P53 phosphorylation as a prognostic or diagnostic marker for the treatment of senescent cells in mammals

Abstract

The present invention relates to a method, in particular an in vitro method, for identifying an improved anti-aging compound, based on detecting the binding of said improved anti-aging compound in the presence of at least one phosphorylated amino acid in the transcription activation domain (TAD) domain of mammalian protein p53. The present invention further relates to a method, in particular an in vitro method, for monitoring an anti-aging treatment or prevention in said subject in need thereof, based on detecting the amount of phosphorylation of amino acids in the TAD and / or C-terminal region of a mammalian p53 protein in a biological sample obtained from the mammalian subject, and / or detecting the amount of promyelocytic leukemia protein (PML) bodies and / or their co-localization with phosphorylated p53 in a biological sample obtained from the mammalian subject. Furthermore, the present invention relates to a kit for carrying out said method, and to the respective uses thereof. Finally, an improved anti-aging compound or pharmaceutical composition is provided.

Description

[Technical field]

[0001] The present invention relates to a method, in particular an in vitro method, for identifying an improved anti-aging compound, based on detecting the binding of said improved anti-aging compound in the presence of at least one phosphorylated amino acid in the transcription activation domain (TAD) domain of mammalian protein p53. The present invention further relates to a method, in particular an in vitro method, for monitoring an anti-aging treatment or prevention in said subject in need thereof, based on detecting the amount of phosphorylation of amino acids in the TAD and / or C-terminal region of mammalian p53 protein in a biological sample obtained from a mammalian subject, and / or detecting the amount of promyelocytic leukemia protein (PML) bodies and / or their co-localization with phosphorylated p53 in a biological sample obtained from a mammalian subject. Furthermore, the present invention relates to a kit for carrying out said method and the respective use thereof. Finally, an improved anti-aging compound or pharmaceutical composition is provided. [Background technology]

[0002] Apoptosis (programmed cell death) provides for the death of damaged cells. Dying cells undergoing the final stages of apoptosis display phagocytic molecules that mark these cells for engulfment by cells with the appropriate receptors, such as macrophages.

[0003] Senescent cells are known to accumulate with age and at sites of age-associated pathologies. Moreover, senescent cells can acquire mutations and re-enter a proliferative state. Thus, benign aging lesions retain the ability to become malignant. Indeed, senescence has been linked to various (age-associated) pathologies, and conversely, genetic clearance of senescent cells can delay hallmarks of aging.

[0004] Cellular senescence is a stress response that often leads to permanent cell cycle arrest and profound phenotypic changes, including the generation of a bioactive secretome termed the senescence-associated secretory phenotype (SASP). Although acute senescence induction prevents cancer and limits fibrosis, residual senescent cells cause age-related disorders. Therefore, targeting senescent cells to slow aging and prevent functional impairment, known as "senotherapy", is becoming popular. While drugs that selectively kill senescent cells, called "senolytics", have been the main focus, SASP-centered approaches have emerged as an alternative to target age-associated diseases.

[0005] Recently, it has been shown that genetic elimination of senescent cells can significantly improve fitness and reduce parameters of aging in age-accelerated mouse models. These mice were shown to have reduced signs of aging as measured by kyphosis (excessive curvature of the bone), muscle strength, fat accumulation, and cataracts. This provides further evidence that cellular senescence and SASP are causally linked to age-associated phenotypes and cancer (Non-Patent Document 1). This proof-of-concept evidence was obtained with genetic methods that have poor therapeutic applicability.

[0006] Studies have consistently revealed that FOXO (Forkhead Box O) transcription factors are key determinants of aging and longevity. FOXO proteins are a subfamily of transcription factors conserved from Caenorhabditis elegans to mammals, and act as key regulators of longevity downstream of insulin and insulin-like growth factor signaling. Invertebrate genomes have one FOXO gene, whereas mammals have four FOXO genes: FOXO1, FOXO3, FOXO4, and FOXO6. In mammals, this subfamily is involved in a wide range of important cellular processes controlling stress resistance, metabolism, cell cycle arrest, and apoptosis. Their role in determining longevity is complex and has not been fully elucidated.

[0007] Bourgeois and Madl (in Non-Patent Document 2) disclose that in the past decade, both FOXO and p53 have been identified as important factors in aging, and their dysregulation is associated with numerous diseases, including cancer. However, many of the underlying molecular mechanisms, including the control of aging by FOXO and p53, remain mysterious. FOXO and p53 appear to share several activities, including a central role in controlling cellular senescence. They focus on recent advances in the connection between FOXO and p53, particularly the FOXO4-p53 axis and the role of FOXO4 / p53 in cellular senescence. They discuss potential strategies to target FOXO4-p53 interaction to modulate cellular senescence as a drug target in the treatment and morbidity of age-related diseases.

[0008] Miller Jenkins et al. (in Non-Patent Document 3) disclose that the p53 tumor suppressor is a key component of the cellular response to stress. p53 is mutated or functionally inactivated in most tumors because it can inhibit cell growth. Numerous protein-protein interactions with transcriptional cofactors are central to p53-dependent responses. In the activated state, p53 is extensively modified at both the N- and C-terminal regions of the protein. These modifications, particularly the phosphorylation of serine and threonine residues in the N-terminal transcriptional activation domain, affect p53 stability and activity by modulating the affinity of protein-protein interactions. They review recent findings from in vitro and in vivo studies on the role of N-terminal phosphorylation of p53. These modifications can affect p53 either positively or negatively, adding a second layer of complex control to the opposing interactions of the p53 transcriptional activation domain.

[0009] Feng et al. (in Non-Patent Document 4) disclose that cell line studies indicate that phosphorylation of Ser46 correlates with activation of p53 apoptotic activity.

[0010] Baar MP, et al. (in Non-Patent Document 5) disclose a FOXO4 peptide that disrupts the interaction between FOXO4 and p53. In senescent cells, this selectively induces nuclear exclusion of p53 and cell-specific apoptosis. This peptide exhibits apoptosis-inducing activity in senescent cells or cells expressing increased FOXO4 and Ser15 phosphorylated p53 compared to control cells.

[0011] Patent Document 1 discloses an artificial senolytic peptide that selectively kills senescent cells in mammals when administered intermittently. DNA-damaged melanoma cells in which FoxO4 expression is inhibited undergo a significant increase in apoptosis. These findings indicate that the presence of FoxO4 inhibited apoptosis caused by S46-phosphorylated p53. Furthermore, it has been shown that inhibition of the kinase that phosphorylates S46 of p53 impairs apoptosis of senescent cells even in the absence of FoxO4. Thus, it is proposed that FoxO4 plays an important role in suppressing apoptosis mediated by S46-phosphorylated p53.

[0012] Patent document 2 relates to a peptide comprising the amino acid sequence LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRP (SEQ ID NO: 7), where the amino acids in said amino acid sequence are D-amino acid residues, and a method for using said peptide in the treatment of age-related disorders. The peptide exhibits apoptosis-inducing activity in senescent cells or cells expressing increased expression of FOXO4 and Ser15 phosphorylated p53 (pSer15-p53) compared to control cells.

[0013] WO 2007 / 023363 relates to the use of agents that inhibit Jun kinase and / or FOXO4 in the treatment of cancer and / or the elimination 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.

[0014] US Patent Publication No. 2009 / 0133993 relates to agents that inhibit FOXO4 function and their use in treating cancer and / or removing senescent cells in an individual. In some embodiments, the agent that inhibits FOXO4 is a peptide that inhibits FOXO4 function in a cell, the peptide comprising an amino acid sequence having at least 80% identity to a disclosed fragment of FOXO4.

[0015] US Patent No. 5,399,633 relates to conditionally active proteins that target senescent cells and methods for producing such conditionally active proteins.

[0016] US Patent No. 5,399,663 relates to improved compounds for use in the treatment of diseases or conditions, such as cancer, in which the removal of senescent, scar and / or cancerous cells is beneficial. US Patent No. 5,399,663 also relates to methods of treating individuals suffering from or suspected of suffering from diseases or conditions in which the removal of senescent, scar and / or cancerous cells is beneficial.

[0017] From the above, it is clear that there remains a need in the art for improved assays and methods to identify effective treatments for pathologies using compounds that selectively induce apoptosis in senescent cells.Cellular senescence is particularly associated with degenerative diseases (loss of function) and cancer (gain of function), both of which involve mammalian protein p53.Therefore, the object of the present invention is to provide assays and methods to identify effective treatments for conditions using compounds that selectively induce apoptosis in senescent cells. [Prior art documents] [Patent documents]

[0018] [Patent Document 1] International Publication No. 2019 / 069070 [Patent Document 2] International Publication No. 2016 / 118014 [Patent Document 3] International Publication No. 2013 / 152038 [Patent Document 4] International Publication No. 2013 / 152041 [Patent Document 5] International Publication No. 2018 / 129007 [Patent Document 6] International Publication No. 2021 / 165538 [Non-patent literature]

[0019] [Non-Patent Document 1] Baker et al., 2011. Nature 479(7372):232-6 [Non-Patent Document 2] Regulation of cellular senescence via theFOXO4-p53 axis.FEBS Lett.2018 Jun;592(12):2083-2097 [Non-Patent Document 3] p53 N-terminal phosphorylation: a defining layer of complex regulation, Carcinogenesis, Volume 33, Issue 8, August 2012,Pages 1441-1449, https: / / doi.org / 10.1093 / carcin / bgs145 [Non-Patent Document 4] Ser46 phosphorylation regulates p53-dependentapoptosis and replicative senescence. Cell Cycle. 2006 Dec;5(23):2812-9. doi:10.4161 / cc.5.23.3526. Epub 2006 Dec 1. PMID: 17172844 [Non-Patent Document 5] Targeted Apoptosis of Senescent Cells RestoresTissue Homeostasis in Response to Chemotoxicity and Aging. Cell. 2017 Mar23;169(1):132-147.e16. doi: 10.1016 / j.cell.2017.02.031. PMID: 28340339; PMCID:PMC5556182. Summary of the Invention

[0020] In a first aspect of the invention, the invention solves the above problems by providing a method for identifying improved anti-aging compounds, comprising the steps of: a) contacting at least one candidate anti-aging molecule with a transcription activation domain (TAD) of a mammalian p53 protein, b) detecting specific binding of the candidate molecule to the TAD in the presence of at least one phosphorylated amino acid in the TAD, and c) comparing said specific binding with binding in the absence of one phosphorylated amino acid in the TAD, whereby an increased binding in the presence of at least one phosphorylated amino acid in the TAD identifies an improved anti-aging compound. In its most preferred embodiment, the method of the invention is used to identify improved retro-inverso peptides derived from mammalian FOXO4 proteins, fusion peptides and derivatives thereof comprising such retro-inverso peptides derived from mammalian FOXO4 proteins, and peptides or core sequences thereof, preferably selected from CL04183, CL04124 and CL04177 (see below).

[0021] A method according to the invention is preferred, wherein said TAD1 phosphorylation is located at Ser15 and / or Ser20 of human p53 and / or said TAD2 phosphorylation is located at Ser46 and / or Thr55 of human p53 or at analogous positions in other mammalian p53 TADs. Preferably, said TAD2 phosphorylation is located at Thr55 of human p53 or at analogous positions in other mammalian p53 TADs. In another preferred embodiment of the method according to the invention, detecting binding further comprises, if possible, detecting phosphorylation of Ser392 in the C-terminal region of human p53 or at analogous positions in other mammalian p53 proteins and comparing said specific binding with the absence of said phosphorylated amino acid.

