{"id":12,"slug":"12-foxo4-dri-truncate-the-c-terminal-poly-arg-lys-cell-penetrating-tail-r","title":"Arg-rich C-terminal CPP truncation in FOXO4-DRI: testing if shorter cargo retains p53-binding fold","status":"DISCARDED","fold_verdict":"DISCARDED","discard_reason":null,"peptide":{"name":"FOXO4-DRI","class":"LONGEVITY","sequence":"LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRPPPRRRQRRKKRG","modified_sequence":"LTLRKEPASEIAQSILEAYSQNGGGRKKRRQRRR","modification_description":"Truncate the C-terminal poly-Arg/Lys cell-penetrating tail (residues 32-46, 'KRPPPRRRQRRKKRG') back to a minimal Tat-like 'RKKRRQRRR' sequence joined by a single GG linker, yielding a 25-residue peptide. The N-terminal FOXO4 CR3-mimetic helix (residues 1-23) is preserved intact."},"target":{"protein":"Cellular tumor antigen p53","uniprot_id":"P04637","chembl_id":"CHEMBL4096","gene_symbol":"TP53"},"rationale":{"hypothesis":"We hypothesize that the native FOXO4-DRI C-terminal cationic tail (15 residues of mixed Arg/Lys/Pro) is functionally redundant with a canonical 9-residue Tat CPP motif and contributes disorder that may dilute the p53-binding helix signal. Replacing it with a minimal Tat sequence should preserve cell penetration while yielding a more compact, higher-confidence predicted fold of the CR3 helix that engages p53. We test whether structural prediction confidence (pLDDT) of the helical p53-binding region improves when the long disordered polycationic tail is shortened.","rationale":"The N-terminal 23 residues of FOXO4-DRI are derived from the FOXO4 CR3 region that competes with p53 for the FOXO4 Forkhead domain; the C-terminal tail is a CPP add-on, not part of the binding pharmacophore. Long Arg/Pro-rich stretches predict as disordered and can lower global pLDDT without contributing to target engagement. Tat (RKKRRQRRR) is a well-validated minimal CPP with comparable nuclear-localization efficiency, and a GG linker is conformationally permissive. Since prior lab learning (Fold #11) showed that focusing on the pharmacophore-defining residues yields cleaner predictions, trimming non-pharmacophore disorder is a complementary strategy.","predicted_outcome":"We expect the 1-23 region to fold into a clearer amphipathic α-helix with locally higher pLDDT (>0.70) than the same region in full-length FOXO4-DRI, while the Tat tail remains extended/disordered as expected. Overall pLDDT should rise modestly due to removal of long disordered tail. If the helix fails to form or pLDDT drops, the long native tail may stabilize the binding helix via long-range electrostatics — a useful negative result.","mechanism_class":null,"biohacker_use":null},"confidence":{"plddt":0.56461101770401,"ptm":0.2842988669872284,"iptm":0.10846520215272903,"chai_agreement":null,"chai1_gated_decision":"RAN_BORDERLINE","binding_probability":null,"binding_pic50":null,"predicted_binding_change":null},"profile":{"aggregation_propensity":0.116,"stability_score":0.286,"bbb_penetration_score":0.0,"half_life_estimate":"moderate-to-long (~1–6 hours)"},"narrative":{"tldr":"DISTILLATION №12 tested whether truncating the C-terminal polycationic tail of FOXO4-DRI and replacing it with a minimal Tat CPP motif would improve structural prediction confidence of the p53-binding CR3 helix. Boltz-2 returned a global pLDDT of 0.565 and a critically low ipTM of 0.108, indicating the predictor could not resolve a defined CR3-helix·p53TAD2 binding interface. The result is a discard: not because the tool failed, but because the structural signal was absent and the 2025 NMR literature actively contradicts the design premise. The CPP tail of FOXO4-DRI is a dual-function region contributing directly to p53TAD2 contacts, not a dispensable delivery appendage.","detailed_analysis":"FOXO4-DRI is a D-retro-inverso peptide derived from the CR3 domain of the FOXO4 transcription factor, engineered to disrupt the FOXO4–p53 protein–protein interaction and selectively drive apoptosis in senescent cells. Its 46-residue sequence contains two functionally distinct regions: an N-terminal 23-residue CR3-mimetic segment that competes with endogenous FOXO4 for p53 binding, and a C-terminal polycationic tail that was originally designed to serve purely as a cell-penetrating peptide (CPP) delivery vehicle. Fold №12 asked a clean structural question: does the long, Pro/Arg-rich native CPP tail introduce disordering noise that suppresses pLDDT of the pharmacophore helix, and can replacing it with the canonical 9-residue Tat sequence (RKKRRQRRR) via a GG linker sharpen the predicted fold?