22 22     R K K A S S K I A A A I L D A F S Q N G W A N R R S S C K R P - P P R R R Q R R K CL23 23     R K K A S S K I A A A I L D A F S Q N A W A N R R S S C K R P - P P R R R Q R R K CL24 24     R K K A S S K I A A A I L D A F S Q N   W     R R       K R   - P P R R R Q R R K K R G CL25 25     R K K A S S K I E A A I L D A F S Q N   W     R R       K R   - P P R R R Q R R K K R G CL26 26     R K K A S S K I A A E I L D A F S Q N   W     R R       K R   - P P R R R Q R R K K R G CL27 27     R K K A S S K I E A E I L D A F S Q N   W     R R       K R   - P P R R R Q R R K K R G CL28 28     R K K     S K I A A A I L D A F S Q N   W     R R       K R   -     R R R Q R R K K R G CL29 29     R K K     S K I E A E I L D A F S Q N   W     R R       K R   -     R R R Q R R K K R G CL30 30               A K I A A A I L D A F S Q N   W     R R       K R   -     R R R Q R R K K R G CL31 31               A K I E A A I L D A F S Q N   W     R R       K R   -     R R R Q R R K K R G CL32 53     R K K A S S K I E A E I L D A F S Q N   W     R R       K R   - P P R R R Q R R K K R G CL33 54     R K K     S K I E A E I L D A F S Q N   W     R         K R   -     R R R Q R R K K R G CL34 55               A K I E A A I L D A F S Q N   W     R         K R   -     R R R Q R R K K R G CL35 56               A K I E A E I L D A F S Q N   W     R         K R   -     R R R Q R R K K R G CL36 57               A K I E A A I L D E F S Q N   W     R         K R   -     R R R Q R R K K R G CL37 58     R K K A S J K I A I A I L D A F S Q N   W     R R       K R   - P P R R R Q R R K K R G CL38 59     R K K A S S K I A A A Z L D A F S Q N A W A N R R S S C K R   P P P R R R Q R R K K R A CL39 60               A K I E A A I L D A F S Q N   B     R         K R         R R R Q R R K K R G CL40 61               A K I E A E I L E A F S Q N   B     R         K R         R R R Q R R K K R G CL41 62               A K I E A A Z L D A F S Q N   B     R         K R         R R R Q R R K K R G CL42 63     R K K A S S K I E A E I L D A F S Q N   B     R R       K R     P P R R R Q R R K K R G CL43 64     R K K A S S K I E A E Z L D A F S Q N   B     R R       K R     P P R R R Q R R K K R G CL44 65     R K K A S S K I E A E I Z D A F S Q N   B     R R       K R     P P R R R Q R R K K R G CL45 67     R K K     S K I A A A I L D A F Q N     W     R         K R         R R R Q R R K K R G CL46 68     R K K A S S K I E A A I L D A F S Q N   W     R R       K R     P P R R R Q R R K K R G