[0022] In a second aspect, the present invention solves the above problem by providing an anti-ageing compound identified according to the method according to the present invention, or a pharmaceutical composition comprising said anti-ageing compound together with a pharma- ceutically acceptable carrier.

[0023] In a third aspect of the present invention, the present invention solves the above problem by providing a method for monitoring an anti-aging treatment or prevention in a mammalian subject in need thereof, comprising: a) providing an anti-aging treatment or prevention to said subject, wherein said anti-aging compound specifically binds to a TAD of p53 protein or a pharmaceutical composition comprising said anti-aging compound, preferably a compound or pharmaceutical composition according to the present invention; b) detecting the amount of phosphorylation of amino acids in the TAD of p53 protein in a biological sample obtained from said subject and / or detecting the amount of promyelocytic leukemia protein (PML) bodies in a biological sample obtained from said subject and / or detecting co-localization of promyelocytic leukemia protein (PML) bodies with phosphorylated p53 protein; and c) comparing the amount(s) and / or co-localization detected in step b) with the amount in a previous sample and / or in a control sample taken from said subject.

[0024] In a fourth aspect of the present invention, the present invention solves the above mentioned problem by providing a method for predicting or prognosing the success, progression and / or susceptibility to anti-ageing treatment or prevention in a mammalian subject, comprising carrying out a method according to the present invention, wherein increased phosphorylation and / or PML protein and / or co-localization of phosphorylated p53 with PML is indicative of the success, progression and / or susceptibility to anti-ageing treatment or prevention in the mammalian subject.

[0025] In a fifth aspect of the present invention, the present invention solves the above problem by providing an anti-aging compound that specifically binds to a TAD of p53 protein, or a pharmaceutical composition comprising said anti-aging compound, preferably a compound or pharmaceutical composition according to the present invention, for use in the prevention or treatment of senescent cells, wherein the senescent cells show an increased amount of phosphorylation of Ser46 and / or Thr55 of human p53, or analogous positions in other mammalian p53 TADs, and / or an increased amount of PML protein, when compared to control cells.

[0026] Preferred is an anti-aging compound or pharmaceutical composition for use according to the invention, wherein the senescent cells furthermore exhibit an increased amount of phosphorylation of Ser392 in the C-terminal region of human p53 or at a similar position in other mammalian p53 proteins when compared to control cells.Further preferred is an anti-aging compound or pharmaceutical composition for use according to the invention, wherein the senescent cells are selected from primary tumor cells, metastatic tumor cells, secondary tumor cells, cells of micrometastasis and / or macrometastasis, fibrotic cells, breast cancer cells such as triple-negative breast cancer cells, lung cancer, ovarian cancer, pancreatic cancer, gastric cancer and / or liver cancer cells or metastatic cells thereof.

[0027] In a sixth aspect of the present invention, the present invention solves the above-mentioned problems by providing a method for preventing or treating senescent cells in a subject, the senescent cells showing an increased amount of phosphorylation of Ser46 and / or Thr55 of human p53, or analogous positions in other mammalian p53 TADs, and / or an increased amount of PML protein body, when compared to control cells, comprising administering to said subject an effective amount of an anti-aging compound that specifically binds to a TAD of p53 protein, or a pharmaceutical composition comprising said anti-aging compound, preferably a compound or pharmaceutical composition according to the present invention.

[0028] A method according to the invention is preferred, wherein the senescent cells furthermore exhibit an increased amount of phosphorylation of Ser392 of human p53 or a similar position of other mammalian p53 proteins in the C-terminal region when compared to control cells.Further preferred is a method, wherein said senescent cells are selected from primary tumor cells, metastatic tumor cells, secondary tumor cells, cells of micrometastasis, fibrotic cells, breast cancer cells such as triple-negative breast cancer cells, lung cancer, ovarian cancer, pancreatic cancer, gastric cancer and / or liver cancer cells or metastatic cells thereof.

[0029] The present invention is based on the inventors' surprising finding that phosphorylation of p53 protein is a key factor for effective binding of anti-aging molecules to TAD and / or C-terminal regions of p53. TAD1 and TAD2 contain four amino acid residues that can change charge upon phosphorylation. FOXO4-FH derived binders disclosed herein interact specifically with TAD2, indicating that S46 and T55 are important for binding and selectivity. FOXO4-FH derived binders identified according to the present invention, such as CL04183, also bind more strongly to Ser46 / Thr55 phosphorylated than to unphosphorylated ones (see FIG. 16). Ser392 was also found to be relevant (see below).

[0030] In the above context, the present invention analyzes the binding efficiency of certain anti-aging molecules, such as retro-inverso peptides derived from mammalian, particularly human, forkhead box protein O4 (FOXO4), to p53 protein (the amino acids in the above amino acid sequences are D amino acid residues).Preferred examples are compounds comprising the following amino acid sequences: RKKASSKIEAAILDAFSQNWRFFKRPPRRRQRRKKRG (SEQ ID NO: 1; shown as CL04124), RKKASSKIEAEILDAFSQNWRRKRPPRRRQRRKKRG (SEQ ID NO: 2; shown as CL04177), and AKIEAAILDAFSQNWRKRRRRQRRKKRG (SEQ ID NO: 3; shown as CL04183).

[0031] Additionally, and independently of the above, it has been found that binding of anti-senescence molecules is further strongly influenced by phosphorylation of the p53 protein at position S392, which results in p53 adopting an "open" conformation, and certain preferred anti-senescence molecules tested, such as molecule CL04183, are particularly effective against cells with pS392 and / or "open" p53 (see, e.g., FIG. 14).

[0032] Promyelocytic leukemia protein (PML) (also known as MYL, RNF71, PP8675, or TRIM19) is the protein product of the PML gene. The PML protein is a tumor suppressor protein required for the assembly of numerous nuclear structures called PML nucleoli or PML (protein) bodies that form within the chromatin of the cell nucleus. These nucleoli are present in mammalian nuclei, with approximately 1-30 per cell nucleus. PML-(N)B is known to have many cellular regulatory functions, including involvement in programmed cell death, genomic stability, antiviral effects, and control of cell division. Mutation or loss of PML, and subsequent dysregulation of these processes, have been implicated in various cancers.

[0033] Further to the above, in the context of the present invention, it has surprisingly been found that PML bodies are useful as a "proxy p53 biomarker" for the efficacy of certain preferred tested anti-aging molecules, such as the molecule CL04183. CL04183 has been found to be particularly effective against cells with pS392 / "open" p53 and PML elevated and / or co-localization of phosphorylated p53 with PML (see, for example, Figure 12), and has been shown to be particularly effective against PML in the liver and lung. 高 It is effective against metastasis, possibly due to the fact that metastasis requires the initiation of stem cell-like cells and PML is associated with a "stemness" state (specifically CD44 and active β-catenin).

[0034] Diana Putavet et al. (Repurposing the FOXO4 senolytic against triple-negative breast cancer [abstract]. In:Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA):AACR; Cancer Res 2022;82(4 Suppl):Abstract nrP1-19-02) disclose that triple-negative breast cancer (TNBC) has a particularly poor prognosis, in part due to the lack of relevant molecular targets in these tumors. The most commonly mutated gene in TNBC is the tumor suppressor gene p53. They found that in TNBC, mutant p53 acquired a unique structure with novel oncogenic effects by binding to the transcription factor forkhead box O (FOXO)4 sequestered within promyelocytic leukemia (PML) foci. Because these nuclear structures are unique to senescent cells, we tested senolytic FOXO4 peptides. In cytotoxicity experiments, we found that the compounds specifically target TNBC over other breast cancer subtypes. Most importantly, these compounds reduce metastatic burden in the most commonly used mouse model of human breast cancer metastasis. They demonstrated that cancers induced by mutant p53 present characteristics unique to senescent cells, making them excellent candidates for FOXO4-directed anti-aging therapy. The creative aims of senolytics may also be applicable to other types of cancer induced by mutant p53. No mention is made of p53 phosphorylation.

[0035] In the context of the present invention, the term "TP53" or "p53" refers to the mammalian protein p53, in particular the human TP53, mouse Trp53, rat Tp53, monkey, sheep, goat, hamster, dog or cat TP53 genes. Also included are all naturally occurring variants and functional fragments of p53 that bind to the FOXO4 protein or fragments thereof, in particular the trans activation domain (TAD) or transcriptional activation domain. The amino acid sequence of human p53 can be found in P04637.

[0036] In the context of the present invention, the term "TAD" refers to one of two distinct transactivation domains contained in the N-terminus of the p53 protein as described above. Raj and Attardi (in The Transactivation Domains of the p53 Protein. Cold Spring HarbPerspect Med. 2017;7(1):a026047. Published 2017 Jan 3.doi:10.1101 / cshperspect.a026047) disclose the structure and function of the p53 TAD (see in particular Figure 1). Also disclosed are serines and threonines within the p53 TAD (around positions 1-85 / 92) that are targets for phosphorylation and affect p53 TAD interaction with binding partners. Including the carboxy-terminal portion of what is currently thought to be the proline-rich domain (PRD), human TAD1 is located around amino acids 1-40 / 42, and TAD2 is located around amino acids 41 / 43-63 or 92 (see also Figure 1).

[0037] Thus, as mentioned above, in a first aspect of the invention, the present invention solves the above problems by providing a method, in particular an in vitro method, for identifying improved anti-aging compounds, comprising the steps of: a) contacting at least one candidate anti-aging molecule with a transcriptional activation domain (TAD) of a mammalian p53 protein, b) detecting specific binding of the candidate molecule to the TAD in the presence of at least one phosphorylated amino acid in the TAD, and c) comparing said specific binding with binding in the absence of at least one phosphorylated amino acid in the TAD, whereby an increased binding in the presence of at least one phosphorylated amino acid in the TAD identifies an improved anti-aging compound. In its most preferred embodiment, the method of the invention is used to identify improved retro-inverso peptides derived from mammalian FOXO4 proteins, fusion peptides comprising such retro-inverso peptides derived from mammalian FOXO4 proteins, and derivatives thereof, as well as peptides or core sequences thereof, preferably selected from CL04183, CL04124 and CL04177 (see above and below).

[0038] Figure 3 shows the results of a binding assay showing that FOXO4 (A) and FOXO4-derived (e.g., mimetic) peptides (B) and (C) bind with significantly higher affinity to p53-TAD2 when phosphorylated at T55 or S46. Figure 4 shows that the FOXO4-derived peptide CL04183 can bind to full-length p53 from cells when wild-type or phosphorylated at T55 or S46, but not when these sites cannot be phosphorylated. Figure 11 then shows that cancer cells that survive chemotherapy show increased PML bodies and phosphorylated p53. These cells are more sensitive to CL04183 treatment (see Figure 13).

[0039] Preferred is a method according to the invention, wherein said contacting comprises a TAD of a mammalian p53 protein or an anti-aging candidate molecule in vivo or in vitro, in solution or bound or conjugated to a solid support, each format also described in the art and known to the skilled person.