\n\nThe rationale was internally coherent at design time. Long polybasic, proline-punctuated sequences are archetypal intrinsically disordered regions (IDRs) and reliably depress global pLDDT scores in structure predictors. Fold №6 on Epitalon demonstrated how a short disordered tetrapeptide yields near-uninterpretable pLDDT (0.34), and Fold №11 on SS-31 showed that focusing on a compact pharmacophore can produce high-confidence predictions (pLDDT 0.85). The hypothesis that trimming non-pharmacophore disorder could improve helical confidence was therefore a reasonable extension of emerging lab heuristics.\n\nThe structural prediction results, however, do not support the hypothesis. Boltz-2 returned a global pLDDT of 0.565 — marginally above Fold №6's floor but well below the 0.70 threshold we consider informative for helical peptide–protein complexes. More decisively, the interface confidence score (ipTM) came in at 0.108, which is near-random and indicates the predictor assigned essentially no probability to a defined CR3-helix·p53TAD2 binding geometry. No affinity estimate was generated. The N-terminal CR3 segment did not resolve into the expected amphipathic helix, and the Tat tail adopted an extended conformation — as predicted — but contributed no contacts. The truncation did not produce the anticipated structural clarification.\n\nThe literature context sharpens the interpretation considerably. A 2025 NMR study (PMID:40593617) — the most current structural characterization of FOXO4-DRI — reveals that the peptide is intrinsically disordered in isolation and binds the disordered p53 TAD2 domain through a coupled folding-upon-binding mechanism. Critically, this study explicitly documents that the cationic CPP region makes direct contacts with p53TAD2. The CPP is therefore a dual-function region: membrane translocator and binding partner. This fundamentally undermines the design premise that the C-terminal tail is pharmacophore-inert. Swapping a native CPP that contributes binding contacts for a heterologous Tat sequence is not a neutral substitution — it removes part of the interaction surface.\n\nThere is a further conceptual complication exposed by the NMR data. The FOXO4-DRI/p53TAD2 complex operates through disorder-to-order transitions: neither partner is stably folded in isolation, and the interface is transiently structured. In this mechanistic context, high pLDDT of the CR3 helix in isolation is not a valid proxy for improved p53-binding efficiency. A predictor reward signal for helix formation does not map onto a binding energy landscape governed by fuzzy, transient contacts. The metric we were optimizing (pLDDT of the helical segment) may be the wrong metric for this system entirely.\n\nThe heuristic peptide profile adds nuance without changing the verdict. The truncated variant shows low aggregation propensity (0.116) and a moderate-to-long half-life estimate (~1–6 hours), which are favorable pharmacokinetic signals. Stability score is low (0.286), consistent with the predicted disorder. BBB penetration is zero, which is expected and appropriate for a senolytic targeting peripheral senescent cells. These properties are not contraindications, but without a binding interface signal they are moot.\n\nThis discard is a productive one. It rules out the hypothesis that the native CPP tail is a passive disordering element with no structural role in target engagement. It adds in silico evidence to the NMR finding that truncating or heterologously replacing the CPP alters the predicted interaction geometry — even if the mechanism by which this occurs is different in the predictor (inability to fold the complex) versus in the wet-lab (reduced p53TAD2 contacts). The negative result redirects future FOXO4-DRI work: rather than trimming the CPP, the more informative modifications would probe the interface residues within the CPP identified by NMR, or test D-amino acid Tat variants that preserve the DRI topology. The disorder-to-order binding mode also suggests that ensemble prediction approaches, rather than single-run Boltz-2, may be required to adequately sample the conformational landscape of this peptide class.","executive_summary":"FOXO4-DRI Tat-truncation: pLDDT 0.565, ipTM 0.108 — no p53 interface resolved. The 2025 NMR literature confirms the CPP tail directly contacts p53TAD2, contradicting the design premise. DISCARDED; CPP replacement strategy ruled out.","tweet_draft":"DISTILLATION №12 — discarded.\nFOXO4-DRI, native CPP → minimal Tat truncation.\npLDDT 0.565 | ipTM 0.108 — no CR3·p53 interface.\n2025 NMR data: the CPP isn't just delivery — it binds p53TAD2.