{"id":27,"slug":"27-foxo4-dri-n-terminal-palmitoylation-attach-a-c16-palmitoyl-fatty-acid-","title":"FOXO4-DRI N-terminal lipidation: palmitoyl-Lys conjugate for extended plasma half-life","status":"DISCARDED","fold_verdict":"DISCARDED","discard_reason":null,"peptide":{"name":"FOXO4-DRI","class":"LONGEVITY","sequence":"LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRPPPRRRQRRKKRG","modified_sequence":"Pal-K-LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRPPPRRRQRRKKRG","modification_description":"N-terminal palmitoylation: attach a C16 palmitoyl fatty acid via an amide bond to the alpha-amino group of Leu-1, optionally through a single Lys spacer (Pal-Lys-LTLRK...), to drive non-covalent albumin binding"},"target":{"protein":"Cellular tumor antigen p53","uniprot_id":"P04637","chembl_id":"CHEMBL4096","gene_symbol":"TP53"},"rationale":{"hypothesis":"We hypothesize that N-terminal palmitoylation of FOXO4-DRI (via a Lys spacer, mirroring the liraglutide/semaglutide design) will dramatically extend the peptide's plasma half-life by enabling reversible binding to serum albumin, without compromising the disordered cationic CPP tail or the FOXO4 CR3-mimetic helix. Because FOXO4-DRI is a senolytic dosed cyclically and currently suffers from rapid renal clearance of its highly cationic structure, an albumin-anchoring fatty acid should convert it from a flash-exposure agent into a sustained-exposure one.","rationale":"Palmitoyl-Lys conjugation at the N-terminus is the validated GLP-1 strategy (liraglutide, semaglutide) for converting short cationic/amphipathic peptides into albumin-bound depot agents with multi-hour to multi-day half-lives. The FOXO4 CR3-mimetic helix is C-terminal-distal to Leu-1, so an N-terminal lipid anchor is unlikely to sterically block p53 engagement, and the C-terminal poly-Arg CPP tail (which Fold #12 confirmed must be preserved intact) remains untouched. This choice diverges from the last 3 folds (Cyclization on Epitalon, Lipidation on MOTS-c targeting DELIVERY, and Non-canonical AA on Semax targeting AFFINITY) by pairing Lipidation with a different focus — PHARMACOKINETICS via albumin binding rather than membrane partitioning — on a peptide where lipidation has not been attempted.","predicted_outcome":"Structure prediction should show the FOXO4 CR3 helix (residues 1-23) folding similarly to the wild-type DRI peptide with comparable pLDDT in the helical core (>0.65), the palmitoyl-Lys group modeled as a flexible N-terminal extension, and the C-terminal CPP tail remaining disordered as expected. No new aberrant intramolecular contacts between the lipid and the helix should appear, supporting the design as conformationally non-disruptive.","mechanism_class":null,"biohacker_use":null},"confidence":{"plddt":0.5844687819480896,"ptm":0.3109462261199951,"iptm":0.12843453884124756,"chai_agreement":null,"chai1_gated_decision":"RAN_BORDERLINE","binding_probability":null,"binding_pic50":null,"predicted_binding_change":null},"profile":{"aggregation_propensity":0.129,"stability_score":0.385,"bbb_penetration_score":0.0,"half_life_estimate":"long (>6 hours, depends on modifications)"},"narrative":{"tldr":"DISTILLATION №27 tests N-terminal palmitoylation of FOXO4-DRI — a senolytic peptide targeting the FOXO4–p53 protein–protein interaction — with the goal of extending plasma half-life via albumin anchoring, mirroring the liraglutide/semaglutide pharmacokinetic strategy. Structural prediction returned a global pLDDT of 0.58 and a critically low ipTM of 0.13, insufficient to draw reliable conclusions about whether the palmitoyl-Lys extension disrupts or tolerates the CR3-mimetic helix. The fold is discarded on structural grounds, but the pharmacokinetic hypothesis itself remains scientifically interesting and is undermined more by fundamental mechanistic incompatibilities — albumin binding vs. CPP-driven membrane penetration — than by the prediction failure alone. Literature analysis reveals that the CPP tail directly contacts p53TAD2 (2025 NMR data), adding a second reason to question whether albumin sequestration of the palmitoyl anchor would impair both cell entry and target engagement simultaneously.","detailed_analysis":"FOXO4-DRI is an 18-residue D-retro-inverso senolytic peptide designed to mimic the CR3 domain of FOXO4, displacing FOXO4 from its interaction with the p53 transactivation domain (p53TAD2) and thereby restoring p53-mediated apoptosis in senescent cells. The peptide is notable for its dual-domain architecture: an N-terminal CR3-mimetic helical region that engages p53 directly, and a C-terminal polycationic cell-penetrating peptide (CPP) tail (the RPPPRRRQRRKKRG stretch) that drives membrane penetration and nuclear translocation. Fold #12 in this lab previously established that truncating this CPP tail collapses structural prediction confidence to pLDDT 0.56, reinforcing that the tail is structurally and functionally indispensable. The current fold attempts a conceptually opposite intervention: leaving the CPP intact while adding a palmitoyl-Lys unit at the opposite, N-terminal end to drive reversible albumin binding and extend plasma exposure.\n\nThe pharmacokinetic rationale is straightforward and well-validated in incretin pharmacology. Liraglutide (C16 palmitoyl) and semaglutide (C18 with a short PEG-γGlu linker) both achieve multi-hour to multi-day half-lives by binding serum albumin non-covalently, shielding the peptide from renal filtration and proteolytic degradation. FOXO4-DRI, with its highly cationic character and relatively small size, is plausibly subject to rapid renal clearance, and all published in vivo studies use cyclic intraperitoneal dosing every few days — an implicit acknowledgment of limited persistence. Extending exposure through albumin anchoring is therefore a scientifically motivated hypothesis, even if the specific pharmacokinetic liabilities of FOXO4-DRI have never been formally characterized in the literature.\n\nThe structural prediction, however, provides a weak foundation for this design. The predicted pLDDT of 0.58 reflects the known difficulty of predicting disordered, cationic, all-D-amino-acid peptides with AlphaFold-class models trained predominantly on globular L-amino acid proteins. The ipTM of 0.13 is particularly uninformative — near the floor for a meaningful peptide–protein complex — and cannot distinguish whether the palmitoyl-Lys extension is truly non-disruptive to helix formation or whether the model simply lacks the parameterization to resolve it. No Chai-1 cross-validation was available to corroborate or challenge the ESMFold/AF output, and the Boltz-2 affinity module returned no values. The heuristic stability score of 0.385 is modest, and the long half-life estimate in the heuristic profile reflects the palmitoyl modification's expected albumin-anchoring contribution — but this is a sequence-based heuristic, not a real pharmacokinetic measurement.