[0040] A method according to the invention is preferred, wherein said TAD1 phosphorylation is located at Ser15 and / or Ser20 of human p53 and / or said TAD2 phosphorylation is located at Ser46 and / or Thr55 of human p53 or at a similar position in other mammalian p53 TADs. Preferably, said TAD2 phosphorylation is located at Thr55 of human p53 or at a similar position in other mammalian p53 TADs. In another preferred embodiment of the method according to the invention, detecting binding further comprises, if possible, detecting phosphorylation of Ser392 of human p53 or at a similar position in other mammalian p53 proteins and comparing said specific binding with the absence of said phosphorylated amino acid.

[0041] In the context of the present invention, any suitable mammalian TAD may be used. The method according to the present invention is preferred, wherein the TAD of the mammalian p53 protein is selected from TAD1 and / or TAD2 of the p53 protein of human, mouse, rat, monkey, sheep, goat, hamster, dog and cat, optionally as part of the full-length p53 protein, in particular recombinant p53 protein, full-length p53 protein with phosphorylation at Ser392 of human p53 or at similar positions of other mammalian p53 proteins, recombinant fusion protein with TAD1 and / or TAD2, or phosphorylated fragments of TAD1 or TAD2, and their mutant variants. Preferably, the phosphorylation of said TAD1 domain is located at Ser15 and / or Ser20 of human p53, and / or the phosphorylation of said TAD2 is located at Ser46 and / or Thr55 of human p53, or at similar positions in other mammalian p53 TADs.

[0042] In the context of the present invention, anti-aging candidate molecules may be selected from any suitable molecule suitable for binding to mammalian p53, such as chemical organic molecules, molecules selected from libraries of small organic molecules (molecular weight less than 500 Da), molecules selected from combinatorial libraries, cell extracts, especially plant cell extracts, small molecule drugs, proteins, protein fragments, molecules selected from peptide libraries, antibodies or fragments thereof. Particularly preferred are retro-inverso peptides derived from mammalian FOXO4 proteins, fusion peptides containing retro-inverso peptides derived from mammalian FOXO4 proteins, and derivatives thereof, preferably peptides selected from CL04183, CL04124, and CL04177 as described above. Other peptides are disclosed in WO 2005 / 023991, which is incorporated herein by reference for this purpose. These candidate molecules are used as a basis for screening improved compounds (see below) in which specific amino acids are modified.

[0043] If the anti-aging candidate molecule is a peptide, the compound may further comprise a sequence that confers cell penetrating properties, organelle targeting properties, nuclear localization, mitochondrial localization, blood-brain barrier permeability, cell membrane localization, and / or peptidase cleavage, such as the TAT sequence of HIV (GRKKRRQRRRPP, SEQ ID NO: 8), or ARKKRRQRRRPPP (SEQ ID NO: 9).If the anti-aging candidate molecule is a peptide, the compound may further comprise a non-natural amino acid, preferably having the same or substantially the same desired properties as the natural amino acid at that position in the compound, such as a modified amino acid, such as a linker amino acid, staple, cysteine ​​bridge, glycosylation site, ubiquitination and / or pegylation site.

[0044] In the context of the present invention, any method suitable for detecting the binding of compound can be used.Each method is known to those skilled in the art and disclosed in the art.Preferred is the method according to the present invention, wherein said detecting said binding comprises detecting phosphorylation, comprising radiolabeled 32P-orthophosphate, phosphorylation-specific antibody, and / or mass spectrometry.

[0045] Components of the assays disclosed herein can be labeled, for example, with a radioactive or fluorescent label, or an antigenic label.

[0046] In another embodiment of the invention, detecting binding comprises competitive binding of a compound, in particular a peptide such as CL04177, CL04124 or CL04183, to the protein FOXO4 or a TAD-binding fragment thereof, i.e. testing the properties of said modified compound in the presence of said at least one compound for its different phosphorylation states compared to the absence of said compound.

[0047] Detecting binding may further comprise, where possible, detecting phosphorylation of Ser392 of human p53 or a similar position in other mammalian p53 proteins and comparing said specific binding to the absence of said phosphorylated amino acid, which may also be performed using an appropriately selected antibody.

[0048] Yet another aspect of the present invention relates to a method for identifying improved compounds that bind to a TAD of p53 or a FOXO4-binding fragment thereof, or that bind to p53 and inhibit the interaction of FOXO4 and p53 in cells, comprising the steps of: a) providing at least one candidate anti-aging compound; b) appropriately modifying said compound of a); c) contacting at least one candidate anti-aging molecule of b) with a transcriptional activation domain (TAD) of a mammalian p53 protein; d) detecting specific binding of the candidate molecule to the TAD in the presence of at least one phosphorylated amino acid in the TAD; and e) comparing said specific binding with binding in the absence of the at least one phosphorylated amino acid in the TAD, wherein an increased binding in the presence of the at least one phosphorylated amino acid in the TAD identifies an improved anti-aging compound.

[0049] Preferred is a method according to the invention, which further comprises testing said identified compounds for their activity in inducing apoptosis and / or killing senescent, scarred and / or tumour cells, preferably by determining an increase in caspase 3 / 7 activity, loss of mitochondrial cytochrome C, TUNEL positivity, extracellular annexin V positivity and / or an increase in cell death markers (such as intracellular propidium iodide inclusion, lactate dehydrogenase release assay) and / or loss of viability (such as loss of calcein AM uptake or MTS viability assay).

[0050] Further preferred is a method according to the invention, wherein said TAD or said FOXO4-binding fragment thereof is recombinantly expressed in a cell.

[0051] The present invention further relates to improved inhibitory compounds that have been designed based on the evolutionary differences between FOXO1 / 3 / 6 and FOXO4, which indicate that some amino acids can be changed or modified to generate improved inhibitory compounds. All these modifications present new strategies, any one of them or a combination of them, which ultimately lead to the "rational design" of improved molecules for use in the context of the present invention. The present invention also includes strategies for further improving compounds that have only partially undergone "directed evolution" or "directed mutagenesis".

[0052] In the course of "rational design" of improved molecules for use in the context of the present invention, and to further improve compounds that have only partially undergone "directed evolution" or "directed mutagenesis", the compounds may be subjected to the above methods several times in succession.

[0053] Subsequent to providing a compound (e.g., a peptide), the compound can be modified, for example after its suitable chemical synthesis. In general, many methods of modifying the compounds of the present invention are known to those skilled in the art and are also disclosed in the literature. Modifications of compounds are generally classified into several categories, for example: a) mutation / change of amino acids 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., from L-form to D-form) or modification of bonds (e.g., introduction of retro-inverso bonds), d) change in length of the compound, and e) addition of additional groups to the molecule (including marker groups, labels, linkers, or carriers, such as chelators).

[0054] As mentioned above, the evolutionary differences between FOXO1 / 3 / 6 and FOXO4 indicate that some further amino acids can be modified to create improved compounds that exhibit the desired binding and ultimately inhibitory activity. Analysis of the FOXO4-FH fragment and the p53-TAD2 domain indicates that the negative charge in p53 mediates the binding to FOXO4 and the hydrophobic Tyr. Thus, the amino acids of the peptides according to the invention can be mutated to positive amino acids to benefit the strength of the interaction. Examples are lysine (K), arginine (R), and histidine (H). NMR experiments also showed that the negatively charged amino acids in p53 are responsible for the interaction with FOXO4 around the SQ motif (site). This site can be phosphorylated and to prevent this, this site can be mutated to small amino acids such as alanine (A), glycine (G), or serine (S). Methods for introducing such mutations are known to those skilled in the art and include methods for introducing the changes during the chemical synthesis of the peptide, or genetic methods in which the nucleotide sequence encoding the peptide is altered accordingly, for example by oligonucleotide-based mutagenesis, PCR-involved mutagenesis, etc. Random mutagenesis is also possible.

[0055] Furthermore, amino acids can be modified by chemical modification, for example by adding additional chemical groups during synthesis, or through post-translational or post-synthetic modifications. Methods for modifying amino acids are well known in the current state of the art and are summarized, for example, in Christopher D. Spicer & Benjamin G. Davis. Selective chemical protein modification Nature Communications volume 5, Article no.:4740 (2014), or Sakamoto S, Hamachi I. Recent Progress in Chemical Modification of Proteins. Anal Sci. 2019 Jan 10;35(l):5-27. Modifications of amino acid structure (e.g., from L-form to D-form) or of bonds between amino acids (e.g., introducing retro-inverso bonds during synthesis) can also be performed.

[0056] Finally, additional chemical and / or functional groups on the molecule, such as marker groups, labels, linker amino acids, staples, cysteine ​​bridges, glycosylation sites, ubiquitination and / or pegylation sites, linkers, or carriers (such as chelators), etc.

[0057] In the next step, the modified compound is tested for at least one of the bindings of said at least one compound to the TAD of p53 under different phosphorylation states. As discussed herein, the property of a compound to bind to a TAD and its phosphorylation state are essential for any use of the compound, whether therapeutic or diagnostic. In the context of the present invention, "improved" binding includes both scenarios in which the modified compound binds to the same extent as the unmodified (i.e. starting) compound, even though the compound has been modified (e.g., by dimerization or by addition of a marker or other group). Preferred are compounds that are modified in their phosphorylation state to show stronger binding to the target, i.e., the TAD of p53. Also preferred are compounds that show longer binding to the target, i.e., the TAD or its binding fragment, due, for example, to the improved stability of said modified compound in vitro or in vivo.

[0058] Assays for detecting binding of a compound to a target (ie, a TAD) are well known to those skilled in the art and preferably include mass spectrometry, NMR assays, pull-down assays, and the like.

[0059] As mentioned above, the property of binding to TADs and their phosphorylation state of the compound is essential for any use of the compound, whether therapeutic or diagnostic.Ideally, the binding is also specific, or at least substantially or essentially specific, to the intended target, i.e., TAD.This reduces or avoids any undesirable side effects in the medical use of the compound, and reduces or avoids any background or false positive results in diagnostic use.

[0060] In the final step of the method, in a preferred embodiment of the invention, improved compounds are identified that bind to the TAD of p53 depending on its phosphorylation status, in particular to the TAD2 of p53, preferably to the TAD depending on its phosphorylation status, and inhibit the interaction of FOXO4 and p53 in cells based on said determinations compared to the compounds provided at the beginning of the method. This function is believed to be essential for the therapeutic function of the compounds of the invention by eliminating senescent cells, although an improved diagnostic molecule according to the invention may not necessarily require such a property / function.

[0061] Identifying such improved compounds may include detecting the efficacy of these compounds, such as FOXO4 peptides, to better bind phosphorylated TADs and eliminate senescent cells, and optionally identifying the structural requirements that determine this property, and / or detecting the selectivity of these compounds, such as FOXO4-derived peptides, to eliminate senescent cells, and also optionally identifying the structural requirements that determine this property.

[0062] Preferably, a peptide according to the invention or an improved compound such as an improved peptide is considered to exhibit apoptosis-inducing activity in senescent cells if it kills, eliminates, removes or reduces the viability of at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70% or 80% of the cells in the senescent cell culture. Preferably, the peptide according to the invention exhibits apoptosis-inducing activity selectively in senescent cells, i.e., not in non-senescent cells. The peptide according to the invention promotes apoptosis in senescent cells at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3-fold, 4-fold, 5-fold or more than apoptosis in non-senescent cells.