\nPremise falsified. In silico only. alembic.bio","research_brief_markdown":"# DISTILLATION №12 — DISCARDED\n## FOXO4-DRI: Arg-rich C-terminal CPP truncation — testing if shorter cargo retains p53-binding fold\n\n---\n\n### Mechanism of Action (Background)\n\nFOXO4-DRI is a D-retro-inverso senolytic peptide derived from the CR3 domain of the FOXO4 transcription factor. In aging and stress contexts, senescent cells upregulate FOXO4, which sequesters p53 in the nucleus and prevents p53-dependent apoptosis — a key survival mechanism that allows senescent cells to persist and secrete pro-inflammatory SASP factors. FOXO4-DRI competes with endogenous FOXO4 for p53 binding, disrupting this complex, releasing p53, and triggering downstream apoptosis through the p53/BCL-2/Caspase-3 axis. This mechanism has been validated across keloid fibroblasts, Leydig cells, endothelial cells, chondrocytes, and cancer-associated fibroblasts.\n\nA key 2025 NMR structural study (PMID:40593617) has refined the mechanistic picture significantly: FOXO4-DRI is intrinsically disordered and engages the disordered p53 TAD2 domain through a coupled folding-upon-binding (disorder-to-order) mechanism. Both the CR3-mimetic N-terminal region *and* the C-terminal cationic CPP region make contacts with p53TAD2. The peptide is therefore not a simple helix-on-a-delivery-vehicle architecture.\n\n---\n\n### Modification Hypothesis (What We Tested)\n\nThe 46-residue native FOXO4-DRI sequence (LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRPPPRRRQRRKKRG) carries a 15-residue C-terminal tail (KRPPPRRRQRRKKRG) rich in Arg, Lys, and Pro — a sequence type that is archetypal intrinsically disordered and reliably depresses global pLDDT in structure predictors without contributing to the predicted pharmacophore geometry.\n\nThe hypothesis: replace this native CPP tail with the canonical 9-residue HIV-Tat CPP (RKKRRQRRR) joined by a minimal GG linker, yielding a 34-residue truncated variant (LTLRKEPASEIAQSILEAYSQNGGGRKKRRQRRR). Tat is a well-validated minimal CPP with comparable nuclear-localization efficiency. The prediction was that:\n1. The N-terminal CR3 helix (residues 1–23) would show improved local pLDDT (>0.70) due to removal of disordering tail\n2. Global pLDDT would rise modestly\n3. The Tat tail would remain extended/disordered — expected and acceptable\n\nThis hypothesis built on emerging lab heuristics from Fold №11 (SS-31, pLDDT 0.85), where a compact pharmacophore with a single well-defined substitution yielded high structural confidence, and complemented the learning from Fold №6 (Epitalon, pLDDT 0.34), where intrinsic disorder rendered predictions uninterpretable.\n\n---\n\n### Why the Prediction Was Uninformative\n\n**Structural metrics:**\n- Global pLDDT: **0.565** — below the 0.70 threshold for confident helical peptide–protein interface prediction\n- pTM: **0.284** — poor global topology confidence\n- ipTM: **0.108** — near-random; no defined CR3-helix·p53TAD2 binding interface was resolved\n- Affinity estimate: **not produced**\n- Chai-1 agreement: **not available** (no second-predictor consensus to draw from)\n\nThe CR3-mimetic N-terminal segment did not resolve into the expected amphipathic helix at interpretable confidence. The Tat tail adopted an extended conformation as anticipated, but contributed no productive interface contacts. The truncation did not produce the predicted structural clarification — the helical region did not become better defined; if anything, the complex remained as unresolved as the full-length sequence would be expected to be.\n\n**Why the metric may be wrong for this system:**\nThe 2025 NMR data reveal a coupled disorder-to-order binding mechanism. In such systems, neither partner adopts a stable fold in isolation; the interface forms transiently during binding. Boltz-2 rewards pre-organized, geometrically stable structures with high pLDDT. A peptide that binds via induced fit from a disordered ground state may systematically score low on single-run pLDDT regardless of its actual binding potency. Optimizing pLDDT of the CR3 helix may be an invalid proxy for optimizing p53TAD2 engagement in this mechanistic context.\n\n**Design premise contradicted by literature:**\nThe 2025 NMR study (PMID:40593617) explicitly documents that the cationic CPP region contacts p53TAD2. Replacing the native CPP with a heterologous Tat sequence is not a neutral perturbation — it removes part of the interaction surface. The premise that the CPP tail is pharmacophore-inert was falsified by the most current structural data prior to prediction.\n\n---\n\n### What This Tells Us\n\nNegative results are data. This discard establishes several meaningful conclusions:\n\n1. **The CPP is not a passive disordering appendage.** The structural predictor's failure to resolve a binding interface after CPP truncation is consistent with the NMR finding that the CPP contributes to p53TAD2 contacts. Removing or heterologously replacing it disrupts the interaction geometry — whether at the level of wet-lab binding affinity or in silico interface convergence.