Fox04-DRI

Research Overview

FOXO4-DRI serves as an innovative research tool for investigating cellular senescence, senescent cell clearance mechanisms, and potential senolytic therapeutic approaches in laboratory settings. This modified peptide represents a significant advancement in senescence research by providing a targeted mechanism for investigating how disruption of specific protein-protein interactions can selectively induce apoptosis in senescent cells while sparing normal, healthy cells.

Cellular senescence represents a fundamental biological process where cells enter permanent growth arrest in response to various stressors including DNA damage, telomere shortening, oncogene activation, and oxidative stress. While senescence serves protective functions by preventing damaged cell proliferation and cancer development, accumulation of senescent cells contributes to aging, chronic inflammation through senescence-associated secretory phenotype (SASP), tissue dysfunction, and age-related diseases. FOXO4-DRI provides researchers with a tool to investigate selective removal of these senescent cells.

The peptide’s design leverages the critical role of FOXO4-p53 interaction in maintaining senescent cell viability. In senescent cells, FOXO4 transcription factor binds to p53, sequestering it away from pro-apoptotic gene promoters and preventing the cell death that would normally result from p53 activation. FOXO4-DRI was specifically engineered to competitively disrupt this interaction, liberating p53 to activate apoptotic pathways selectively in senescent cells. Research protocols examine these mechanisms in cell culture systems, tissue models, and preclinical aging models.

The D-retro-inverso modification distinguishes FOXO4-DRI from conventional peptides. This chemical modification involves reversing the peptide sequence (retro) and using D-amino acids instead of L-amino acids (inverso), creating a peptide with enhanced stability against enzymatic degradation while maintaining the three-dimensional structure and biological activity necessary for target interaction. This approach provides researchers with improved peptide stability for experimental applications.

Molecular Characteristics

Complete Specifications:

• CAS Registry Number: Not assigned (proprietary modified peptide)
• Molecular Weight: 2,957.2 Da
• Molecular Formula: Not publicly disclosed (contains D-amino acids)
• Peptide Classification: D-retro-inverso modified peptide, senolytic compound
• Appearance: White to off-white lyophilized powder
• Solubility: Water, bacteriostatic water, phosphate buffered saline, DMSO

The D-retro-inverso modification involves using D-amino acids (mirror images of natural L-amino acids) arranged in reverse sequence compared to the original peptide. This structural approach maintains spatial orientation of critical side chains while providing dramatic enhancement in proteolytic stability. Proteases evolved to cleave peptide bonds between L-amino acids and generally cannot efficiently cleave bonds between D-amino acids, resulting in extended biological half-life and improved experimental utility.

The molecular weight of 2,957.2 Da places FOXO4-DRI in the range allowing cellular penetration while maintaining sufficient size for specific protein-protein interaction disruption. The peptide’s design targets the specific interface between FOXO4 transcription factor and p53 tumor suppressor protein, requiring precise structural features to achieve competitive inhibition of this critical interaction in senescent cells.

Mechanism of Action in Research Models

FOXO4-DRI’s investigated mechanism provides insight into senescent cell biology and potential senolytic approaches:

FOXO4-p53 Interaction in Senescence:

In normal cells, p53 activation leads to either cell cycle arrest (allowing DNA repair) or apoptosis (if damage is irreparable). However, senescent cells maintain high p53 levels without undergoing apoptosis, a paradox explained by the FOXO4-p53 interaction. FOXO4 transcription factor, upregulated in senescent cells, physically binds p53 and sequesters it at DNA damage sites, preventing p53 from accessing pro-apoptotic gene promoters.

Competitive Disruption Mechanism:

FOXO4-DRI competes with endogenous FOXO4 for p53 binding, disrupting the FOXO4-p53 complex. This liberation of p53 allows it to translocate to pro-apoptotic gene promoters (PUMA, NOXA, BAX) and activate apoptotic cascades. Research indicates this mechanism shows selectivity for senescent cells, which rely on FOXO4-p53 interaction for survival, versus normal cells where this interaction is less critical.

Senescent Cell Selectivity:

Laboratory studies investigate the selectivity of FOXO4-DRI for senescent versus proliferating cells. Research suggests senescent cells exhibit increased sensitivity to FOXO4-DRI-induced apoptosis compared to normal cells, potentially due to:

• Higher baseline p53 levels in senescent cells
• Greater dependence on FOXO4-p53 interaction for survival
• Elevated DNA damage response activation in senescent cells
• Distinct chromatin structure and gene expression profiles

Cell Penetration and Localization:

Despite its relatively large size (2,957.2 Da), FOXO4-DRI demonstrates cell penetration capacity investigated in research models. The D-retro-inverso modification may contribute to membrane permeability through altered charge distribution and conformational properties. Intracellular localization and nuclear access are critical for disrupting the nuclear FOXO4-p53 interaction.