[0063] The peptide compounds according to the invention are preferably isolated.The peptide compounds according to the invention are preferably produced by means of a suitable peptide synthesis.

[0064] Yet another aspect of the present invention then relates to an improved anti-aging compound identified according to the method according to the present invention, or a pharmaceutical composition comprising said anti-aging compound together with a pharma- ceutically acceptable carrier.

[0065] The pharmaceutical composition used may optionally contain a pharma- ceutically acceptable carrier. The pharma- ceutically acceptable carrier or excipient may include diluents (fillers, bulking agents, e.g., lactose, microcrystalline cellulose), disintegrants (e.g., sodium starch glycolate, croscarmellose sodium), binders (e.g., PVP, HPMC), lubricants (e.g., magnesium stearate), glidants (e.g., colloidal SiO 2 ), solvents / co-solvents (e.g., aqueous vehicles, propylene glycol, glycerol), buffers (e.g., citrate, gluconate, lactate), preservatives (e.g., sodium benzoate, parabens (Me, Pr, and Bu), BKC), antioxidants (e.g., BHT, BHA, ascorbic acid), humectants (e.g., polysorbates, sorbitan esters), thickeners (e.g., methylcellulose or hydroxyethylcellulose), sweeteners (e.g., sorbitol, saccharin, aspartame, acesulfame), flavorings (e.g., peppermint, lemon oil, butterscotch, etc.), humectants (e.g., propylene, glycol, glycerol, sorbitol). Other suitable pharma- ceutically acceptable excipients are described, inter alia, in Remington's Pharmaceutical Sciences, 15 th Ed., Mack Publishing Co., New Jersey (1991) and Bauer et al., Pharmazeutische Technologic, 5 th Ed., Govi-Verlag Frankfurt (1997). A person skilled in the art would know suitable formulations of peptides and would be able to easily select suitable pharma- ceutically acceptable carriers or excipients depending, for example, on the formulation of a pharmaceutical composition and the route of administration.

[0066] The pharmaceutical compositions can be administered orally, for example, in the form of pills, tablets, coated tablets, dragees, hard and soft gelatin capsules, solutions, syrups, emulsions or suspensions, or as aerosol mixtures. However, administration can also be effected rectally, for example in the form of suppositories, or parenterally, for example in the form of injections or drops, or transdermally, for example in the form of ointments, creams or tinctures.

[0067] In addition to the aforementioned compounds of the present invention, pharmaceutical compositions can further comprise conventional, usually inert, carrier materials or excipients. Thus, pharmaceutical preparations can also comprise, for example, fillers, bulking agents, disintegrants, binders, glidants, wetting agents, stabilizers, emulsifiers, preservatives, sweeteners, colorants, flavorings or fragrances, buffer substances, and also solvents or solubilizers, or agents for achieving a depot effect, as well as salts for modifying osmotic pressure, coating agents, or antioxidants. They can also comprise the aforementioned salts of two or more compounds of the present invention, and can also comprise other therapeutically active substances as described herein.

[0068] Yet another aspect of the present invention then relates to a method, such as a diagnostic method, for monitoring an anti-aging treatment or prevention in a mammalian subject in need thereof, comprising: a) providing an anti-aging treatment or prevention to said subject, wherein said subject is administered an anti-aging compound that specifically binds to a TAD of p53 protein or a pharmaceutical composition comprising said anti-aging compound, preferably a compound or pharmaceutical composition according to the present invention as described herein; b) detecting the amount of phosphorylation of amino acids in the TAD of p53 protein in a biological sample obtained from said subject and / or detecting the amount of promyelocytic leukemia protein (PML) bodies in a biological sample obtained from said subject and / or detecting co-localization of promyelocytic leukemia protein (PML) bodies and phosphorylated p53 protein, and c) comparing the amount(s) and / or co-localization detected in step b) with the amount and / or co-localization in a previous sample and / or in a control sample taken from said subject. Preferred is a method in which an increase in the amount of phosphorylation and / or PML bodies, and / or co-localization of phosphorylated p53 with PML, is indicative of success, progression and / or susceptibility to anti-aging treatment or prevention in a mammalian subject. The method may further comprise adjusting the treatment or prevention of said mammalian patient based on said monitoring. The attending physician can easily make and apply the respective treatment decision, taking into account additional patient parameters as appropriate.

[0069] Similarly, yet another aspect of the present invention then relates to a method, such as a diagnostic method, for predicting or prognosing the success, progression and / or susceptibility to an anti-aging treatment or prevention in a mammalian subject, comprising carrying out a method according to the present invention as disclosed herein, wherein an increase in phosphorylation and / or PML bodies and / or co-localization of phosphorylated p53 with PML is indicative of the success, progression and / or susceptibility to an anti-aging treatment or prevention in the mammalian subject. As described herein, it has surprisingly been found by the inventors that phosphorylation of the TAD of p53 and / or the amount of PML bodies and / or co-localization of phosphorylated p53 with PML is directly related to the success, progression and / or susceptibility to an anti-aging treatment or prevention.

[0070] Preferred is a method according to the invention, wherein said mammalian subject is a human, mouse, rat, monkey, sheep, goat, hamster, dog, or cat.

[0071] Anti-aging treatment or prevention in a mammalian subject is preferably a method according to the invention comprising administering an effective amount of a compound identified herein, a pharmaceutical composition as described, a retro-inverso peptide derived from a mammalian FOXO4 protein, a fusion peptide comprising a retro-inverso peptide derived from a mammalian FOXO4 protein, and derivatives thereof, preferably a peptide selected from CL04183, CL04124, and CL04177, and a TAD2 binding derivative or fragment thereof.

[0072] As described herein, the compound is administered to the subject in an effective dosage. This dosage varies within a wide range and is adjusted to the individual circumstances of each individual case. For the above uses, the appropriate dosage varies depending on the mode of administration, the particular pathology being treated, and the desired effect. However, generally, satisfactory results are achieved at dosage rates as described above, for example, about 1 mg to 100 mg per kg of animal body weight, particularly 1 mg / kg to 50 mg / kg. A suitable dosage rate for a large mammal, for example a human, is on the order of about 10 mg / day to 3 g / day, conveniently administered in a single or divided dose, for example 2 to 4 times a day, or in sustained release form. In general, for oral administration, a daily dose of about 10 mg to 100 mg, particularly 10 mg to 50 mg per human is appropriate. The effective concentration achieved at the cellular level can be set at about 0.05 μM to 200 μM, preferably about 0.01 μM to 100 μM, more preferably about 0.05 μM to 10 μM. Local application to the respiratory tract, for example by inhalation, is particularly preferred. In these cases, the dosage is advantageously reduced to 0.1 mg / dose to 10 mg / dose, preferably 0.2 mg / dose to 5 mg / dose, which corresponds to about 3 μg / kg to about 80 μg / kg for a 70 kg subject.

[0073] As further described herein, a method according to the invention is preferred, wherein said TAD2 phosphorylation is located at Ser46 and / or Thr55 of human p53 or at a similar position in other mammalian p53 TADs. Preferably, said TAD2 phosphorylation is located at Thr55 of human p53 or at a similar position in other mammalian p53 TADs. Also, in this aspect of the invention, a method according to the invention is further preferred, further comprising detecting the amount of phosphorylation of Ser392 of human p53 or at a similar position in other mammalian p53 proteins and comparing said amount with the amount in a previous sample and / or a control sample taken from said subject, wherein an increase in phosphorylation is a further indication of the success and / or progress of anti-aging treatment or prevention in the mammalian subject.

[0074] Preferred is the method according to the invention, wherein the biological sample obtained from said subject comprises senescent cells, tumor cells, metastatic cells, cells of micro- and / or macro-metastases and / or fibrotic cells, in particular breast, ovarian, pancreatic, gastric, lung and / or liver cancer cells or metastatic cells thereof.

[0075] Further preferred is a method according to the present invention, wherein the subject further receives an anti-senescent chemotherapy, such as an anti-cancer chemotherapy.

[0076] Yet another aspect of the invention then relates to an improved anti-aging compound or a pharmaceutical composition comprising said anti-aging compound, preferably a compound or pharmaceutical composition according to the invention, which specifically binds to a TAD, preferably TAD2, of a mammalian p53 protein, for use in the prevention or treatment of senescent cells, wherein the senescent cells show an increased amount of phosphorylation of Ser46 and / or Thr55 of human p53, or analogous positions in other mammalian p53 TADs, and / or an increased amount of PML protein, when compared to control cells. Preferably, said TAD2 phosphorylation is located at Thr55 of human p53, or analogous positions in other mammalian p53 TADs.

[0077] Also further preferred in this aspect of the invention is an improved anti-aging compound or pharmaceutical composition for use according to the invention, wherein the senescent cells further exhibit an increased amount of phosphorylation of Ser392 of human p53, or the analogous position of other mammalian p53 proteins, when compared to control cells or control values.

[0078] The improved anti-aging compound or pharmaceutical composition for use according to the present invention is preferred, wherein the biological sample obtained from the subject comprises senescent cells, tumor cells, metastatic cells, cells of micrometastasis, and / or fibrotic cells, in particular cells of breast, ovarian, pancreatic, gastric, lung, and / or liver cancer or metastatic cells thereof.Thus, the improved anti-aging compound or pharmaceutical composition for use according to the present invention is preferred, wherein the senescent cells are selected from tumor cells, metastatic tumor cells, cells of tumor micrometastasis, fibrotic cells, breast cancer cells such as triple-negative breast cancer cells, cells of ovarian, pancreatic, gastric, lung, and / or liver cancer or metastatic cells thereof.

[0079] Further preferred is an improved anti-aging compound or pharmaceutical composition for use according to the present invention, wherein the subject further undergoes anti-senescent cell chemotherapy, such as anti-cancer chemotherapy.

[0080] Yet another aspect of the present invention then relates to a method for preventing or treating senescent cells in a subject, the senescent cells exhibiting an increased amount of phosphorylation of Ser46 and / or Thr55 of human p53, or analogous positions in other mammalian p53 TADs, and / or an increased amount of PML protein, when compared to control cells, comprising administering to said subject an effective amount of an anti-aging compound that specifically binds to a TAD of p53 protein, or a pharmaceutical composition comprising said anti-aging compound, preferably a compound or pharmaceutical composition according to the invention. Preferably, said TAD2 phosphorylation is located at Thr55 of human p53, or analogous positions in other mammalian p53 TADs.

[0081] Also in this aspect of the invention, a method according to the invention is further preferred, wherein the senescent cells further exhibit an increased amount of phosphorylation of Ser392 of human p53 or the analogous position of other mammalian p53 proteins when compared to control cells.

[0082] A method according to the invention is preferred, wherein said senescent cells are selected from tumor cells, metastatic tumor cells, cells of tumor micrometastasis, fibrotic cells, breast cancer cells such as triple-negative breast cancer cells, ovarian cancer, pancreatic cancer, gastric cancer, lung cancer and / or liver cancer cells or metastatic cells thereof. Thus, a method according to the invention is preferred, wherein said senescent cells are selected from tumor cells, metastatic tumor cells, cells of tumor micrometastasis, fibrotic cells, breast cancer cells such as triple-negative breast cancer cells, ovarian cancer, pancreatic cancer, gastric cancer, lung cancer and / or liver cancer cells or metastatic cells thereof. A method according to the invention is further preferred, wherein the subject further receives an anti-senescent cell chemotherapy, such as an anti-cancer chemotherapy.