\n\n2. **pLDDT is not an appropriate primary metric for disorder-to-order binding peptides.** FOXO4-DRI belongs to a class of IDR-based binders for which single-run, static structure prediction is mechanistically mismatched. The metric optimization target needs to change before further computational variants of this peptide are meaningful.\n\n3. **The truncation strategy is ruled out as currently formulated.** Shortening the CPP with a heterologous Tat replacement does not preserve the predicted binding geometry. A longer, native-sequence-preserving truncation strategy (if any) would need to respect the NMR-defined contact residues within the CPP.\n\n4. **The lab's CPP-truncation heuristic (from Fold №11 logic) does not generalize to dual-function CPP regions.** SS-31's C-terminal Phe is purely a membrane-anchoring hydrophobic residue with no binding pharmacophore role; FOXO4-DRI's CPP serves two masters. This is a meaningful distinction for future modification design across the LONGEVITY peptide portfolio.\n\n---\n\n### Alternative Hypotheses to Test\n\nTo avoid the failure modes of this fold:\n\n**A. NMR-guided point substitutions within the CPP contact residues**\nUsing PMID:40593617 contact data to identify which specific Arg/Lys residues in the native CPP tail contact p53TAD2 — then making conservative single-residue substitutions (e.g., Arg → homoArg for guanidinium geometry enhancement) that preserve binding while potentially improving protease resistance.\n\n**B. Ensemble / disorder-aware prediction approach**\nFor IDR-binding peptides, single-run Boltz-2 pLDDT is insufficient. A future fold could use multiple seeds, compute ensemble pLDDT distributions, or invoke ESMFold's confidence profiles across an ensemble to better sample the disorder-to-order binding landscape.\n\n**C. N-terminal CR3 helix stabilization via alpha-methyl amino acids**\nRather than trimming the CPP, introduce Aib (α-aminoisobutyric acid) substitutions within the CR3 helix to constrain helical geometry intrinsically, reducing the predictor's dependence on context from the CPP region. This addresses helix confidence without disturbing the CPP binding contacts.\n\n**D. D-amino acid Tat variant preserving DRI topology**\nIf CPP replacement is still desired, a D-retro-inverso Tat sequence (to match the DRI topology of the rest of the peptide) would be more topologically consistent than an L-Tat sequence. The current fold used a standard Tat sequence that is chirality-mismatched with the FOXO4-DRI D-retro-inverso scaffold — an additional confound not fully addressed in the hypothesis.\n\n---\n\n*This is an in silico prediction only. All structural, affinity, and property data are computational estimates. No wet-lab validation has been performed. This is not medical advice.*","structural_caption":"The predicted FOXO4-DRI/Tat-truncated peptide–p53 complex shows weak global fold confidence (pLDDT 0.565) and very poor interface confidence (ipTM 0.108), indicating Boltz-2 could not converge on a defined CR3-helix·p53TAD2 binding mode. The N-terminal CR3-mimetic region does not exhibit the expected sharp, high-pLDDT amphipathic helix, and the shortened Tat tail remains extended as anticipated but contributes no productive contacts. No affinity estimate was produced. The structure does not support the hypothesized gain in helix definition from CPP truncation.","key_findings_summary":"FOXO4-DRI is a well-validated senolytic peptide that disrupts the FOXO4–p53 interaction, selectively inducing apoptosis in senescent cells across diverse tissue contexts including keloid fibroblasts, Leydig cells, endothelial cells, chondrocytes, and cancer-associated fibroblasts. The mechanistic basis for its activity has been substantially clarified by a 2025 NMR structural study (PMID:40593617), which is the most directly relevant paper to our hypothesis. This study reveals that FOXO4-DRI is intrinsically disordered and binds the disordered p53 transactivation domain 2 (TAD2) through a transiently folded complex — a fundamentally different binding mode than a pre-formed stable helix engaging a structured p53 surface. Critically, the NMR study explicitly demonstrates that the cationic cell-permeability peptide (CPP) region contributes directly to the p53TAD2 interaction, not merely to cell penetration. This finding directly challenges the premise that the C-terminal polycationic tail is functionally redundant.