Research Applications

Cellular Senescence Investigation

FOXO4-DRI serves as a valuable tool for fundamental senescence research:

• Senescence Marker Studies: Investigation of senescence-associated markers (SA-β-gal, p16, p21, γH2AX) and their modulation following senescent cell removal
• SASP Characterization: Examination of senescence-associated secretory phenotype components (IL-6, IL-8, MMPs) and their reduction following senolytic treatment
• Senescent Cell Identification: Research on distinguishing senescent from quiescent or terminally differentiated cells
• Senescence Induction Models: Studies using various senescence-inducing stimuli (replicative, stress-induced, oncogene-induced, therapy-induced) followed by selective clearance
• Molecular Pathway Analysis: Investigation of pathways maintaining senescent cell viability and mechanisms of selective senescent cell death

Research protocols typically employ primary cell cultures undergoing replicative senescence, stress-induced premature senescence models, and oncogene-induced senescence to investigate FOXO4-DRI effects on senescent cell populations.

Aging Research and Geroscience

FOXO4-DRI provides researchers with tools to investigate the role of senescent cells in aging processes:

• Aging Model Studies: Examination of senescent cell accumulation in naturally aged animal models and effects of selective clearance
• Tissue Function Investigation: Research on how senescent cell removal impacts age-related tissue dysfunction
• Regenerative Capacity Studies: Investigation of tissue regenerative potential following senescent cell clearance
• Healthspan Research: Analysis of functional parameters (physical performance, metabolic function, cognitive performance) in aging models with senolytic intervention
• Lifespan Studies: Longitudinal research examining survival outcomes in aging models with senescent cell clearance protocols

Laboratory studies investigate these questions using aged animal models, naturally senescent cell populations in tissues, and longitudinal experimental designs to assess long-term outcomes of senescent cell clearance.

Age-Related Disease Models

Research applications extend to disease models where senescent cell accumulation contributes to pathology:

• Metabolic Disease Research: Investigation of senescent cell roles in diabetes, obesity, fatty liver disease, and metabolic syndrome models
• Cardiovascular Research: Studies on senescent cell contributions to atherosclerosis, cardiac dysfunction, and vascular aging
• Neurodegenerative Disease Models: Examination of senescent cell accumulation in Alzheimer’s, Parkinson’s, and other neurodegenerative conditions
• Osteoarthritis Research: Investigation of senescent chondrocyte roles in joint degeneration and effects of selective clearance
• Pulmonary Disease Models: Research on senescent cells in COPD, pulmonary fibrosis, and age-related lung dysfunction
• Kidney Disease Studies: Examination of senescence in chronic kidney disease and aging-related renal dysfunction

Experimental models combine disease induction protocols with senolytic interventions to investigate causal relationships between senescent cell burden and disease pathology.

Cancer Research Applications

FOXO4-DRI research extends to cancer biology and treatment:

• Therapy-Induced Senescence: Investigation of senescent cell accumulation following chemotherapy and radiation therapy
• Treatment Resistance Studies: Research on how senescent cancer cells or senescent stromal cells contribute to therapy resistance
• Tumor Microenvironment Research: Examination of senescent cells in tumor microenvironment and their effects on cancer progression
• Combination Therapy Studies: Investigation of senolytic compounds combined with conventional cancer treatments
• Secondary Effect Prevention: Research on preventing adverse effects of therapy-induced senescence through selective clearance

Laboratory protocols examine cancer cell lines induced to senescence, tumor-bearing animal models with therapy-induced senescence, and co-culture systems examining senescent cell effects on proliferating cancer cells.

Regenerative Medicine Research

FOXO4-DRI provides tools for investigating senescence effects on regenerative processes:

• Stem Cell Function Studies: Research on how senescent cell clearance affects stem cell proliferation, differentiation, and regenerative capacity
• Tissue Repair Research: Investigation of regenerative responses to injury following senescent cell removal
• Wound Healing Studies: Examination of senescent cell roles in chronic wounds and effects of selective clearance on healing
• Progenitor Cell Research: Studies on tissue-specific progenitor cell function in context of senescent cell burden
• Tissue Engineering Applications: Investigation of senescent cell effects on engineered tissue function and integration

Research models include stem cell assays, tissue injury and repair models, and regenerative capacity assessments in context of controlled senescent cell populations.

FOXO4-p53 Interaction Studies

The peptide serves as a research tool for investigating this specific protein-protein interaction:

• Interaction Mapping: Characterization of FOXO4-p53 binding interface and structural requirements
• Functional Consequences: Investigation of biological outcomes when FOXO4-p53 interaction is disrupted
• Cell Type Specificity: Research on how this interaction varies across different cell types and contexts
• Post-translational Modifications: Studies on how FOXO4 and p53 modifications affect their interaction
• Alternative Interaction Partners: Examination of how FOXO4-DRI affects other FOXO4 or p53 interactions

Biochemical approaches including co-immunoprecipitation, fluorescence resonance energy transfer (FRET), proximity ligation assays, and structural biology techniques are employed to characterize FOXO4-p53 interaction disruption.