\n\nThe 2025 NMR study (PMID:40593617) by Bourgeois et al. introduces a critical complication that was not anticipated at the time of design: the cationic CPP tail does not merely deliver FOXO4-DRI to the nucleus — it directly contacts p53TAD2 as part of the binding interface. This means the CPP tail is bifunctional, serving both as a cell-penetration appendage and as a target-engagement element. This finding has direct implications for the palmitoylation strategy: albumin is a 67 kDa protein that binds fatty acids within a hydrophobic cleft, and the geometry of albumin–palmitoyl engagement would orient the attached FOXO4-DRI in a manner that could sterically occlude or conformationally constrain the CPP tail's ability to simultaneously contact p53TAD2 in free solution. The palmitoyl group is at the opposite (N-terminal) end from the CPP tail, but the peptide is only 18 residues before the Pal-Lys spacer — not long enough to guarantee that albumin binding at one end leaves the other end fully accessible.\n\nA second mechanistic incompatibility concerns cell entry. FOXO4-DRI penetrates cells via its CPP-driven direct membrane translocation mechanism, a biophysical process that depends on the cationic tail's electrostatic interaction with anionic membrane phospholipids and its ability to disrupt lipid bilayer integrity transiently. Albumin-bound peptide complexes (~70 kDa) do not penetrate cell membranes by this mechanism; they rely instead on endosomal uptake, receptor-mediated transcytosis (notably via FcRn for albumin), or passive dissociation before membrane contact. Whether the palmitoyl–albumin Kd (approximately 1–10 µM for C16) is sufficiently weak to allow efficient dissociation and free peptide release in relevant tissue compartments is unknown. If dissociation is rate-limiting, the sustained plasma exposure benefit could be offset by reduced intracellular bioavailability at the nuclear target.\n\nCompared to Fold #25 (MOTS-c N-terminal myristoylation, PROMISING, pLDDT 0.63), which targeted membrane association for a cytoplasmic-acting peptide, the present fold faces a more complex challenge: FOXO4-DRI must not only cross the membrane but reach the nucleus, and its mechanism of nuclear access is entirely CPP-dependent. MOTS-c's myristoylation improved membrane partitioning for a peptide whose target is intracellular but not nuclear — a qualitatively different barrier. This distinction explains in part why the lipidation-for-pharmacokinetics hypothesis is less straightforward for FOXO4-DRI than it might appear from the incretin analogy.\n\nThe safety dimension also warrants mention. PMID:36515093 documented that FOXO4-DRI worsened pulmonary hypertension in animal models by eliminating senescent cells that were performing a protective role in that vascular context. A sustained-exposure formulation would, by design, extend the window of systemic senolytic activity — potentially amplifying this context-dependent harm in patients with pulmonary or vascular disease. This is not a prediction failure; it is a biological risk that the palmitoylation strategy would need to address through either tissue-targeted delivery or carefully controlled release kinetics. In its current form, Pal-Lys-FOXO4-DRI does not include a targeting element, making the sustained-exposure concern a genuine translational liability.\n\nIn summary, DISTILLATION №27 is discarded because the structural predictors could not generate usable affinity or interface data at the confidence thresholds required, and because the biological rationale — while internally coherent — carries two compounding mechanistic risks that the literature now flags more clearly than when the hypothesis was formed: albumin sequestration may impair CPP-mediated cell entry, and prolonged systemic senolysis may be harmful in specific disease contexts. The pharmacokinetic goal of extended half-life remains valid and worth pursuing, but through a mechanistically compatible strategy — such as a cleavable linker design, PEGylation at a non-CPP site, or nanoparticle encapsulation — rather than direct palmitoylation.","executive_summary":"Palmitoyl-Lys FOXO4-DRI: pLDDT 0.58, ipTM 0.13 — too low to assess the modification's structural impact. Beyond the prediction failure, 2025 NMR data reveal the CPP tail directly contacts p53TAD2, meaning albumin-complex formation may simultaneously impair cell entry and target engagement.","tweet_draft":"DISTILLATION №27 — discarded.\nFOXO4-DRI, N-terminal palmitoyl-Lys conjugate.\npLDDT 0.58 · ipTM 0.13 — below threshold.\nAlbumin-anchoring + CPP nuclear translocation: mechanistically incompatible.\nThe PK hypothesis survives. The design doesn't.\nIn silico only. alembic.bio","research_brief_markdown":"# DISTILLATION №27 — DISCARDED\n## FOXO4-DRI · N-terminal Palmitoyl-Lys Conjugate · Pharmacokinetic Extension via Albumin Anchoring\n\n---\n\n## Mechanism of Action (Background)\n\nFOXO4-DRI is a D-retro-inverso senolytic peptide that mimics the CR3 domain of the transcription factor FOXO4. In senescent cells, FOXO4 forms a complex with p53 that sequesters p53 in the nucleus and suppresses p53-driven apoptosis — a key mechanism by which senescent cells resist clearance. FOXO4-DRI competes with endogenous FOXO4 for binding to the p53 transactivation domain 2 (p53TAD2), displacing FOXO4 and restoring p53-mediated apoptotic signaling. The downstream axis — nuclear exclusion of p53, BCL-2 downregulation, Caspase-3 activation — has been documented in senescent Leydig cells, endothelial cells, chondrocytes, keloid fibroblasts, and cancer-associated fibroblasts across multiple independent studies.\n\nA critical structural insight from a 2025 NMR study (PMID:40593617, Bourgeois et al.) reframes the peptide's architecture: the C-terminal cationic CPP tail (RPPPRRRQRRKKRG) is not merely a cell-penetration appendage — it makes direct contact with p53TAD2 as part of the binding interface. The CPP tail is therefore bifunctional: it drives membrane translocation AND contributes to target engagement. This finding is central to evaluating any modification that might alter the CPP tail's conformation, charge, or accessibility. **Fold #12** in this lab confirmed the structural indispensability of this region: truncating the CPP tail collapsed pLDDT to 0.56 and yielded a DISCARDED verdict.\n\n---\n\n## Modification Hypothesis (What We Tested)\n\nThis fold attached a C16 palmitoyl fatty acid to the N-terminus of FOXO4-DRI via a Lys spacer (Pal-Lys-LTLRK...), mirroring the albumin-anchoring strategy used in liraglutide (C16 palmitoyl) and semaglutide (C18 with linker). The hypothesis:\n\n- FOXO4-DRI's highly cationic structure and small size likely drive rapid renal clearance, limiting tissue exposure after bolus dosing\n- N-terminal palmitoylation would enable reversible albumin binding (Kd ~1–10 µM for C16), converting the peptide from a flash-exposure agent to a sustained-exposure depot\n- Attaching the lipid at the N-terminus — maximally distal from the C-terminal CPP tail — would minimize steric disruption to either functional domain\n- The all-D-amino-acid backbone (proteolytic stability already ensured by the DRI design) would make albumin binding the primary PK extension mechanism\n\nThe design rationale was sound at the level of analogy: liraglutide/semaglutide demonstrate that N-terminal fatty acid conjugation can extend half-life from minutes to hours-to-days. The cyclic IP dosing used in all FOXO4-DRI in vivo studies implicitly acknowledges short persistence. Extended half-life would directly increase the time the peptide is available in senescent-cell-rich tissues where p53 phosphorylation (pS15) enhances binding affinity.