SS-31

Research Overview

SS-31 (Elamipretide, MTP-131, Bendavia) serves as a valuable research tool for investigating mitochondrial dysfunction and protection mechanisms in laboratory settings. This aromatic-cationic tetrapeptide represents the first member of the Szeto-Schiller peptide series developed specifically for mitochondrial targeting through cardiolipin interactions. Research applications have expanded across cardiovascular disease models, neurodegenerative research, metabolic dysfunction studies, and ischemia-reperfusion injury investigations.

The peptide’s designation SS-31 reflects its development by Szeto and Schiller laboratories, with the compound being the 31st in a series of mitochondria-targeted peptides. Unlike earlier mitochondrial therapies that relied on lipophilic cations requiring intact membrane potential, SS-31 concentrates in mitochondria through cardiolipin binding interactions that remain effective even in depolarized mitochondria. This property makes SS-31 particularly valuable for investigating mitochondrial dysfunction in pathological states where membrane potential is compromised.

SS-31 research demonstrates the peptide’s ability to stabilize cardiolipin, protect cristae structure, enhance ATP production, reduce mitochondrial ROS generation, and preserve mitochondrial function across diverse experimental models. Studies document SS-31’s effects in cardiac ischemia models, heart failure research, neurodegenerative disease models, skeletal muscle dysfunction, renal injury models, and age-related mitochondrial decline investigations.

Molecular Characteristics

Complete Specifications:

• CAS Registry Number: 736992-21-5
• Molecular Weight: 640.7 Da
• Molecular Formula: C₃₂H₄₉N₉O₅
• Amino Acid Sequence: D-Arg-Dmt-Lys-Phe-NH₂ (D-arginine, 2′,6′-dimethyltyrosine, lysine, phenylalanine)
• Chemical Class: Aromatic-cationic tetrapeptide
• Cardiolipin Affinity: High selectivity for cardiolipin binding
• Appearance: White to off-white lyophilized powder
• Solubility: Water, saline, phosphate buffered saline, cell culture media

The peptide’s four-amino acid structure incorporates critical design elements for mitochondrial targeting. The D-arginine residue provides cationic charge and proteolytic resistance. The 2′,6′-dimethyltyrosine (Dmt) contributes aromatic character and hydrophobic interactions. Together with lysine (cationic) and phenylalanine (aromatic), the sequence creates an alternating charge-aromatic-charge-aromatic pattern. This amphipathic structure enables selective binding to cardiolipin’s unique dimeric structure with four acyl chains.

The C-terminal amidation (-NH₂) protects against carboxypeptidase degradation. The incorporation of D-amino acid provides additional proteolytic stability. These structural features contribute to SS-31’s enhanced stability in biological systems compared to typical peptides.

Mitochondrial Targeting Mechanism

Cardiolipin Binding Specificity:

SS-31’s mechanism of action centers on selective cardiolipin binding in mitochondrial inner membranes. Cardiolipin represents approximately 20% of inner membrane phospholipids and plays critical roles in:

• Cristae structure maintenance
• Electron transport chain complex assembly and stability
• ATP synthase dimerization and efficiency
• Mitochondrial fission and fusion dynamics
• Cytochrome c retention in intermembrane space

Cardiolipin’s unique dimeric structure with four fatty acid chains creates distinct binding sites. SS-31’s alternating cationic-aromatic sequence complements cardiolipin’s negatively charged headgroups and hydrophobic acyl chains. Binding studies demonstrate nanomolar affinity constants and high selectivity over other phospholipids.

Mitochondrial Accumulation:

SS-31 demonstrates remarkable mitochondrial concentration:

• Mitochondrial:cytoplasmic ratio exceeds 1000:1 in many cell types
• Concentration occurs independent of membrane potential (unlike lipophilic cations)
• Effective in both healthy and depolarized mitochondria
• Cell-permeable without requiring specialized uptake mechanisms
• Retains activity in pathological conditions affecting membrane potential

This selective accumulation enables targeted investigation of mitochondrial-specific effects without significant cytoplasmic or nuclear interactions.