Pharmacokinetic Profile in Research Models

FOXO4-DRI pharmacokinetic characterization informs experimental design:

Enhanced Stability Profile:

• D-retro-inverso modification provides dramatic enhancement in proteolytic stability
• Resistance to peptidase degradation extends biological half-life compared to L-amino acid peptides
• Stability in biological fluids (serum, plasma) substantially improved over conventional peptides
• Extended experimental window for investigating biological effects

Cellular Penetration:

• Cell penetration capacity demonstrated in research models despite relatively large molecular weight
• Nuclear localization required for FOXO4-p53 interaction disruption
• Penetration efficiency may vary across cell types and experimental conditions
• Research protocols should consider cellular uptake kinetics in experimental design

Administration Routes:

• Multiple routes investigated in preclinical research: intraperitoneal, intravenous, subcutaneous
• Route selection influences pharmacokinetics, biodistribution, and experimental outcomes
• Local versus systemic administration considerations depending on research objectives

Duration of Action:

• Biological effects may persist beyond detectable peptide presence
• Apoptotic cascade activation creates sustained biological response
• Experimental protocols should consider temporal dynamics of senescent cell clearance

These pharmacokinetic characteristics inform dosing strategies, timing of assessments, and interpretation of experimental results in senescence research.

Laboratory Handling and Storage Protocols

Lyophilized Powder Storage:

• Store at -20°C to -80°C in original sealed vial
• Protect from light exposure and moisture
• Desiccated storage environment essential for modified peptide stability
• Stability data available for 12+ months at -20°C
• Avoid repeated temperature fluctuations

Reconstitution Guidelines:

• Reconstitute with sterile water, bacteriostatic water (0.9% benzyl alcohol), PBS (pH 7.0-7.4), or DMSO
• For aqueous solvents: Add slowly down vial side to minimize foaming
• For DMSO: May require brief warming and gentle mixing for complete dissolution
• Gentle swirling motion recommended (avoid vigorous shaking)
• Allow complete dissolution before use (typically 2-5 minutes)
• Final concentration should be determined based on experimental requirements
• pH monitoring recommended to ensure stability (optimal pH 6.5-7.5 for aqueous solutions)

Reconstituted Solution Storage:

• Short-term storage: 4°C for up to 7 days in sterile conditions
• Long-term storage: -20°C or -80°C in single-use aliquots
• Aliquoting strongly recommended to prevent repeated freeze-thaw cycles
• Maximum 2-3 freeze-thaw cycles to maintain peptide integrity
• DMSO solutions may be stored at -20°C for extended periods
• Document reconstitution date and concentration for laboratory records

Handling Precautions:

• Modified peptides may exhibit different solubility profiles than natural peptides
• Work in clean environment to prevent contamination
• Use sterile technique when preparing solutions for cell culture applications
• Consider peptide aggregation potential at high concentrations
• Vortex briefly if precipitation occurs upon thawing

Quality Assurance and Analytical Testing

Each FOXO4-DRI batch undergoes comprehensive analytical characterization ensuring research-grade quality:

Purity Analysis:

• High-Performance Liquid Chromatography (HPLC): ≥98% purity
• Analytical method: Reversed-phase HPLC with UV detection at 220nm
• Multiple peak integration ensuring accurate purity determination
• Related substance analysis identifying impurities and degradation products
• Stability-indicating HPLC methods for detecting time-dependent changes

Structural Verification:

• Mass Spectrometry (ESI-MS or MALDI-TOF): Confirms molecular weight 2,957.2 Da
• High-resolution mass spectrometry for accurate mass determination
• MS/MS fragmentation analysis for structural characterization
• Verification of D-amino acid incorporation where applicable
• Peptide content determination by weight

Contaminant Testing:

• Bacterial endotoxin: <5 EU/mg by Limulus Amebocyte Lysate (LAL) method
• Heavy metals: ICP-MS analysis confirming levels below detection limits
• Residual solvents: GC analysis for TFA, acetonitrile, DMSO within acceptable limits
• Water content: Karl Fischer titration (<8% for lyophilized material)
• Sterility testing for products requiring sterile handling

Functional Testing:

• Bioactivity assays may be performed on select batches
• Senescent cell selective toxicity assays in research models
• Comparison to reference standards
• Stability studies under recommended storage conditions

Documentation:

• Comprehensive Certificate of Analysis (COA) provided with each batch
• Batch genealogy documenting manufacturing and testing history
• Third-party analytical verification available upon request
• Stability data for recommended storage conditions
• Material safety data sheet (MSDS) for safety information
• Handling and storage recommendations specific to modified peptide

Research Considerations

Experimental Design Factors:

Researchers should carefully consider multiple factors when designing FOXO4-DRI experiments:

1. Senescent Cell Population Characterization: Confirm senescence induction using multiple markers (SA-β-gal, p16, p21, SASP factors, senescence morphology) before senolytic treatment. Heterogeneity in senescent cell populations may influence response to FOXO4-DRI.