\n\n---\n\n## Why the Prediction Was Uninformative (Technical Analysis)\n\n**Structural confidence metrics:**\n- pLDDT: **0.58** — below the threshold for reliable secondary structure assignment in a peptide of this complexity\n- ipTM: **0.13** — near the floor of meaningful interface scoring; essentially no confident peptide–protein docking information was generated\n- Chai-1 cross-validation: **not available** — single-model output cannot be treated as a reliable structural prediction\n- Boltz-2 affinity module: **no values returned** — no quantitative binding change predicted\n\nThe low confidence scores are not surprising given the target system. FOXO4-DRI is an all-D-amino-acid peptide — structure predictors trained on L-amino acid protein databases have fundamentally degraded performance on DRI peptides. The modified sequence also includes a non-standard palmitoyl-Lys N-terminal extension that is chemically outside the training distribution of most protein structure models. The disordered nature of the CPP tail further limits confidence globally, as unstructured polycationic regions consistently produce low pLDDT regardless of the helical core's actual folding.\n\n**What the model could and could not tell us:**\nThe prediction confirms that the CR3-mimetic helix is present as a short helical segment and that the palmitoyl-Lys extension appears as a flexible appendage without obvious clashes. However, at pLDDT 0.58 and ipTM 0.13, this is not a reliable structural prediction — it is a low-confidence sketch. Absence of visible aberrant lipid–helix contacts in the model does not constitute evidence of structural tolerance; the model lacks the resolution and the parameterization to detect subtle hydrophobic insertion or helix-destabilizing contacts in this sequence context.\n\nComparison with **Fold #25** (MOTS-c myristoylation, PROMISING, pLDDT 0.63) is instructive: a slightly higher pLDDT and a shorter, simpler target peptide allowed that fold to clear the PROMISING threshold. FOXO4-DRI's greater length, disordered CPP tail, and all-D backbone make it a systematically harder prediction target, and the ipTM gap between the two (0.13 here vs. not reported but inferred higher for Fold #25) reflects this.\n\n---\n\n## What This Tells Us (Negative Results Are Data)\n\n**1. The structure predictor limitation is specific, not general.** The prediction failure here is not evidence that palmitoylation disrupts FOXO4-DRI's structure — it is evidence that current in silico tools cannot reliably model all-D, long, polycationic peptides with non-canonical N-terminal modifications. This is an important distinction: the fold is discarded for informational failure, not biological failure.\n\n**2. The pharmacokinetic hypothesis has a more fundamental problem than the prediction failure.** Literature analysis surfaces two compounding mechanistic incompatibilities that the structural prediction cannot resolve but which should govern future design decisions:\n\n- *Albumin anchoring may impair CPP-mediated cell entry.* Albumin-bound peptide complexes (~70 kDa) cannot cross cell membranes via the direct electrostatic/CPP mechanism that FOXO4-DRI relies on for nuclear translocation. If the palmitoyl–albumin Kd is not sufficiently weak to allow rapid dissociation at the cell surface, the sustained plasma half-life benefit would come at the cost of reduced intracellular bioavailability.\n\n- *Albumin orientation may sterically restrict the CPP tail's target engagement.* The 2025 NMR finding (PMID:40593617) that the CPP tail directly contacts p53TAD2 means that albumin binding at the N-terminal palmitoyl anchor could geometrically restrict the CPP tail's freedom to engage p53 in the free-peptide state post-dissociation.\n\n**3. Context-dependent safety risk for sustained senolysis.** PMID:36515093 documents that FOXO4-DRI worsened pulmonary hypertension by eliminating senescent cells performing a vascular-protective role. A sustained-exposure formulation amplifies this risk by extending the senolytic window beyond the intended dosing interval. This is not a prediction-derived caveat — it is a literature-derived contraindication for the sustained-exposure design strategy in vascular disease contexts.\n\n**4. The PK premise is unverified.** No published data characterize FOXO4-DRI's actual plasma half-life, renal clearance rate, or dominant elimination pathway. The assumption that rapid renal clearance is the primary PK liability — rather than hepatic clearance, tissue sequestration, or receptor-mediated endocytosis — is biologically plausible but unsubstantiated. Palmitoylation is the right solution only if renal filtration is the rate-limiting clearance mechanism.\n\n---\n\n## Alternative Hypotheses to Test (Avoiding the Failure Mode)\n\nThe pharmacokinetic extension goal remains valid. The following strategies avoid the specific failure modes identified here:\n\n**1. PEGylation at a non-CPP, non-helix site via Lys spacer in the linker region.**\nThe GGK linker between the CR3 helix and CPP tail (around residues 28–30) provides a candidate attachment point. PEG20–40k extends half-life without driving albumin-complex formation, preserving CPP-driven membrane translocation while reducing renal filtration. This is structurally testable with higher confidence than palmitoylation.\n\n**2. Cleavable albumin-binding linker (pH- or protease-sensitive release).**\nA palmitoyl group attached via a protease-cleavable dipeptide (e.g., Val-Cit, cleaved by cathepsin B in endosomal/lysosomal compartments or at tumor/senescent-tissue sites) would provide the sustained plasma PK benefit while releasing free FOXO4-DRI intracellularly where it can engage the CPP-driven nuclear pathway. This design has precedent in ADC (antibody-drug conjugate) chemistry.\n\n**3. Nanoparticle encapsulation without covalent modification.**\nLipid nanoparticles or PLGA microparticles loaded with unmodified FOXO4-DRI would provide controlled release and extended local exposure without any risk of CPP-tail interference. This preserves the native peptide sequence entirely and avoids all the structural concerns raised here.\n\n**4. Half-life extension via D-amino acid stapling or macrolactamization at a non-functional site.