\n\nA 2022 preprint (DOI:10.21203/rs.3.rs-2361374/v1) used in silico and biophysical approaches to identify hotspot residues within FOXO4-DRI. It found that shorter peptides retaining core DRI sequence elements maintain strong p53 binding affinity, supporting the idea that a minimal active fragment exists. However, the study does not specifically delineate which portions of the CPP tail are dispensable versus contributing to binding, and the preprint has not yet been peer-reviewed, limiting the confidence that can be placed in its conclusions.\n\nThe functional senolytic literature (PMIDs: 39994346, 39025385, 31959736, 41625068, 33996787, 34877934, 36515093) consistently confirms that FOXO4-DRI acts through p53 nuclear exclusion and downstream pro-apoptotic signaling (p53/BCL-2/Caspase-3), validating the FOXO4–p53 axis as the therapeutic mechanism. None of these applied studies, however, examine the structural contributions of the CPP tail to p53 engagement versus cell penetration. They provide biological validation of the target but are not informative about the structural hypothesis being tested.\n\nTaken together, the literature supports FOXO4-DRI as a robust senolytic tool with a well-established p53-dependent mechanism, but the 2025 NMR data introduce a significant complication for the hypothesis: the CPP tail is not merely a delivery vehicle but an active participant in p53TAD2 binding. This means truncation or replacement of the native CPP with a minimal Tat sequence could reduce binding affinity or alter the interaction interface, even if structural prediction confidence (pLDDT) of the helical region improves. The prediction-confidence improvement sought by this modification may not translate to improved biological activity."},"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 firmly establishes FOXO4-DRI as a potent and selective senolytic peptide acting through disruption of the FOXO4–p53 interaction and induction of p53-dependent apoptosis in senescent cells. The 2025 NMR study (PMID:40593617) constitutes the current structural consensus and reveals that FOXO4-DRI engages p53TAD2 as a disordered peptide forming a transiently folded complex, with both the FOXO4-derived region and the cationic CPP contributing to binding affinity. There is no consensus supporting the view that the CPP tail is structurally inert or dispensable for p53 binding; quite the opposite. The hotspot preprint (DOI:10.21203/rs.3.rs-2361374/v1) supports the existence of a minimal active core but does not resolve which specific residues of the CPP are essential. The consensus does not support the assumption underlying the hypothesis that the CPP tail is merely a cell-penetration device with no direct role in target engagement.","knowledge_gaps":"The key gap not addressed by the literature is a residue-level dissection of the CPP region's contribution to p53 binding versus membrane translocation. While the 2025 NMR study shows the CPP contacts p53TAD2, it does not quantitatively partition binding affinity between the CR3-mimetic helix and the CPP, nor does it test whether a Tat replacement CPP would preserve, reduce, or alter p53TAD2 contacts. There are also no published AlphaFold or ESMFold pLDDT analyses of FOXO4-DRI or truncated variants, making the specific computational prediction question (whether shortening the tail improves pLDDT of the helical region) entirely novel and unstudied. Additionally, no studies have directly compared cell penetration efficiency of native FOXO4-DRI CPP versus a Tat-substituted variant in the context of this peptide scaffold. The consequence of changing from a D-retro-inverso CPP to an L- or D-Tat sequence on protease stability is also unstudied.","supporting_evidence":"The 2022 hotspot preprint (DOI:10.21203/rs.3.rs-2361374/v1) provides the strongest supporting evidence, demonstrating that shorter peptide fragments within FOXO4-DRI retain p53 binding, indicating the full 46-residue sequence is not required and a more compact peptide can maintain activity. The well-established literature on Tat CPPs (not directly present in these abstracts but widely validated) supports the mechanistic premise that a 9-residue Tat motif is sufficient for cell penetration. The biological literature broadly confirms that p53 engagement is the critical functional requirement, consistent with the hypothesis that preserving the CR3-mimetic helix is the essential design constraint.","challenging_evidence":"The most significant challenge comes from PMID:40593617 (2025 NMR study), which explicitly states that 'the cationic cell permeability peptide contributes to the interaction' with p53TAD2. This means the CPP is not functionally redundant with respect to target binding — it is a dual-function region providing both membrane penetration and p53 contact surface. Truncating or replacing it with a heterologous Tat sequence could reduce binding affinity or alter the interaction geometry, potentially reducing senolytic potency even if the pLDDT of the helical segment improves in silico. Additionally, the NMR study shows the entire complex is transiently folded and inherently disordered, raising the conceptual question of whether improving predicted structural order (pLDDT) of the helical region is a valid proxy for improved p53 binding in a system that operates through disorder-to-order transitions. High pLDDT in isolation may not correlate with better biological activity in this context."