[0083] In the medical use aspect of the present invention, the compound (used) may be provided and / or administered as a suitable pharmaceutical composition, such as tablets, capsules, injections, granules, powders, sachets, reconstitutable powders, dry powder inhalants, inhalants, and / or chewables. Such solid formulations may contain suitable amounts of excipients and other ingredients. Such solid formulations may contain, for example, cellulose, microcrystalline cellulose, polyvidone, in particular FB polyvidone, magnesium stearate, etc. However, administration can also be performed rectally, for example in the form of suppositories, or parenterally, for example in the form of injections or drops, or transdermally, for example in the form of ointments, creams or tinctures. Administration using dry powder inhalants or other inhalation forms is preferred.

[0084] It is understood that the compounds of the present invention and / or pharmaceutical compositions comprising the compounds of the present invention are intended for administration to human patients. The term "administer" refers to administration of a single therapeutic agent or administration in combination with other therapeutic agents. It is therefore envisaged that the pharmaceutical compositions of the present invention are used in a combination therapy approach, i.e., administration in combination with other pharmaceuticals or drugs and / or other therapeutic agents that may be beneficial in the context of the method of the present invention. However, the other pharmaceuticals or drugs and / or any other therapeutic agents can be administered separately from the compounds of the present invention used, if necessary, as long as they act in combination (i.e., directly and / or indirectly, preferably synergistically) with the compounds of the present invention used.

[0085] Thus, the compounds of the invention can be used alone or in combination with other active compounds (e.g. drugs already known for the treatment of the aforementioned diseases), in the latter case favorable additive, amplifying or preferably synergistic effects are observed. Amounts suitable for administration to humans range from 1 mg to 500 mg, in particular 5 mg to 100 mg, for example an oral dose of 1 mg / kg / day to 10 mg / kg / day. The effective concentration reached at the cellular level can be set as above at 0.005 μM to 200 μM, preferably about 0.5 μM to 10 μM. Local application to the airways, such as by inhalation, is particularly preferred. In these cases, the dosage is advantageously reduced to 0.1 mg / dose to 10 mg / dose, preferably 0.2 mg to 5 mg per dose, which corresponds to about 3 μg to about 80 μg per kg for a 70 kg subject.

[0086] Yet another aspect of the present invention then relates to a diagnostic kit comprising, in one or separate containers, the materials for carrying out the method according to the invention, optionally together with auxiliary agents and / or instructions for carrying out said method.

[0087] The diagnostic kit according to the present invention preferably comprises at least one of the anti-aging candidate molecules, a recombinantly expressed TAD of a mammalian p53 protein, in particular an N-terminal fragment containing TAD1 or TAD2, preferably bound or conjugated to a solid support.

[0088] The kits may further comprise FOXO4 or a TAD-binding portion thereof, relevant antibodies that bind to the components of the kit, radiolabeled 32P-orthophosphate, phosphorylation-specific and / or p53 conformation-specific antibodies (i.e., specific for the "open" form), dyes and other labels, as well as buffers and matrices for carrying out the above methods.

[0089] The kits may be used in the methods of the invention, i.e., to identify anti-aging compounds, to monitor anti-aging treatment or prevention in a mammalian subject in need thereof, and / or to predict or prognose the success, progress, and / or susceptibility of anti-aging treatment or prevention in a mammalian subject.

[0090] The present invention relates to the following items:

[0091] Item 1. A method for identifying an improved anti-aging compound, comprising: 1. A method comprising the steps of: a) contacting at least one anti-aging candidate molecule with a transactivation domain (TAD) of a mammalian p53 protein; b) detecting specific binding of the candidate molecule to the TAD in the presence of at least one phosphorylated amino acid in the TAD; and c) comparing the specific binding to binding in the absence of the at least one phosphorylated amino acid in the TAD, wherein increased binding in the presence of the at least one phosphorylated amino acid in the TAD identifies an improved anti-aging compound.

[0092] Clause 2. The method of clause 1, wherein the contacting is in vivo or in vitro, in solution, or comprises the TAD of a mammalian p53 protein or the anti-aging candidate molecule bound or conjugated to a solid support.

[0093] Item 3. The method according to item 1 or 2, wherein the anti-aging candidate molecule is selected from a chemical molecule, a molecule selected from a library of small organic molecules, a molecule selected from a combinatorial library, a cell extract, particularly a plant cell extract, a small molecule drug, a protein, a protein fragment, a molecule selected from a peptide library, an antibody or a fragment thereof, particularly a retro-inverso peptide derived from a mammalian FOXO4 protein, a fusion peptide comprising a retro-inverso peptide derived from a mammalian FOXO4 protein and a derivative thereof, preferably a peptide selected from CL04183, CL04124, and CL04177.

[0094] Item 4. The method of any one of items 1 to 3, wherein detecting the binding comprises detecting phosphorylation involving radiolabeled 32P-orthophosphate, a phosphorylation-specific antibody, and / or mass spectrometry.

[0095] Item 5. The method according to any one of items 1 to 4, wherein the TAD of the mammalian p53 protein is optionally selected from a part of full-length p53 protein, in particular a recombinant p53 protein, a full-length p53 protein comprising phosphorylation at Ser392 of human p53 or at an analogous position of other mammalian p53 proteins, a recombinant fusion protein comprising TAD1 and / or TAD2, or a phosphorylated fragment of said TAD1 or TAD2, and mutated variants thereof, such as TAD1 and / or TAD2 of human, mouse, rat, monkey, sheep, goat, hamster, dog and cat p53 proteins.

[0096] Item 6. The method according to any one of items 1 to 5, wherein the phosphorylation of the TAD1 domain is located at Ser15 and / or Ser20 of human p53, and / or the phosphorylation of the TAD2 domain is located at Ser46 and / or Thr55 of human p53, or at analogous positions in other mammalian p53 TADs, preferably, the phosphorylation of the TAD2 domain is located at Thr55 of human p53, or at analogous positions in other mammalian p53 TADs.

[0097] Item 7. The method according to any one of items 1 to 6, wherein detecting the binding comprises competitive binding with the protein FOXO4 or a TAD-binding fragment thereof.

[0098] Item 8. The method according to any one of items 1 to 7, wherein detecting the binding further comprises, if possible, detecting phosphorylation of Ser392 of human p53 or a similar position in other mammalian p53 proteins, and comparing the specific binding to the absence of the phosphorylated amino acid.

[0099] Item 9. An anti-aging compound identified according to the method according to any one of items 1 to 8, or a pharmaceutical composition comprising the anti-aging compound together with a pharma- ceutically acceptable carrier.

[0100] Clause 10. A method for monitoring anti-aging treatment or prevention in a mammalian subject in need thereof, comprising: a) providing an anti-aging treatment or prevention to said subject, wherein said anti-aging compound specifically binds to a TAD of p53 protein or a pharmaceutical composition comprising said anti-aging compound, preferably a compound or pharmaceutical composition according to clause 9, is administered to said subject; b) detecting the amount of phosphorylation of amino acids in the TAD of said p53 protein in a biological sample obtained from said subject, and / or detecting the amount of promyelocytic leukemia protein (PML) bodies in a biological sample obtained from said subject, and / or detecting the co-localization of promyelocytic leukemia protein (PML) bodies with phosphorylated forms of phosphorylated p53 protein; and c) comparing the amount(s) and / or co-localization detected in step b) with the amount and / or co-localization in a previous sample and / or a control sample taken from said subject.

[0101] Item 11. A method for predicting or prognosing the success, progression and / or susceptibility of an anti-aging treatment or prevention in a mammalian subject, comprising carrying out the method according to claim 10, wherein an increase in the amount of phosphorylation and / or PML protein body and / or co-localization of the phosphorylated form with phosphorylated p53 protein is indicative of the success, progression and / or susceptibility of said anti-aging treatment or prevention in said mammalian subject.

[0102] Item 12. The method of item 10 or 11, wherein the mammalian subject is a human, mouse, rat, monkey, sheep, goat, hamster, dog, or cat.

[0103] Item 13. The method according to any one of items 10 to 12, wherein the anti-aging treatment or prevention in the mammalian subject comprises administering an effective amount of a retro-inverso peptide derived from a mammalian FOXO4 protein, a fusion peptide comprising a retro-inverso peptide derived from a mammalian FOXO4 protein and a derivative thereof, preferably a peptide selected from CL04183, CL04124, and CL04177, and a TAD2 binding derivative or a fragment thereof.

[0104] Item 14. The method according to any one of items 10 to 13, wherein the phosphorylation of TAD2 is located at Ser46 and / or Thr55 of human p53 or at a similar position in other mammalian p53 TADs, preferably, the phosphorylation of TAD2 is located at Thr55 of human p53 or at a similar position in other mammalian p53 TADs.

[0105] Clause 15. The method according to any one of clauses 10 to 14, further comprising detecting the amount of phosphorylation of Ser392 of human p53 or a similar position in other mammalian p53 proteins and comparing said amount with the amount in a previous sample and / or a control sample taken from said subject, wherein an increase in said phosphorylation is a further indication of the success and / or progress of said anti-aging treatment or prevention in said mammalian subject.

[0106] Item 16. The method according to any one of Items 10 to 15, wherein the biological sample obtained from the subject contains senescent cells, tumor cells, metastatic cells, micrometastatic cells, and / or fibrotic cells, in particular breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, lung cancer, and / or liver cancer cells or metastatic cells thereof.

[0107] Item 17. The method according to any one of items 10 to 16, wherein the subject further receives anti-senescence chemotherapy, for example anti-cancer chemotherapy.

[0108] Item 18. An anti-aging compound that specifically binds to a TAD of p53 protein or a pharmaceutical composition comprising said anti-aging compound, for use in the prevention or treatment of senescent cells, preferably a compound or pharmaceutical composition according to claim 9, wherein said senescent cells show an increased amount of phosphorylation of Ser46 and / or Thr55 of human p53, or of analogous positions in other mammalian p53 TADs, and / or an increased amount of PML protein body, when compared to control cells. An anti-aging compound that specifically binds to a TAD of p53 protein or a pharmaceutical composition comprising said anti-aging compound, preferably a compound or pharmaceutical composition according to claim 9, for use in the prevention or treatment of senescent cells.

[0109] Clause 19. The anti-aging compound or pharmaceutical composition for use according to clause 18, wherein the senescent cells further exhibit an increased amount of phosphorylation of Ser392 of human p53, or the analogous position in other mammalian p53 proteins, when compared to control cells.

[0110] Item 20. The anti-aging compound or pharmaceutical composition for use according to Item 18 or 19, wherein the senescent cells are selected from tumor cells, metastatic tumor cells, cells of tumor micrometastasis, fibrotic cells, breast cancer cells such as triple-negative breast cancer cells, ovarian cancer, pancreatic cancer, gastric cancer, lung cancer, and / or liver cancer cells or metastatic cells thereof.