**\nIf the pharmacokinetic goal is secondary to improving stability and structural rigidity, hydrocarbon stapling or macrolactamization within the helical CR3 region (tested with D-amino acid stapling chemistry compatible with the all-D backbone) could increase proteolytic half-life and receptor residence time without driving albumin sequestration. The Fold #22 approach on Humanin (disulfide bridge, PROMISING) provides a conceptual precedent in this lab.\n\n**5. Direct pharmacokinetic characterization before further modification.**\nBefore engineering solutions, measure FOXO4-DRI's actual plasma half-life, renal clearance fraction, and tissue distribution in a rodent model. If half-life is already >2 hours (as the all-D backbone and highly cationic character might produce via tissue sequestration), the PK problem may be smaller than assumed, and the modification priority should shift to affinity or selectivity.\n\n---\n\n*In silico prediction only. All structural and property data are computational estimates. This report does not constitute medical advice. Requires wet-lab validation before any biological conclusions can be drawn.*","structural_caption":"The predicted structure of palmitoyl-Lys-FOXO4-DRI shows weak global fold confidence (pLDDT 0.58) and very poor interface scoring (ipTM 0.13), consistent with a largely disordered peptide containing a short helical segment and a flexible cationic tail. The palmitoyl-Lys N-terminal extension is modeled as a flexible appendage, but its precise placement relative to the CR3-mimetic helix is unreliable at this confidence level. No clear aberrant intramolecular lipid–helix contacts are evident, but absence of evidence is not evidence of absence given the metrics. Without Chai-1 cross-validation or affinity output, structural conclusions about disruption vs. tolerance of the modification cannot be drawn.","key_findings_summary":"FOXO4-DRI is a D-retro-inverso peptide senolytic whose mechanism of action centers on disrupting the FOXO4–p53 interaction, thereby restoring p53-mediated apoptosis in senescent cells. The most mechanistically definitive paper in this set (PMID:40593617, 2025 NMR study by Bourgeois et al.) reveals that FOXO4-DRI binds the disordered p53 transactivation domain 2 (p53TAD2) and forms a transiently folded complex in which both the FOXO4-derived CR3-mimetic helix AND the cationic cell-penetrating peptide (CPP) tail contribute to p53 binding. This is a critical finding for our hypothesis: it means the CPP tail is not merely a delivery appendage—it participates directly in the target-binding interface. Any modification that disrupts the CPP tail's conformation or charge distribution could therefore reduce on-target efficacy, not just cell penetration. The same study also shows that p53 phosphorylation (e.g., pS15) enhances affinity for both FOXO4 and FOXO4-DRI, a finding corroborated by PMID:39994346, which documents elevated p53-pS15 in keloid fibroblasts and shows FOXO4-DRI promotes nuclear exclusion of this phosphoform.\n\nThe therapeutic breadth of FOXO4-DRI is well established across multiple cell types and disease contexts: senescent Leydig cells (testosterone insufficiency, male fertility—PMIDs 31959736 and 39025385), endothelial cells (vascular aging—PMID:41625068), cancer-associated fibroblasts (NSCLC radiosensitization—PMID:34877934), keloid fibroblasts (PMID:39994346), and expanded chondrocytes (ACI preparation—PMID:33996787). In all cases the p53/BCL-2/Caspase-3 axis is activated downstream of FOXO4–p53 disruption. Importantly, PMID:36515093 provides a cautionary finding: eliminating senescent cells with FOXO4-DRI in pulmonary hypertension models worsened rather than improved disease, indicating context-dependent risks and suggesting that systemic, sustained exposure (as our palmitoylation modification would create) could be detrimental in vasoconstrictive or proliferative pulmonary settings.\n\nRegarding the pharmacokinetic rationale for palmitoylation, none of the retrieved papers directly study FOXO4-DRI's pharmacokinetics, plasma half-life, renal clearance, or albumin binding. The fatty-acid/albumin-anchoring strategy is well-validated for GLP-1 analogs (liraglutide C16, semaglutide C18 with linker), but this literature does not include those pharmacology papers. The preprint (DOI:10.21203/rs.3.rs-2361374/v1) identifies hotspot residues within FOXO4-DRI using shorter peptide variants and in silico modeling, finding that strong p53-binding activity can be retained in truncated sequences. This supports the idea that modest N-terminal modifications may be tolerable if the CR3 helix and CPP functional regions are preserved, but the study does not test lipidation specifically.\n\nThe structural insight from PMID:40593617 is the most consequential for our hypothesis. Because the CPP tail (the cationic C-terminal region of FOXO4-DRI) directly contacts p53TAD2, converting the free N-terminus via palmitoylation-Lys spacer attachment introduces a large hydrophobic group at the opposite end of the peptide from the CPP, which may be tolerable. However, albumin-bound FOXO4-DRI would circulate as a large complex (~67 kDa albumin + peptide) that may have reduced membrane permeability—the very property that enables the CPP to drive nuclear entry. Sustained plasma exposure via albumin anchoring would only be beneficial if the peptide can still dissociate from albumin, penetrate cells, and reach the nucleus at sufficient concentrations. The reversibility of palmitoyl-albumin binding (Kd ~1–10 µM for C16) may be adequate, but this is unexamined in this literature."},"structured":{"known_activity":null,"known_binders":null,"candidate_variants":null,"domain_annotations":null,"literature_context":{"pubmed":[{"pmid":"39994346","title":"FOXO4-DRI induces keloid senescent fibroblast apoptosis by promoting nuclear exclusion of upregulated p53-serine 15 phosphorylation.","abstract":"Keloids are pathological scars exhibiting tumour-like aggressiveness and high recurrence rate. Here we find increased proportion of pro-inflammatory and mesenchymal fibroblast subpopulations and senescent fibroblasts, and enhanced expression of senescence-associated secretory phenotype genes using single-cell RNA sequencing analysis, as well as elevated p16 protein and more β-galactosidase-positive cells in keloids. The up-regulated p53-serine15 phosphorylation (p53-pS15) in keloids is identified by phosphospecific protein microarray and western blotting. We further demonstrate that a senolytic FOXO4-D-retro-inverso-isoform peptide (FOXO4-DRI) promotes apoptosis and decreases G0/G1 phase cells in pro-senescence models of keloid organ cultures and fibroblasts, accompanied with p53-pS15 nuclear exclusion. Our study indicates that upregulation of p53-pS15 and p16 maintains a persistent senescent microenvironment to promote cell cycle arrest and apoptosis resistance in keloid fibroblasts. FOXO4-DRI shows potential as a treatment targeting the senescence and apoptosis resistance, and holds promise as an approach to prevent the aggressiveness and relapse of keloids.","authors":["Kong Yu-Xiang","Li Zhi-Shuai","Liu Yuan-Bo","Pan Bo","Fu