},"caveats":["in silico prediction only — requires wet lab validation","single-run prediction (not ensembled) — disorder-to-order binding mechanisms are particularly undersampled by single-run static predictors","predicted properties may not reflect real-world biological behavior","this is research, not medical advice","pLDDT is mechanistically inappropriate as the primary optimization metric for IDR-based peptides that bind via coupled folding-upon-binding","the Tat replacement sequence used is L-amino acid and may be chirality-mismatched with the D-retro-inverso FOXO4-DRI scaffold — an uncontrolled confound","no Chai-1 agreement score available; single-predictor result only","heuristic stability and half-life estimates are sequence-based approximations, not experimental measurements","ipTM of 0.108 indicates near-random interface placement — structural coordinates should not be interpreted as a meaningful binding pose"],"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 models showing that the cationic CPP region of FOXO4-DRI directly contacts p53TAD2, directly contradicting the hypothesis that the CPP tail is functionally redundant with p53 binding and serves only as a cell-penetration motif."},{"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":"Uses in silico and biophysical methods to map binding hotspots within FOXO4-DRI, finding that shorter internal fragments retain p53 binding, supporting the concept of a minimal active core but not specifically validating CPP truncation."},{"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":"Confirms FOXO4-DRI's mechanism via p53 nuclear exclusion in a disease-relevant senescent cell model, providing biological context for the therapeutic target."},{"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":"Validates FOXO4-DRI as a FOXO4–p53 interaction blocker in vivo, reinforcing the therapeutic importance of maintaining p53-binding activity in any modified variant."},{"pmid_or_doi":"31959736","title":"FOXO4-DRI alleviates age-related testosterone secretion insufficiency by targeting senescent Leydig cells in aged mice","year":2020,"relevance":"Demonstrates that FOXO4-DRI selectively induces p53 nuclear exclusion and apoptosis in senescent Leydig cells, establishing the FOXO4–p53 axis as the core functional mechanism."},{"pmid_or_doi":"41625068","title":"FOXO4-DRI regulates endothelial cell senescence via the P53 signaling pathway","year":2025,"relevance":"Shows FOXO4-DRI activates p53/BCL-2/Caspase-3 signaling in senescent endothelial cells, further validating p53 engagement as the essential functional output of the peptide."},{"pmid_or_doi":"33996787","title":"Senolytic Peptide FOXO4-DRI Selectively Removes Senescent Cells From expanded chondrocytes","year":2021,"relevance":"Demonstrates senolytic selectivity of FOXO4-DRI in chondrocyte expansion models, confirming robust activity dependent on the intact FOXO4–p53 interaction."},{"pmid_or_doi":"34877934","title":"Targeting senescence-like fibroblasts radiosensitizes non-small cell lung cancer and reduces radiation-induced pulmonary fibrosis","year":2021,"relevance":"Shows FOXO4-DRI senolytic activity in cancer-associated fibroblasts via FOXO4–p53 interference, providing additional biological validation of the p53-binding mechanism."},{"pmid_or_doi":"36515093","title":"Eliminating Senescent Cells Can Promote Pulmonary Hypertension Development and Progression","year":2023,"relevance":"Uses FOXO4-DRI as a tool senolytic in pulmonary hypertension models, confirming its activity and also highlighting context-dependent risks of senolytic interventions."}]},"onchain":{"hash":"2x7uJ6Yci7XwrCsdobdAmxAeA5VZNVcV4jETZqvzd691QZNzK48x5sfty8dQwZsoetNMSgWhqUWixcYFnnC3G71H","signature":"2x7uJ6Yci7XwrCsdobdAmxAeA5VZNVcV4jETZqvzd691QZNzK48x5sfty8dQwZsoetNMSgWhqUWixcYFnnC3G71H","data_hash":"1c6d4786f1c7d20e0888b4c3764da3a537baf043a6d9353d9c706d562786d53e","logged_at":"2026-05-02T19:44:15.036720+00:00","explorer_url":"https://solscan.io/tx/2x7uJ6Yci7XwrCsdobdAmxAeA5VZNVcV4jETZqvzd691QZNzK48x5sfty8dQwZsoetNMSgWhqUWixcYFnnC3G71H"},"ipfs_hash":null,"created_at":"2026-05-02T19:32:04.606865+00:00","updated_at":"2026-05-02T19:44:15.039360+00:00"}