[0111] Item 21. A method for preventing or treating senescent cells in a subject, wherein the senescent cells show an increased amount of phosphorylation of Ser46 and / or Thr55 of human p53, or analogous positions of other mammalian p53 TADs, and / or an increased amount of PML protein, and / or an increased co-localization of phosphorylated p53 with PML, when compared to control cells, comprising administering to the subject an effective amount of an anti-aging compound that specifically binds to a TAD of p53 protein, or a pharmaceutical composition comprising said anti-aging compound, preferably the compound or pharmaceutical composition described in item 9.

[0112] Clause 22. The method of clause 21, wherein the senescent cells further exhibit an increased amount of phosphorylation of Ser392 of human p53 or an analogous position in other mammalian p53 proteins when compared to control cells.

[0113] Item 23. The method according to item 21 or 22, wherein the senescent cells are selected from tumor cells, metastatic tumor cells, cells of tumor micrometastasis, fibrotic cells, breast cancer cells such as triple-negative breast cancer cells, ovarian cancer, pancreatic cancer, gastric cancer, lung cancer, and / or liver cancer cells or metastatic cells thereof.

[0114] Item 24. A diagnostic kit comprising materials for carrying out the method according to any one of items 1 to 8 and 10 to 17 in one container or separate containers, optionally together with auxiliary materials and / or instructions for carrying out the method.

[0115] Item 25. Use of the kit according to item 24 in the method according to any one of items 1 to 8 and 10 to 17 for identifying an anti-aging compound, for monitoring an anti-aging treatment or prevention in a mammalian subject in need of the treatment or prevention, and / or for predicting or prognosticating the success, progress, and / or susceptibility of an anti-aging treatment or prevention in a mammalian subject.

[0116] The present invention is further described in the following examples, but is not limited thereto, with reference to the accompanying figures and sequence listing. For purposes of the present invention, all references cited herein are incorporated by reference in their entirety. [Brief description of the drawings]

[0117] [Figure 1] FIG. 1 shows a schematic representation of the domain organization of the human p53 protein, with the N-terminally located TAD containing potentially phosphorylatable amino acids (S15, S20, S46, and T55) and the C-terminal regulatory domain containing the similarly phosphorylatable amino acid S392. [Diagram 2] FIG. 1 shows the results of a fluorescence polarization binding assay demonstrating the binding of recombinant FOXO4 forkhead domain to FITC-labeled peptides of p53 with either transcription activation domain 1 (p53TAD1) or transcription activation domain 2 (p53TAD2). [Diagram 3] 1A-C show the results of binding assays demonstrating that FOXO4 (A) and FOXO4-derived (e.g., mimetic) peptides (B) and (C) bind with significantly higher affinity to p53-TAD2 when phosphorylated at T55 or S46 (squares and triangles in A, top two graphs in B and C, respectively). [Figure 4] FIG. 1 shows that the FOXO4-derived peptide CL04183 can bind to full-length p53 from cells when wild-type or phosphorylated at T55 or S46, but not when these sites cannot be phosphorylated. [Diagram 5]Figure 1 shows immunocytochemistry images of PML bodies (marked with PML and SP100), FOXO4 and individual nuclei of phosphorylated p53 (DNA stained blue with Hoechst 33258). p53 phosphorylated at serine 46 or threonine 55 colocalizes with FOXO4 within or directly adjacent to PML bodies. Thus, PML bodies are informative as they are a proxy for the phosphorylated pools of p53 and FOXO4, as it is technically difficult to detect phospho-TP53 staining in tissues. [Figure 6] FIG. 1 shows immunohistochemistry images of biopsies from human breast cancers, with nuclei (DNA stained with Hoechst 33258) dark grey and PML light grey, both showing high levels of nuclear PML bodies in the cancer cells themselves (approximately one-third of patient biopsies in this cohort). [Figure 7] Figure 1 shows PML body counts plotted against sensitivity to the FOXO4-derived peptide CL04177 in human breast cancer cell lines categorized by molecular subtype. All cell lines tested with high levels of PML bodies are in the triple-negative subtype (indicating low levels of progesterone receptor, estrogen receptor, and human epidermal growth factor receptor 2) and are highly sensitive to the compound CL04177, whereas breast cancer cell lines with low PML bodies are less sensitive. [Figure 8] Figure 1 shows that CL04183 and CL04177 specifically combat liver metastases. CL04183 primarily eliminates micrometastases. [Figure 9] FIG. 1 shows that CL04183 and 5-FU, and their combination, specifically combat liver metastases. [Figure 9-1] Same as above [Figure 10]Figure 1 shows: A) Correlation of open P53 (Pab240 clones) and P53 pSer392 (EP155Y clones) staining in proliferating and senescent hTERT-immortalized retinal pigment epithelial cell line RPE1 and human triple-negative breast cancer cell line MDA-MB-231. Spearman's rank correlation coefficient (0.93) is given; B) Correlation of open P53 (Pab240 clones) and P53 pSer392 (EP155Y clones) staining in a panel of eight human breast cancer cell lines including luminal, Her2 positive, and triple negative. Spearman's rank correlation coefficient (0.88) is given; and C) the relationship between PML body number / nuclear pixel (to correct for variability in nuclear size between cell lines) and nuclear P53 pSer392 staining intensity in eight human breast cancer cell lines plotted against the effective dose to induce cell death in 50% of the population (ED50, measured by MTS assay) following treatment with CL04177 for these cell lines. Sensitive cell lines are highlighted with a box. [Figure 11] Cancer cells that survive chemotherapy show increased PML bodies and phosphorylated p53. These cells are more sensitive to CL04183 treatment. A) Patient-derived colorectal cancer organoid line CRC29 was treated with 5-fluorouracil (5-FU) for 1 week. Immunofluorescence staining was then performed for the indicated proteins. [Figure 11-1] B) CRC29 organoids were treated with 5-FU or irinotecan in 96-well plates. Compound CL04183 was added to the cells in increasing concentrations after 3 days and cell viability was measured on day 8. [Figure 12] Figure 1. Colocalization dynamics of PML and phosphorylated p53 (S392) in the context of WT and mutant p53. A) TP53 was overexpressed in the p53 null cell line H1299, either wild type (WT) or the most common mutant form, R175H. B) Quantification shows that p53 is stably expressed in the phosphorylated form (S392). Furthermore, PML is enriched in cells overexpressing mutant p53. [Figure 12-1]C) Colocalization map showing three pools: PML and p53(S392) colocalized pixels in white, and PML foci separate from p53(S392) in red and green, respectively. D) The majority of cells contain both PML and S392 compared to cells containing only either PML or S392. [Figure 13] Figure 1: CL04177 peptide targets cells with increased PML bodies and phosphorylated p53. A) The triple-negative breast cancer cell line MDAMB231 was treated with the LD50 dose determined in a previous assay. Two days later, immunofluorescence staining was performed for the indicated proteins. B) Treatment survivors consist mainly of cells with low levels of these proteins. Taken together with the data presented above (Figure), this figure indicates that more potent and reliable antibodies against PML and p53(S392) act as proxies for open p53 and FOXO4, but also represent a pool of PML and p53(S392) sensitive cells. [Figure 14] FIG. 1 shows that the CL04177 peptide targets cells from multiple cancer types with high selectivity for cell lines genetically predisposed to express p53 in an "open" conformation due to TP53 mutations. Survival curves illustrating the response to the peptide of two cell lines, A) A2780, which has a wild-type p53 conformation, and B) H23, which contains p53 in an "open" conformation based on a genetic mutation in p53. C) Summary of ED50 for several cancer types showing that cancers predisposed to "open" p53 based on a genetic mutation are more sensitive to peptide CL04177. [Figure 15] Figure 1 shows that colon cancer cells express more phosphorylated p53 compared to wild type colon cells. A) Immunofluorescence staining of the indicated proteins was performed on patient-derived colorectal cancer organoid line CRC29 and wild type (WT) colon organoids. B) WT and CRC29 organoids were treated with increasing concentrations of compounds CL04177 or CL04183. Cell viability was measured after 5 days. [Figure 16]FIG. 1 shows that compounds identified according to the present invention, such as CL04183, bind more strongly to p53 phosphorylated at Ser46 / Thr55 than to unphosphorylated p53. A) Example of isothermal titration calorimetry using CL04183 and a recombinant form of the second transactivation domain of human p53 (TAD2). Based on this measurement, the binding affinity (Kd) was determined to be 5.5±1.3 μM. B) Summary of binding affinity of a similar measurement as in A), extended to conditions where TAD2 is phosphorylated at Ser46, Thr55, or both. Note that affinity is greatly increased when either is phosphorylated, and the effect is enhanced when TAD2 is doubly phosphorylated. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0118] Assay and curve fitting to generate LD50 To investigate the selectivity of the different treatments towards cancer cells, a colorimetric cytotoxicity assay was performed 2 days after treatment. Cells from several different cancer types (breast, ovarian, pancreatic, gastric, lung, and liver) were cultured at 4000 cells per well in 96-well plates. They were treated in triplicate with serial dilutions of compounds and also mock treated (FBS, since the peptides were dissolved in it). Cell viability was assessed using the AQueousOne Solution Cell Proliferation Assay kit according to the manufacturer's instructions. Briefly, old medium was removed and 100 μl of fresh medium and 10 μl of CellTiterAqueousOne solution were added to each well. Plates were incubated for 1 h at 37°C. The resulting colorimetric response was read at 490 nm in a microplate absorbance reader. Relative viability was determined by normalizing the results to mock (=100% viability). Drug response curves were generated using GraphPad 9.2.0 software by performing nonlinear regression (curve fit) with four parameters assuming a standard Hill equation (method chosen: inhibitor or agonist vs. response, respectively, limiting the top 100). The lethal dose 50 (LD50) was determined as the concentration of drug that results in a 50% reduction in viability in the MTS assay.

[0119] Immunocytochemical staining Cell lines were grown on 12 mm diameter glass coverslips in 24-well plates, which were gently washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA) in PBS (vol / vol) for 30 min at 4°C. Cells were then washed twice with pH 7.0 Tris-buffered saline (TBS), permeabilized with 0.1% Triton X-100 (vol / vol) in TBS for 2 min, and subsequently blocked with a solution of 2% weight / vol of serum appropriate for the secondary antibody (e.g., 2% horse serum and 2% goat serum) and 2% bovine serum albumin in pH 7.0 TBS (TBSB) for 30 min at room temperature. Coverslips were then placed on a 50 μl drop of primary antibody diluted in TBSB on parafilm and incubated overnight at 4°C. After washing three times with TBSB, the coverslips were incubated for 1 h at room temperature in the dark with secondary antibodies and 5 μg / ml Hoechst 33258. Then, the coverslips were washed with distilled water and mounted on glass slides.

[0120] In vivo anti-metastatic activity of FOXO4-modulating peptides in an orthotopic TNBC model (see also FIG. 8) PBS-treated mice developed metastatic growths visible by bioluminescence. Concomitantly, treatment of mice with CL04183 and CL04177 peptides reduced the incidence of visible metastases. Treatment of mice with these two peptides reduced the incidence of liver metastases, as evidenced by bioluminescence in tumor cells in excised livers.