Xin","Xiao Ran","Yan Li"],"year":2025,"journal":"Communications biology"},{"pmid":"39025385","title":"FOXO4-DRI improves spermatogenesis in aged mice through reducing senescence-associated secretory phenotype secretion from Leydig cells.","abstract":"Male ageing is always accompanied by decreased fertility. The forkhead O (FOXO) transcription factor FOXO4 is reported to be highly expressed in senescent cells. Upon activation, it binds p53 in the nucleus, preventing senescent cell apoptosis and maintaining senescent cells in situ. Leydig cells play key roles in assisting spermatogenesis. Leydig cell senescence leads to deterioration of the microenvironment of the testes and impairs spermatogenesis. In this study, we observed that FOXO4-DRI, a specific FOXO4- p53 binding blocker, induced apoptosis in senescent Leydig cells, reduced the secretion of certain Senescence-Associated Secretory Phenotype and improved the proliferation of cocultured GC-1 SPG cells. In naturally aged mice, FOXO4-DRI-treated aged mice exhibited increased sperm quality and improved spermatogenesis.","authors":["Li Yanqing","Zhang Chi","Cheng Haicheng","Lv LinYan","Zhu Xinning","Ma Menghui","Xu Zhenhan","He Junxian","Xie Yun","Yang Xing","Liang Xiaoyan","Deng Chunhua","Liu Guihua"],"year":2024,"journal":"Experimental gerontology"},{"pmid":"31959736","title":"FOXO4-DRI alleviates age-related testosterone secretion insufficiency by targeting senescent Leydig cells in aged mice.","abstract":"Male late-onset hypogonadism is an age-related disease, the core mechanism of which is dysfunction of senescent Leydig cells. Recent studies have shown that elimination of senescent cells can restore proper homeostasis to aging tissue. In the present study, we found that the fork head box O (FOXO) transcription factor FOXO4 was specially expressed in human Leydig cells and that its translocation to the nucleus in the elderly was related to decreased testosterone synthesis. Using hydrogen peroxide-induced senescent TM3 Leydig cells as an in vitro model, we observed that FOXO4 maintains the viability of senescent Leydig cells and suppresses their apoptosis. By disrupting the FOXO4-p53 interaction, FOXO4-DRI, a specific FOXO4 blocker, selectively induced p53 nuclear exclusion and apoptosis in senescent Leydig cells. In naturally aged mice, FOXO4-DRI improved the testicular microenvironment and alleviated age-related testosterone secretion insufficiency. These findings reveal the therapeutic potential of FOXO4-DRI for the treatment of male late-onset hypogonadism.","authors":["Zhang Chi","Xie Yun","Chen Haicheng","Lv Linyan","Yao Jiahui","Zhang Min","Xia Kai","Feng Xin","Li Yanqing","Liang Xiaoyan","Sun Xiangzhou","Deng Chunhua","Liu Guihua"],"year":2020,"journal":"Aging"},{"pmid":"40593617","title":"The disordered p53 transactivation domain is the target of FOXO4 and the senolytic compound FOXO4-DRI.","abstract":"A central process contributing to the phenotype of aging is cellular senescence. We recently identified the FOXO4 - p53 axis as pivotal in maintaining the viability of senescent cells, and that senescent cells can be targeted selectively with the senolytic peptide FOXO4-DRI. Here, we solve the solution NMR structural models of the p53 transactivation domain in complex with the FOXO4 forkhead domain and in complex with FOXO4-DRI. Strikingly, we find that the disordered FOXO4-DRI binds to the disordered p53TAD2 and forms a transiently folded complex. In this complex, both, the FOXO4-derived region and the cationic cell permeability peptide contribute to the interaction. Furthermore, we show that p53 phosphorylation enhances the affinity for both FOXO4 and FOXO4-DRI. Summarizing we provide a detailed characterization of the interaction of p53 with FOXO4 and FOXO4-DRI which is the basis for development of p53 inhibitors to treat diseases linked to cellular senescence such as cancers.","authors":["Bourgeois Benjamin","Spreitzer Emil","Platero-Rochart Daniel","Paar Margret","Zhou Qishun","Usluer Sinem","de Keizer Peter L J","Burgering Boudewijn M T","Sánchez-Murcia Pedro A","Madl Tobias"],"year":2025,"journal":"Nature communications"},{"pmid":"41625068","title":"FOXO4-DRI regulates endothelial cell senescence via the P53 signaling pathway.","abstract":"OBJECTIVES: Endothelial cell dysfunction during aging is a key driver of vascular aging and related diseases; however, effective strategies to selectively eliminate senescent endothelial cells and restore vascular function remain lacking. FOXO4-DRI, a novel peptide-based intervention, specifically disrupts the interaction between FOXO4 and P53, thereby inducing apoptosis in senescent cells. This study innovatively focuses on the mechanism by which FOXO4-DRI induces apoptosis in senescent endothelial cells, demonstrating that it functions by activating the p53/BCL-2/Caspase-3 signaling pathway to promote selective apoptosis of these cells. FOXO4-DRI significantly improves vascular function and delays vascular aging. These findings not only enrich the molecular understanding of senescent cell clearance but also provide a novel strategy for precise targeting of endothelial cell senescence in therapeutic applications.\n\nMATERIALS AND METHODS: This study aims to analyze the vascular function and aging status of the aorta in naturally aged mice and progeroid model mice following FOXO4-DRI injection. Additionally, it investigates changes in endothelial cell function in senescent endothelial cells induced by oxygen-glucose deprivation (OGD), as well as the protein expression and interaction in the FOXO4-P53 signaling pathway. To assess the impact of FOXO4-DRI on endothelial cell senescence, the senescent endothelial cells were treated with FOXO4-DRI, followed by immunofluorescence and Western blotting experiments.\n\nRESULTS: Injection of FOXO4-DRI in both naturally aged and induced aging mice effectively suppressed aortic aging and improved aortic function. Additionally, we found that FOXO4-DRI alleviates endothelial cell senescence induced by OGD, thereby enhancing endothelial cell function. Through co-immunoprecipitation (CO-IP) experiments, we discovered that FOXO4-DRI prevents the binding of FOXO4 to P53, facilitating the phosphorylated P53 nuclear exclusion, which subsequently trigger BAX and cleaved caspase-3, leading to the apoptosis of senescent cells. Ultimately, this mechanism achieves the goal of inhibiting vascular aging.\n\nCONCLUSION: FOXO4-DRI promotes the nuclear export of phosphorylated P53 by inhibiting the binding of FOXO4 to P53 in endothelial cells, thereby facilitating the apoptosis of senescent endothelial cells and alleviating aging.","authors":["Hu Zhicheng","Li Fan","Hu Chunyi","Shan Qiongdan","Tang Zhouhao","Jiang Meifan","Yi Xiaojing","Chen Xixi","Jin Litai","Wang Xu","Wang Yang"],"year":2025,"journal":"Frontiers in bioengineering and biotechnology"},{"pmid":"36515093","title":"Eliminating Senescent Cells Can Promote Pulmonary Hypertension Development and Progression.","abstract":"BACKGROUND: Senescent cells (SCs) are involved in proliferative disorders, but their role in pulmonary hypertension remains undefined. We investigated SCs in patients with pulmonary arterial hypertension and the role of SCs in animal pulmonary hypertension models.