2. Concentration and Timing: Published research reports various concentration ranges depending on cell type, senescence induction method, and experimental system. Dose-response studies recommended for each specific experimental context. Consider temporal dynamics of apoptosis induction (typically hours to days).

3. Selectivity Assessment: Include parallel experiments with non-senescent cells (proliferating, quiescent) to evaluate selectivity of effects. Senolytic selectivity is a critical parameter distinguishing these compounds from general cytotoxic agents.

4. Cell Type Considerations: FOXO4-DRI effects may vary across cell types due to differences in p53 status, FOXO4 expression, apoptotic pathway integrity, and cellular penetration. Initial characterization recommended for each cell type.

5. Senescence Induction Method: Different senescence-inducing stimuli (replicative, oncogene-induced, stress-induced, therapy-induced) may create senescent cells with varying characteristics affecting FOXO4-DRI sensitivity.

6. SASP Analysis: Comprehensive characterization of senescence-associated secretory phenotype components provides insight into mechanisms. Cytokine arrays, qPCR panels, and proteomics approaches can characterize SASP changes following senescent cell clearance.

7. Apoptosis Confirmation: Employ multiple apoptosis detection methods (annexin V, caspase activation, TUNEL, DNA fragmentation) to confirm mechanism. Distinguish from other cell death modalities (necrosis, necroptosis, autophagy).

8. Long-term Effects: In vitro and in vivo studies should consider long-term consequences of senescent cell clearance including effects on remaining cell populations, tissue function, and potential compensatory responses.

Control Strategies:

Appropriate controls are essential for interpreting FOXO4-DRI research:

• Vehicle Controls: Matched solvent controls for each experimental condition
• Scrambled Peptide Controls: Non-functional peptide sequences as negative controls
• Alternative Senolytics: Comparison with other senolytic compounds (dasatinib+quercetin, navitoclax, fisetin) to assess relative efficacy and mechanisms
• Time Course Controls: Untreated senescent cells maintained for equivalent durations
• Cell Viability Controls: Non-senescent cells treated identically to assess selectivity

In Vivo Research Considerations:

For preclinical animal research:

• Appropriate animal models with senescent cell accumulation (naturally aged, progeroid models, disease models)
• Senescent cell detection and quantification in tissues (immunohistochemistry, flow cytometry, molecular markers)
• Dosing strategies (frequency, duration, route) based on objectives and pharmacokinetics
• Comprehensive outcome assessments (molecular, cellular, tissue, functional, systemic)
• Long-term monitoring for safety, efficacy, and potential adverse effects
• Ethical considerations and appropriate IACUC approvals

Compliance and Safety Information

Regulatory Status:
FOXO4-DRI is provided as a research chemical for in-vitro laboratory studies and preclinical research only. This modified peptide has not been approved by the FDA for human therapeutic use, dietary supplementation, or medical applications. Senolytic compounds remain in early research and development phases.

Intended Use:

• In-vitro cell culture studies investigating cellular senescence
• In-vivo preclinical research in approved animal models with appropriate oversight
• Laboratory investigation of FOXO4-p53 interaction and senescent cell biology
• Mechanism of action studies for senolytic therapeutic approaches
• Academic and institutional research applications in aging biology and geroscience

NOT Intended For:

• Human consumption or administration
• Therapeutic treatment or diagnosis
• Dietary supplementation or anti-aging products
• Veterinary therapeutic applications without appropriate oversight
• Any use outside of controlled research settings

Safety Protocols:
Researchers should follow comprehensive safety practices when handling FOXO4-DRI:

• Use appropriate personal protective equipment (lab coat, gloves, safety glasses)
• Handle in well-ventilated areas or biological safety cabinet for cell culture applications
• Follow institutional biosafety and chemical safety guidelines
• Dispose of waste according to local regulations for biological and chemical waste
• Avoid skin contact and inhalation of powder
• Wash thoroughly after handling
• Consult material safety data sheet (MSDS) for comprehensive safety information
• Train personnel in proper handling procedures before use
• Maintain spill response materials and protocols
• Document handling and usage for institutional safety records

Research Ethics:
Research involving senolytic compounds should consider:

• Appropriate institutional approvals (IACUC for animal research)
• Ethical implications of aging intervention research
• Responsible communication of research findings
• Acknowledgment of early-stage research status
• Transparency regarding limitations and uncertainties

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