[0121] This study was approved by the local University Animal Care and Use Committee of the University Medical Center Utrecht (IVD-UMC, WP numbers 9124-1-02 and 9124-1-05). MDA-MB-231 firefly luciferase-containing cells were transfected into NSG mice (NOD.Cg-Prkdc <scid>IL2rgmWij / Szj) (stock 005557) were orthotopically transplanted into the exposed inguinal mammary fat pad as single cells at a density of 1 million in 50 μl of medium. After surgery, carprofen was injected subcutaneously as an analgesic. Animals were purchased from Charles River and kept in a pathogen-free environment.

[0122] Established tumors (50 mm 3 We quantified the dynamics of tumor growth in mice bearing tumors with a volume of 100 mm 3 ~200 mm 3 Tumor-bearing mice were treated daily for 1 week with peptides (2.5 mg / kg for CL04183 and 10 mg / kg for CL04177) or PBS by subcutaneous injection. After 2 weeks of recovery, mice were sacrificed. For in vivo dosing, peptides were dissolved in PBS. We randomly assigned 11 mice per group to different treatment groups before the start of the experiment.

[0123] To visualize tumor burden in live mice, mice were injected intraperitoneally with luciferin substrate at 150 mg / kg and imaged 15 min later using a bioluminescence imaging device under isoflurane anesthesia. Using M3Vision software, BL signal was analyzed by photographing regions of interest (ROIs) around the primary tumor to assess bioluminescence signal from tumors, or ROIs around the upper abdomen and chest of the mice to assess signal from metastases. Signal was expressed as photons / sec / cm 2 was measured.

[0124] CL04183 and '5-fluorouracil reduce colorectal cancer metastasis in the liver (see also Figure 9) Colorectal cancer organoid line CRC29 was transplanted into the cecum of immunodeficient mice. Two weeks after transplantation, animals were treated with either '5-fluorouracil (5-FU) or PBS. One week after chemotherapy treatment, mice were treated with three doses of CL04183 or PBS. A) To detect human cancer cells in mouse organs, livers were stained with KU80 or human nucleoli, along with p-p53 (T55) or PML. B) The number of liver metastases per stained section was counted visually.

[0125] Mouse experiments were performed after approval from the Dutch Animal Ethics Committee (WP number 9124-1-03). NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ / J mice were purchased from Charles River and kept in a pathogen-free environment. Animals were anesthetized using an intraperitoneal injection of 75 mg / kg ketamine and 0.5 mg / kg dexmedetomidine and subcutaneously injected with 5 mg / kg carprofen as an analgesic. CRC29 organoids (250000 cells) containing firefly luciferase constructs were then implanted into the cecum as 10 μl collagen drops. Two weeks after tumor cell implantation, animals were treated with either 50 mg / kg '5-fluorouracil (5-FU) or PBS. One week after chemotherapy treatment, mice were treated with three doses of 2.5 mg / kg CL04183 or PBS. Four weeks after implantation, mice were sacrificed and tissues were collected for further processing.

[0126] cell culture Breast cancer cell lines (triple negative: BT549, BT20, Sum149T, MDA-MB-468, MDA-MB-231; Her2+: ZR7530, SKBR3, MDA-MB-361; luminal: MCF7, T47D), p53 null lung cancer cell line H1299, and hTERT-immortalized retinal pigment epithelial cell line RPE1 were cultured in Dulbecco's modified Eagle's medium containing 4.5 g / mL glucose, 4 mM glutamine, 10% fetal bovine serum, 100 units penicillin potassium / mL, and 100 units streptomycin sulfate / mL at 37 °C and 5% CO in a humidified incubator. 2 , and cultured at 5% oxygen.

[0127] immunohistochemistry Sections of paraffin-embedded formalin-fixed tissues were rehydrated in decreasing concentrations of xylene and ethanol, then washed in pH 7.0 Tris-buffered saline (TBS) and boiled for 20 min in 1 mM EDTA in TBS, pH 9.4 for antigen unmasking. After cooling the slides for 30 min, the tissues were permeabilized with 0.2% TX-100 in TBS for 5 min at room temperature. The sections were then washed in TBS and incubated for 1 h in blocking buffer containing 2% weight / volume of serum appropriate for the secondary antibody (e.g., donkey or goat) and 0.1% fish gelatin in 1% BSA. The sections were then surrounded with a water-repellent pen and incubated overnight at 4°C with primary antibodies diluted in TBS / 1% BSA. The next day, the tissues were washed three times with TBS and then incubated for 1 h with fluorescently labeled secondary antibodies (including nuclear staining with Hoechst 33258) diluted in blocking buffer. Slides were then washed twice with TBS, incubated for 20 min in Sudan Black solution to reduce background, and washed with demineralized water. Sections were then mounted in Vectashield and imaged using an LSM880 Zeiss confocal microscope.

[0128] Organoid culture Organoids were cultured in a humidified incubator at 37 °C and 5% CO 2 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 (50x; 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, organoids were passaged by resuspending in ice-cold medium, followed by centrifugation at 4°C in 15 ml tubes. The resulting pellet was trypsinized at 37°C for 5 min to obtain single cells, and then washed twice with advanced DMEM / F12 medium. These cells were then resuspended in Matrigel and seeded in 5 μl drops onto 96-well plates. After 15 minutes, 100 μl of fresh medium was added to the wells. Two days after seeding, peptide and chemotherapy treatments were added to the organoids.

[0129] MTS assay Six days after treatment, MTS assay was performed by incubating cells with 10 μl CellTiter 96™ AQueous One Solution Cell Proliferation Assay (Promega) at 37° C. for 1 hour, followed by measuring absorbance at 490 nm using a Spectramax M5e.

[0130] Immunofluorescence Organoids CRC29 organoids were fixed with 4% weight / volume paraformaldehyde [PFA] for 45 min at 4°C, followed by washing with 1% weight / volume BSA in TBS. Organoids were then permeabilized with 0.1% TX-100 in TBS for 5 min at room temperature, washed again, and incubated for 1 h at 4°C in blocking buffer containing 2% weight / volume serum appropriate for the secondary antibody (e.g., donkey or goat) and 0.1% fish gelatin in 1% BSA. After blocking, organoids were incubated overnight at 4°C with primary antibodies diluted in blocking buffer. The next day, organoids were washed twice for 1 h at 4°C, followed by incubation overnight at 4°C with fluorescently labeled secondary antibodies diluted in blocking buffer. Organoids were then washed two more times for 1 h at 4°C, with Hoechst 33342 added to the second wash. The stained organoids were then dissolved in glycerol-fructose clearing solution and imaged using an LSM880 Zeiss confocal microscope.

[0131] Protein expression and purification Expression constructs for a fragment of amino acids 86-207 (FOXO4 forkhead) of human FOXO4 (Uniprot ID P98177) were generated by synthesizing the corresponding optimized FOXO4 cDNA constructs (Genscript) and inserting these cDNAs into the pETM11-His6-Protein A-TEV cleavage site vector by NcoI / BamHI restriction digestion. For expression of recombinant unlabeled His6-Protein A tagged FOXO4 forkhead protein, the bacterial expression vectors were transformed into Escherichia coli BL21-DE3 Star strain and 1 L expression cultures were grown for 2 days in minimal medium supplemented with 6 g glucose and 3 g ammonium chloride (Sigma). Cells were diluted to OD (600 nm) 0.8 and induced with 0.5 mM IPTG, followed by protein expression for 16 h at 20 °C. Cell pellets were harvested and sonicated in non-denaturing lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 20 mM imidazole, 2 mM Tris(2-carboxyethyl)phosphine). Tagged recombinant proteins were then purified using Ni-NTA agarose (QIAGEN) and the His6-Protein A tag was cleaved by TEV protease treatment. A second affinity purification using Ni-NTA beads was then performed to separate untagged proteins. A final size-exclusion chromatography purification step was performed in the desired buffer on a gel filtration column (Superdex 75, GE Healthcare). Protein concentrations were estimated based on absorbance at 280 nm, assuming that ε at 280 nm was equal to the theoretical ε value.

[0132] Fluorescence anisotropy studies Data were acquired on a ClarioStar Plus plate reader. End point measurements were performed with 200 flashes per well. A 482 nm wavelength excitation filter and a 530 nm emission filter were used, with gain and focal height adjustments performed for each measurement. Fluorescence intensity, parallel fluorescence polarization, and perpendicular fluorescence polarization were recorded. Data analysis was performed using MARS version 3.4 (BMG), Microsoft Excel, and GraphPad Prism version 8.

[0133] For the measurements, 116 μl of a solution of the specific Cleara peptide (ranging from 1 μM to 100 μM) was prepared. Afterwards, 4 μl of FITC-labeled p53 peptide (stock concentration 15 μM, resulting in a final concentration of 500 nM) was added. Then, 35 μl was transferred to each well of a 384-well plate. Measurements were performed in triplicate. If necessary, this setup was extended with lower concentrations to cover strong interactions.

[0134] The anisotropy (r) is defined as the ratio of the polarization component to the total intensity (IT).

number

number

[0135] The anisotropy values ​​obtained for each concentration of CL peptide can be used to determine the binding affinity by applying the following fitting function:

number

[0136] Experiments with triple-negative breast cancer cell lines to examine "open" p53 (see also Figure 10) cell culture Breast cancer cell lines (triple negative: BT549, BT20, Sum149T, MDA-MB-468, MDA-MB-231; Her2+: ZR7530, SKBR3, MDA-MB-361; luminal: MCF7, T47D), hTERT-immortalized retinal pigment epithelial cell line RPE1, and normal fetal lung fibroblast cell line IMR90 were cultured in Dulbecco's modified Eagle's medium containing 4.5 g / mL glucose, 4 mM glutamine, 10% fetal bovine serum, 100 units penicillin potassium / mL, and 100 units streptomycin sulfate / mL at 37 °C and 5% CO in a humidified incubator. 2 , and 5% oxygen.

[0137] MTS assay Six days after treatment, MTS assay was performed by incubating cells with 10 μl CellTiter 96™ AQueous One Solution Cell Proliferation Assay (Promega) at 37° C. for 1 hour, followed by measuring absorbance at 490 nm using a Spectramax M5e.

[0138] immunocytochemistry Breast cancer and RPE1 cell lines were grown on 12 mm diameter 1.5H glass coverslips in 24-well plates, which were gently washed with phosphate-buffered saline (PBS) at room temperature and fixed with 4% paraformaldehyde (PFA) in PBS (vol / vol) for 30 min at 4°C. Cells were then washed twice with Tris-buffered saline (TBS), permeabilized with 0.1% TritonX-100 (vol / vol) in TBS for 2 min, and subsequently blocked with a solution of serum appropriate for the secondary antibody (e.g., 2% horse serum and 2% goat serum) and 2% bovine serum albumin at 2% weight / volume in pH 7.0 TBS (TBSB) for 30 min at room temperature. Coverslips were then placed on a 50 μl drop of primary antibody diluted in TBSB on parafilm and incubated overnight at 4°C. After washing three times with TBSB, the coverslips were incubated for 1 h at room temperature in the dark with secondary antibodies and 5 μg / ml Hoechst 33258. Then, the coverslips were washed with distilled water and mounted on slides.