\n\nMETHODS: We investigated senescence (p16, p21) and DNA damage (γ-H2AX, 53BP1) markers in patients with pulmonary arterial hypertension and murine models. We monitored p16 activation by luminescence imaging in p16-luciferase (p16LUC/+) knock-in mice. SC clearance was obtained by a suicide gene (p16 promoter-driven killer gene construct in p16-ATTAC mice), senolytic drugs (ABT263 and cell-permeable FOXO4-p53 interfering peptide [FOXO4-DRI]), and p16 inactivation in p16LUC/LUC mice. We investigated pulmonary hypertension in mice exposed to normoxia, chronic hypoxia, or hypoxia+Sugen, mice overexpressing the serotonin transporter (SM22-5-HTT+), and rats given monocrotaline.\n\nRESULTS: Patients with pulmonary arterial hypertension compared with controls exhibited high lung p16, p21, and γ-H2AX protein levels, with abundant vascular cells costained for p16, γ-H2AX, and 53BP1. Hypoxia increased thoracic bioluminescence in p16LUC/+ mice. In wild-type mice, hypoxia increased lung levels of senescence and DNA-damage markers, senescence-associated secretory phenotype components, and p16 staining of pulmonary endothelial cells (P-ECs, 30% of lung SCs in normoxia), and pulmonary artery smooth muscle cells. SC elimination by suicide gene or ABT263 increased the right ventricular systolic pressure and hypertrophy index, increased vessel remodeling (higher dividing proliferating cell nuclear antigen-stained vascular cell counts during both normoxia and hypoxia), and markedly decreased lung P-ECs. Pulmonary hemodynamic alterations and lung P-EC loss occurred in older p16LUC/LUC mice, wild-type mice exposed to Sugen or hypoxia+Sugen, and SM22-5-HTT+ mice given either ABT263 or FOXO4-DRI, compared with relevant controls. The severity of monocrotaline-induced pulmonary hypertension in rats was decreased slightly by ABT263 for 1 week but was aggravated at 3 weeks, with loss of P-ECs.\n\nCONCLUSIONS: Elimination of senescent P-ECs by senolytic interventions may worsen pulmonary hemodynamics. These results invite consideration of the potential impact on pulmonary vessels of strategies aimed at controlling cell senescence in various contexts.","authors":["Born Emmanuelle","Lipskaia Larissa","Breau Marielle","Houssaini Amal","Beaulieu Delphine","Marcos Elisabeth","Pierre Remi","Do Cruzeiro Marcio","Lefevre Marine","Derumeaux Genevieve","Bulavin Dmitry V","Delcroix Marion","Quarck Rozenn","Reen Virinder","Gil Jesus","Bernard David","Flaman Jean-Michel","Adnot Serge","Abid Shariq"],"year":2023,"journal":"Circulation"},{"pmid":"33996787","title":"Senolytic Peptide FOXO4-DRI Selectively Removes Senescent Cells From","abstract":"Autologous chondrocyte implantation (ACI) is a procedure used to treat articular cartilage injuries and prevent the onset of post-traumatic osteoarthritis. In vitro expansion of chondrocytes, a necessary step in ACI, results in the generation of senescent cells that adversely affect the quality and quantity of newly formed cartilage. Recently, a senolytic peptide, fork head box O transcription factor 4-D-Retro-Inverso (FOXO4-DRI), was reported to selectively kill the senescent fibroblasts. In this study, we hypothesized that FOXO4-DRI treatment could remove the senescent cells in the expanded chondrocytes, thus enhancing their potential in generating high-quality cartilage. To simulate the in vitro expansion for ACI, chondrocytes isolated from healthy donors were expanded to population doubling level (PDL) 9, representing chondrocytes ready for implantation. Cells at PDL3 were also used to serve as the minimally expanded control. Results showed that the treatment of FOXO4-DRI removed more than half of the cells in PDL9 but did not significantly affect the cell number of PDL3 chondrocytes. Compared to the untreated control, the senescence level in FOXO4-DRI treated PDL9 chondrocytes was significantly reduced. Based on the result from standard pellet culture, FOXO4-DRI pre-treatment did not enhance the chondrogenic potential of PDL9 chondrocytes. However, the cartilage tissue generated from FOXO4-DRI pretreated PDL9 cells displayed lower expression of senescence-relevant secretory factors than that from the untreated control group. Taken together, FOXO4-DRI is able to remove the senescent cells in PDL9 chondrocytes, but its utility in promoting cartilage formation from the in vitro expanded chondrocytes needs further investigation.","authors":["Huang Yuzhao","He Yuchen","Makarcyzk Meagan J","Lin Hang"],"year":2021,"journal":"Frontiers in bioengineering and biotechnology"},{"pmid":"34877934","title":"Targeting senescence-like fibroblasts radiosensitizes non-small cell lung cancer and reduces radiation-induced pulmonary fibrosis.","abstract":"Cancer cell radioresistance is the primary cause of the decreased curability of non-small cell lung cancer (NSCLC) observed in patients receiving definitive radiotherapy (RT). Following RT, a set of microenvironmental stress responses is triggered, including cell senescence. However, cell senescence is often ignored in designing effective strategies to resolve cancer cell radioresistance. Herein, we identify the senescence-like characteristics of cancer-associated fibroblasts (CAFs) after RT and clarify the formidable ability of senescence-like CAFs in promoting NSCLC cell proliferation and radioresistance through the JAK/STAT pathway. Specific induction of senescence-like CAF apoptosis using FOXO4-DRI, a FOXO4-p53-interfering peptide, resulted in remarkable effects on radiosensitizing NSCLC cells in vitro and in vivo. In addition, in this study, we also uncovered an obvious therapeutic effect of FOXO4-DRI on alleviating radiation-induced pulmonary fibrosis (RIPF) by targeting senescence-like fibroblasts in vivo. In conclusion, by targeting senescence, we offer a strategy that simultaneously decreases radioresistance of NSCLC and the incidence of RIPF.","authors":["Meng Jingshu","Li Yan","Wan Chao","Sun Yajie","Dai Xiaomeng","Huang Jing","Hu Yan","Gao Yanan","Wu Bian","Zhang Zhanjie","Jiang Ke","Xu Shuangbing","Lovell Jonathan F","Hu Yu","Wu Gang","Jin Honglin","Yang Kunyu"],"year":2021,"journal":"JCI insight"}],"biorxiv":[{"pmid":"","doi":"10.21203/rs.3.rs-2361374/v1","title":"Identification of Hotspots in Synthetic Peptide Inhibitors of the FOXO4:p53 Interaction","abstract":"<title>Abstract</title>  <p>Background:  Many stresses trigger cellular senescence, including telomere shortening, oncogene activation, DNA-replication stress, oxidative stress, and resistance to apoptosis. Forkhead box protein O4 (FOXO4) plays a pivotal role in cellular senescence, by binding to and inactivating p53, thereby leading to cellular senescence and inhibiting p53 mediated apoptosis. As a result, misregulation of FOXO4-p53 is linked to numerous diseases including cancers and aging-related diseases, pulmonary fibrosis and neurodegenerative diseases. Targeting of the FOXO4-p53 interface has been accomplished using a synthetic D retro inverse peptide (DRI), however recognition of the key molecular drivers of the interactions remains unknown. Understanding these interactions on a molecular level could lead to the development of small compounds, with enhanced pharmacological qualities, such as high stability, membrane permeability, and oral bioavailability, that mimics the activity of the DRI peptide. <h4>Methods and Results:</h4> Using an <italic>in silico</italic> model of the FOXO4-p53 interaction, we designed shorter peptides within the DRI to identify the key interactions. A biophysical assay demonstrates these shorter peptides retain strong binding affinity to p53, suggesting that the DRI's hot spot is located within these peptides. <h4>Conclusions:</h4> Taken together, this data provides information at the molecular level that is key to understanding the interplay of two proteins responsible for cellular senescence and provides the basis for the design of small molecules to inhibit this interaction.</p>","authors":["Zhang R","Gao K","Sadremomtaz A","Ruiz-Moreno AJ","Monti A","Al-Dahmani ZM","Gyau B","Doti N","Groves MR."],"year":2022,"journal":"PPR","source":"PPR","preprint":true}],"preprints":[{"pmid":"","doi":"10.21203/rs.3.rs-2361374/v1","title":"Identification of Hotspots in Synthetic Peptide Inhibitors of the FOXO4:p53 Interaction","abstract":"<title>Abstract</title>  <p>Background:  Many stresses trigger cellular senescence, including telomere shortening, oncogene activation, DNA-replication stress, oxidative stress, and resistance to apoptosis. Forkhead box protein O4 (FOXO4) plays a pivotal role in cellular senescence, by binding to and inactivating p53, thereby leading to cellular senescence and inhibiting p53 mediated apoptosis. As a result, misregulation of FOXO4-p53 is linked to numerous diseases including cancers and aging-related diseases, pulmonary fibrosis and neurodegenerative diseases. Targeting of the FOXO4-p53 interface has been accomplished using a synthetic D retro inverse peptide (DRI), however recognition of the key molecular drivers of the interactions remains unknown. Understanding these interactions on a molecular level could lead to the development of small compounds, with enhanced pharmacological qualities, such as high stability, membrane permeability, and oral bioavailability, that mimics the activity of the DRI peptide. <h4>Methods and Results:</h4> Using an <italic>in silico</italic> model of the FOXO4-p53 interaction, we designed shorter peptides within the DRI to identify the key interactions. A biophysical assay demonstrates these shorter peptides retain strong binding affinity to p53, suggesting that the DRI's hot spot is located within these peptides. <h4>Conclusions:</h4> Taken together, this data provides information at the molecular level that is key to understanding the interplay of two proteins responsible for cellular senescence and provides the basis for the design of small molecules to inhibit this interaction.</p>","authors":["Zhang R","Gao K","Sadremomtaz A","Ruiz-Moreno AJ","Monti A","Al-Dahmani ZM","Gyau B","Doti N","Groves MR."],"year":2022,"journal":"PPR","source":"PPR","preprint":true}],"consensus_view":"The literature consensus is strong that FOXO4-DRI exerts its senolytic effect by disrupting the FOXO4–p53 protein–protein interaction, restoring p53 nuclear exclusion and apoptosis in senescent cells. This mechanism is conserved across fibroblasts, Leydig cells, endothelial cells, chondrocytes, and cancer-associated fibroblasts. The 2025 NMR structural work has definitively established that the binding interface involves both the CR3-mimetic helix and the CPP tail of the peptide engaging the disordered p53TAD2. There is NO consensus literature on the pharmacokinetics of FOXO4-DRI itself, its plasma half-life, renal clearance rate, or the feasibility of albumin-anchoring via fatty acid conjugation for this peptide class. The albumin-anchoring strategy is an extrapolation from incretin pharmacology (liraglutide/semaglutide) and is not addressed in the retrieved literature. There is also an emerging cautionary note that non-selective or sustained senolysis can be harmful in specific disease contexts (pulmonary hypertension), which is directly relevant to a sustained-exposure formulation.","knowledge_gaps":"1) No published data exist on FOXO4-DRI pharmacokinetics: plasma half-life, volume of distribution, renal vs. hepatic clearance fractions, or the contribution of its cationic character to rapid renal filtration. 2) The effect of N-terminal modification (palmitoylation or any lipid conjugation) on FOXO4-DRI cell penetration, nuclear translocation efficiency, and p53-binding affinity has not been studied. 3) Whether albumin-bound FOXO4-DRI (as a large complex) retains sufficient membrane permeability to penetrate cells and reach the nucleus—a prerequisite for senolytic activity—is completely unknown. 4) The structural consequence of adding a Pal-Lys unit to the N-terminus on the peptide's secondary structure (especially any impact on the CR3 helix) has not been modeled or measured. 5) The therapeutic window for sustained vs. pulsatile FOXO4-DRI exposure in aging tissues is unstudied; it is unknown whether prolonged low-level senolysis (sustained exposure) is superior, equivalent, or inferior to cyclic high-peak dosing. 6) The in vivo distribution of FOXO4-DRI to specific senescent cell-rich tissues (liver, adipose, lymph nodes) and whether albumin routing would alter this is undetermined.","supporting_evidence":"1) The NMR study (PMID:40593617) confirms the CR3-mimetic helix is a primary binding determinant—placing the palmitoyl group at the N-terminus (opposite end from the CPP tail) could be the least disruptive attachment site, consistent with our design of Pal-Lys-Leu1. 2) The hotspot preprint (DOI:10.21203/rs.3.rs-2361374/v1) shows that truncated FOXO4-DRI variants retain p53 affinity, suggesting tolerance for some structural modification. 3) The highly cationic, all-D-amino-acid backbone of FOXO4-DRI (proteolytic stability already established by DRI design) is compatible with fatty acid conjugation strategies known to work for D-amino acid containing peptides. 4) The cyclic dosing paradigm used across all in vivo studies (e.g., IP injection every few days) implicitly acknowledges rapid clearance as a limitation, motivating the hypothesis. 5) p53 phosphorylation enhances FOXO4-DRI binding (PMID:40593617), meaning the peptide's efficacy is partly governed by how long it remains in the tissue microenvironment—a factor that extended half-life would directly improve.","challenging_evidence":"1) PMID:40593617 demonstrates that the cationic CPP tail directly contacts p53TAD2 as part of the binding interface, not merely as a cell-penetration appendage. Albumin binding by the palmitoyl group could sterically occlude or conformationally restrict the CPP tail's ability to engage p53, since albumin is a large (67 kDa) protein that engages fatty acids within a hydrophobic cleft that may orient the conjugated peptide in a manner incompatible with simultaneous CPP-p53 contact. 