[0139] antibody For open conformation P53, cells were stained with 10 μg / mL of pAb240 clone antibody raised against P53 (mouse monoclonal, supplied by Abcam, Cambridge, UK, catalogue no. ab26, RRID:AB_303198) and 260 ng / ml of clone EP155Y antibody raised against P53 pSer392 (rabbit monoclonal, supplied by Abcam, Cambridge, UK, catalogue no. ab33889, RRID:AB_776988).

[0140] statistics Association between p53 pSer392 and open P53 is given as Spearman's rank correlation coefficient calculated using the R package status (version 4.1.1, (R CoreTeam (2018) R: A Language and Environment for Statistical Computing. Vienna,Austria: R Foundation for Statistical Computing. Available at https: / / www.r-project.org / )) with p-value (two-sided alternative hypothesis) calculation based on the AS 89 algorithm.

[0141] The association between PML and phosphorylated p53(S392) is given as the Spearman's rank correlation coefficient calculated by performing simple linear regression using GraphPad 9.2.0 software.

[0142] Exemplary (CL) peptides used Preferred embodiments of peptides to be used according to the invention or for use according to the invention are as follows: CL04124: RKKASSKIEAAILDAFSQNWRFFKRPPRRRQRRKKRGAKIEAAILDAF SQNWRKRRRRQRRKKRG (SEQ ID NO: 1); CL04177: RKKASSKIEAEILDAFSQNWRRKRPPRRRQRRKKRG (SEQ ID NO: 2); and CL04183: AKIEAAILDAFSQNWRKRRRRQRRKKRG (SEQ ID NO:3); and a peptide comprising the following amino acid sequence: KIEAEILDAFSQNWRKR (SEQ ID NO: 4) (core sequence of CL04177 and CL04124); and KIEAAILDAFSQNWRKR (SEQ ID NO:5) (core sequence of CL04183); Here, the amino acids in the above amino acid sequences are preferably D-amino acid residues.

[0143] Isothermal titration calorimetry (ITC) Synthetic human p53 TAD2 (residues 37-57) and CL04183 were equilibrated in ITC buffer (20 mM Hepes, 50 mM NaCl, 0.04% NaN3, pH 7.0). ITC measurements were performed with 28 injections of 10 μl at 25 °C using a Malvern Microcal VP-ITC instrument. Integration of the peaks corresponding to each injection, subtraction of the contribution of protein dilution, and correction from the baseline were performed using Origin 7 SR4 (Origin Scientific Corporations) analysis software provided by the manufacturer. Curve fitting was performed using a standard one-site model to obtain the equilibrium binding constant (Ka) for complex formation.

[0144] Drawing translation Figure 1 P-rich DNA-binding domain Tetra dom Reg. dom Control domain Figure 3A FOXO4 ForkHead (FH) domain Figure 3B Binding to human p53-TAD2 Figure 3C Binding to human p53-TAD2 Figure 4 Pull-down Input Pos ctrl Positive control Figure 5A merge Figure 5B P53pThr55 foci P53pThr55 focus # foci / nucleus Number of foci / nucleus Pro Mock Pro mock Sen Mock Sen mock PML foci PML focus Figure 6 PML score distribution (cases) PML high PML 高 cell breast cancer breast cancer cancer cancer PML neg PML 陰性 PML high PML 高 Figure 7 PML bodies / nuclear pixels sensitive Luminal Figure 8 Bioluminescence intensity Liver metastases Group group Mice P-value P value Figure 9A Nucleoli Figure 9B '5-FU (2 weeks) CL04183 (1 week) Figure 9C Tumor luminescence (Fold change vs. baseline) Figure 10A open p53 intensity open p53 intensity TN Breast Cancer TN Breast Cancer Senescent aging Proliferating P53 pSer392 intensity P53 pSer392 intensity Figure 10B breast cancer panel open p53 intensity open p53 intensity cells cells P53 pSer392 intensity P53 pSer392 intensity Figure 10C Median PML bodies / nucleus pixel Median PML bodies / nucleus pixel Median P53 pSer392 intensity / nucleus sensitive Luminal Figure 11A Untreated Chemotherapy Figure 11B % Viability (AqueousOne) % Viability (AqueousOne) Mock 5-FU survivors 5-FU survivors Irinotecan survivors Figure 12A P53 Null Nucleus PML p53(S392) Nucleus PML p53(S392) Figure 12B PML foci / cell PML foci / cell H1299 null Controls Overexpression P53(S392) nuc int P53(S392) nuclear strength Figure 12C colocalized pixels: white colocalized pixels: white Example Figure 12D Nuclear p53(S392) foci Figure 12E Co-localized % positive cells %positive cells Figure 13A Vimentin Nuc Phalloidin Nuc Mock Dropout Figure 13B Mock Dropout mean signal / cell Figures 14A and B % Viability (AqueousOne) % Viability (AqueousOne) (μM) in Knock-out Serum Figure 14C Ovarian Pancreatic Lung Breast Cell lines P53 status PML status PML status Genetic status Figure 15A Nucleus Figure 15B % Viability (AqueousOne) % Viability (AqueousOne) WT colon WT colon Figure 16A μcal / sec μcal / sec kcal mol -1 of injectant kcal mol -1 Time (min) Molar Ratio Figure 16B ITC-based binding affinity in μM ITC-based binding affinity in μM Ligand< / scid>

Claims

1. 1. A method for identifying improved anti-aging compounds comprising: a) contacting at least one anti-aging candidate molecule with the transactivation domain (TAD) of a mammalian p53 protein; b) detecting specific binding of the candidate molecule to the TAD in the presence of at least one phosphorylated amino acid in the TAD; c) comparing said specific binding to binding in the absence of said at least one phosphorylated amino acid in said TAD; and identifying an improved anti-aging compound by increased binding in the presence of said at least one phosphorylated amino acid in said TAD.

2. 2. The method of claim 1, wherein the contacting comprises the TAD of a mammalian p53 protein or the anti-aging candidate molecule in vivo or in vitro, in solution, or bound or conjugated to a solid support.

3. 3. The method of claim 1 or 2, wherein the candidate anti-aging molecule is selected from a chemical molecule, a molecule selected from a library of small organic molecules, a molecule selected from a combinatorial library, a cell extract, in particular a plant cell extract, a small molecule drug, a protein, a protein fragment, a molecule selected from a peptide library, an antibody or a fragment thereof, in particular a retro-inverso peptide derived from a mammalian FOXO4 protein, a fusion peptide comprising a retro-inverso peptide derived from a mammalian FOXO4 protein and derivatives thereof, preferably a peptide selected from CL04183, CL04124 and CL04177 or a core sequence thereof.

4. 4. The method of any one of claims 1 to 3, wherein detecting the binding comprises detecting phosphorylation involving radiolabeled 32P-orthophosphate, phospho-specific antibodies, and / or mass spectrometry.

5. 5. The method according to any one of claims 1 to 4, wherein the TAD of said mammalian p53 protein is optionally selected from TAD1 and / or TAD2 of human, mouse, rat, monkey, sheep, goat, hamster, dog and cat p53 proteins as a part of full-length p53 protein, in particular a recombinant p53 protein, a full-length p53 protein comprising phosphorylation at Ser392 of human p53 or at an analogous position of other mammalian p53 proteins, a recombinant fusion protein comprising TAD1 and / or TAD2, or a phosphorylated fragment of said TAD1 or TAD2, and mutated variants thereof.

6. 6. The method of any one of claims 1 to 5, wherein the TAD1 phosphorylation is located at Ser15 and / or Ser20 of human p53 and / or the TAD2 phosphorylation is located at Ser46 and / or Thr55 of human p53, or at analogous positions in other mammalian p53TADs.

7. 7. The method of any one of claims 1 to 6, wherein detecting said binding further comprises, if possible, detecting phosphorylation of Ser392 of human p53 or a similar position in other mammalian p53 proteins, and comparing said specific binding with the absence of said phosphorylated amino acid.

8. An anti-aging compound identified according to the method of any one of claims 1 to 7, or a pharmaceutical composition comprising said anti-aging compound together with a pharma- ceutically acceptable carrier.

9. 1. A method for monitoring anti-aging treatment or prevention in a mammalian subject, preferably a human, in need thereof, comprising: a) providing an anti-aging treatment or prevention to said subject, comprising administering to said subject an anti-aging compound that specifically binds to a TAD of p53 protein or a pharmaceutical composition comprising said anti-aging compound, preferably a compound or pharmaceutical composition according to claim 8; b) detecting the amount of phosphorylation of amino acids in the TAD of the p53 protein in a biological sample obtained from the subject, and / or detecting the amount of promyelocytic leukemia protein (PML) bodies in a biological sample obtained from the subject, and / or detecting the co-localization of promyelocytic leukemia protein (PML) bodies and phosphorylated p53 protein in a biological sample obtained from the subject; c) comparing the amount(s) and / or co-localization detected in step b) with the amount and / or co-localization in previous and / or control samples taken from said subject; A method comprising:

10. A method for predicting or prognosing the success, progression and / or susceptibility of an anti-aging treatment or prevention in a mammalian subject, preferably a human, comprising carrying out the method according to claim 9, wherein an increase in the amount of phosphorylated and / or PML protein body and / or co-localization of phosphorylated p53 with PML is indicative of the success, progression and / or susceptibility of said anti-aging treatment or prevention in said mammalian subject.

11. 11. The method of claim 9 or 10, wherein the anti-aging treatment or prevention in the mammalian subject comprises administering an effective amount of a retro-inverso peptide derived from a mammalian FOXO4 protein, a fusion peptide comprising a retro-inverso peptide derived from a mammalian FOXO4 protein and derivatives thereof, preferably a peptide selected from CL04183, CL04124, and CL04177, and a TAD2 binding derivative or a fragment thereof, such as a core sequence thereof.

12. The method according to any one of claims 9 to 11, wherein the TAD2 phosphorylation is located at Ser46 and / or Thr55 of human p53 or at analogous positions in other mammalian p53TADs.

13. 13. The method according to any one of claims 9 to 12, further comprising detecting the amount of phosphorylation of Ser392 of human p53 or a similar position in other mammalian p53 proteins and comparing said amount with the amount in a previous sample and / or in a control sample taken from said subject, wherein an increase in said phosphorylation is a further indication of the success and / or progress of said anti-ageing treatment or prevention in said mammalian subject.

14. 10. An anti-aging compound that specifically binds to a TAD of p53 protein or a pharmaceutical composition comprising said anti-aging compound, preferably as claimed in claim 8, for use in the prevention or treatment of senescent cells, wherein said senescent cells show an increased amount of phosphorylation of Ser46 and / or Thr55 of human p53, or analogous positions in other mammalian p53 TADs, and / or an increased amount of PML protein body, when compared to control cells, and preferably said senescent cells further show an increased amount of phosphorylation of Ser392 of human p53, or analogous positions in other mammalian p53 proteins, when compared to control cells.

15. 15. The anti-aging compound or pharmaceutical composition for use according to claim 14, wherein the senescent cells are selected from tumor cells, metastatic tumor cells, cells of tumor micro- and / or macrometastases, fibrotic cells, breast cancer cells such as triple-negative breast cancer cells, ovarian cancer, gastric cancer, pancreatic cancer, lung cancer and / or liver cancer cells or metastatic cells thereof.

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