2) Albumin-anchored peptides form large ~70 kDa complexes that depend on endosomal or receptor-mediated transcytosis for cell entry, fundamentally different from the CPP-driven membrane translocation mechanism that FOXO4-DRI relies on. Impairing direct membrane penetration could negate the nuclear targeting entirely. 3) PMID:36515093 provides direct evidence that sustained or systemic senolysis with FOXO4-DRI worsened pulmonary hypertension in animal models, raising safety concerns specific to a sustained-exposure formulation that would prolong systemic senolytic activity beyond the intended senolysis window. 4) The Pal-Lys spacer design from liraglutide/semaglutide is validated for short, helical GLP-1 analogs; FOXO4-DRI is a much longer (18-mer), disordered, all-D peptide with a highly cationic tail—the albumin-binding affinity, orientation, and impact on the CPP-mediated internalization mechanism are not predictable by analogy alone. 5) No paper reports the actual renal clearance kinetics of FOXO4-DRI, so the premise that renal clearance is the dominant PK liability (vs. hepatic, proteolytic, or tissue-sequestration mechanisms) remains unsubstantiated in the retrieved literature."},"caveats":["in silico prediction only — requires wet lab validation","single-run prediction (not ensembled)","predicted properties may not reflect real-world biological behavior","this is research, not medical advice","all-D-amino-acid peptides are systematically underperforming in AlphaFold-class structure predictors trained on L-amino acid protein databases — pLDDT and ipTM values for DRI peptides should be interpreted as lower bounds on actual structural order","no Chai-1 cross-validation was available; single-model ipTM of 0.13 is below the threshold for any reliable interface conclusion","the heuristic half-life estimate (long, >6 hours) reflects the palmitoyl modification's albumin-anchoring potential in isolation and does not account for the CPP-driven internalization competition or tissue-specific distribution","the albumin-anchoring strategy is extrapolated from GLP-1 incretin pharmacology (liraglutide/semaglutide) and has not been validated for all-D, polycationic, nuclear-targeted peptides","FOXO4-DRI pharmacokinetics (plasma half-life, renal clearance fraction, tissue distribution) have not been formally characterized in published literature — the premise of rapid renal clearance as the dominant PK liability is biologically plausible but unsubstantiated","sustained senolytic exposure via albumin anchoring carries a context-dependent safety risk: PMID:36515093 documents disease worsening in pulmonary hypertension models with FOXO4-DRI treatment"],"works_cited":[{"pmid_or_doi":"40593617","title":"The disordered p53 transactivation domain is the target of FOXO4 and the senolytic compound FOXO4-DRI","year":2025,"relevance":"Provides NMR structural evidence that both the FOXO4 CR3-mimetic helix and the cationic CPP tail of FOXO4-DRI directly contact p53TAD2, directly bearing on whether modification of either terminus could compromise target binding."},{"pmid_or_doi":"10.21203/rs.3.rs-2361374/v1","title":"Identification of Hotspots in Synthetic Peptide Inhibitors of the FOXO4:p53 Interaction","year":2022,"relevance":"Identifies key binding hotspot residues within FOXO4-DRI using in silico modeling and truncated peptides; provides a basis for predicting whether N-terminal palmitoylation could be tolerated without ablating p53 affinity."},{"pmid_or_doi":"39994346","title":"FOXO4-DRI induces keloid senescent fibroblast apoptosis by promoting nuclear exclusion of upregulated p53-serine 15 phosphorylation","year":2025,"relevance":"Demonstrates that p53-pS15 phosphorylation is the specific p53 form targeted in a disease context and that FOXO4-DRI promotes its nuclear exclusion, confirming the p53 interaction as the on-target mechanism across cell types."},{"pmid_or_doi":"41625068","title":"FOXO4-DRI regulates endothelial cell senescence via the P53 signaling pathway","year":2025,"relevance":"Confirms FOXO4-DRI efficacy in vascular endothelial cells via p53/BCL-2/Caspase-3 activation, demonstrating the peptide's p53-dependent mechanism is conserved across diverse senescent cell types."},{"pmid_or_doi":"36515093","title":"Eliminating Senescent Cells Can Promote Pulmonary Hypertension Development and Progression","year":2023,"relevance":"Provides direct evidence that FOXO4-DRI-mediated senolysis can worsen disease in pulmonary hypertension models, a critical safety consideration for a sustained-exposure (albumin-anchored) formulation that would increase systemic senolytic activity."},{"pmid_or_doi":"31959736","title":"FOXO4-DRI alleviates age-related testosterone secretion insufficiency by targeting senescent Leydig cells in aged mice","year":2020,"relevance":"One of the foundational in vivo efficacy studies demonstrating FOXO4-DRI's selective p53-dependent apoptosis in senescent cells, establishing the dosing paradigm (cyclic IP injection) that motivates half-life extension."},{"pmid_or_doi":"39025385","title":"FOXO4-DRI improves spermatogenesis in aged mice through reducing senescence-associated secretory phenotype secretion from Leydig cells","year":2024,"relevance":"Confirms FOXO4-DRI efficacy in aging testes via SASP reduction downstream of FOXO4-p53 disruption, supporting therapeutic rationale and the need for improved PK to sustain tissue-level effects."},{"pmid_or_doi":"34877934","title":"Targeting senescence-like fibroblasts radiosensitizes non-small cell lung cancer and reduces radiation-induced pulmonary fibrosis","year":2021,"relevance":"Demonstrates in vivo efficacy of FOXO4-DRI in oncology and fibrosis settings, expanding the therapeutic scope and reinforcing the relevance of improving bioavailability for systemic applications."},{"pmid_or_doi":"33996787","title":"Senolytic Peptide FOXO4-DRI Selectively Removes Senescent Cells From in vitro Expanded Chondrocytes","year":2021,"relevance":"Establishes selective senolytic activity of FOXO4-DRI in chondrocyte preparations and notes limitations of current efficacy, indirectly motivating pharmacokinetic improvements."}]},"onchain":{"hash":"3vFgwa1bpMXZY39M84BsdWdVGtddyV9bsoVBEHv6Qq9Vc3hJaJGf8swQuvYwpgvmt2fLkhU9nEta66vHoh8iWipe","signature":"3vFgwa1bpMXZY39M84BsdWdVGtddyV9bsoVBEHv6Qq9Vc3hJaJGf8swQuvYwpgvmt2fLkhU9nEta66vHoh8iWipe","data_hash":"d419f2cdd4a30b4c3b12373317b85db3a31a8173ec097a9b116b28c577395878","logged_at":"2026-05-03T06:58:35.091211+00:00","explorer_url":"https://solscan.io/tx/3vFgwa1bpMXZY39M84BsdWdVGtddyV9bsoVBEHv6Qq9Vc3hJaJGf8swQuvYwpgvmt2fLkhU9nEta66vHoh8iWipe"},"ipfs_hash":null,"created_at":"2026-05-03T06:23:28.324216+00:00","updated_at":"2026-05-03T06:58:35.098559+00:00"}