Pharmacokinetic Profile in Research Models

SS-31 pharmacokinetic characterization in preclinical research reveals important properties for experimental design:

Absorption and Distribution:

• Rapid tissue distribution following administration
• Multiple administration routes investigated: IV, IP, subcutaneous, oral
• Crosses blood-brain barrier (important for neurological research)
• Accumulates preferentially in tissues with high mitochondrial content (heart, brain, kidney, muscle)
• Plasma protein binding: Moderate to low

Metabolism and Elimination:

• Plasma half-life: 2-4 hours in rodent models
• Renal elimination as primary clearance route
• Minimal metabolic degradation due to D-amino acid incorporation
• Tissue residence time exceeds plasma half-life
• Biological effects observed beyond detectable plasma presence

Dosing Considerations for Research:

• Studies utilize broad dose ranges (0.1-10 mg/kg in rodent models)
• Dose selection depends on research model and endpoints
• Both acute and chronic dosing protocols investigated
• Twice-daily dosing common for chronic studies based on half-life
• Route selection influences pharmacokinetic profile

These pharmacokinetic characteristics inform research protocol design, particularly regarding dosing regimens, tissue sampling timing, and duration of biological effects in experimental models.

Research Applications

Cardiovascular Research

SS-31 serves as a research tool for investigating mitochondrial dysfunction in cardiac pathology:

Cardiac Ischemia-Reperfusion Research:

• Investigation of protection mechanisms against ischemia-reperfusion injury
• Studies on mitochondrial membrane potential preservation
• Analysis of ROS generation reduction during reperfusion
• Examination of cytochrome c release prevention
• Research on infarct size reduction mechanisms

Heart Failure Models:

• Studies in pressure-overload heart failure models
• Investigation of diastolic dysfunction and mitochondrial energetics
• Research on cardiac remodeling and fibrosis
• Examination of contractile function preservation
• Analysis of mitochondrial structure (cristae) maintenance

Cardiac Aging Research:

• Investigation of age-related mitochondrial dysfunction in heart
• Studies on mitochondrial ROS and oxidative damage
• Research on diastolic dysfunction in aging
• Examination of mitochondrial quality control mechanisms
• Analysis of cardiac energetics in aged animals

Research protocols employ ex vivo perfused heart systems, in vivo coronary ligation models, pressure-overload models (TAC), and aged animal models to characterize SS-31’s cardioprotective mechanisms.

Neurodegenerative Research

Laboratory studies investigate SS-31 in neurological disease models:

Neurodegenerative Disease Models:

• Alzheimer’s disease model research (APP/PS1 mice, 3xTg mice)
• Parkinson’s disease model investigation (MPTP, 6-OHDA models)
• Huntington’s disease research (R6/2 transgenic models)
• ALS model studies (SOD1 transgenic mice)
• Multiple sclerosis research (EAE models)

Mechanistic Investigations:

• Neuronal mitochondrial function preservation
• Synaptic dysfunction and plasticity research
• Neuroinflammation and microglial activation studies
• Blood-brain barrier penetration verification
• Cognitive function assessment in disease models

Acute Neurological Injury:

• Traumatic brain injury model research
• Stroke and cerebral ischemia investigations
• Spinal cord injury studies
• Optic nerve injury research
• Retinal degeneration models

SS-31’s blood-brain barrier permeability enables investigation of central nervous system mitochondrial dysfunction, distinguishing it from non-penetrant mitochondrial therapies.

Metabolic and Age-Related Research

Research applications extend to metabolic dysfunction and aging:

Metabolic Syndrome Research:

• Diabetes model investigations (Type 1 and Type 2)
• Insulin resistance mechanism studies
• Beta cell function and survival research
• Diabetic cardiomyopathy models
• Diabetic nephropathy research

Skeletal Muscle Research:

• Age-related muscle dysfunction (sarcopenia) studies
• Muscle fatigue and exercise performance research
• Mitochondrial biogenesis pathway investigation
• Metabolic myopathy model research
• Muscle regeneration and repair studies

Aging Research:

• Mitochondrial theory of aging investigations
• Age-related mitochondrial DNA mutations
• Mitochondrial quality control in aging
• Healthspan and functional capacity studies
• Cellular senescence and mitochondrial dysfunction

Laboratory protocols investigate SS-31’s effects on glucose homeostasis, insulin signaling, mitochondrial respiration, ATP production, and age-related functional decline.

Renal Research Applications

SS-31 serves as research tool in kidney injury models:

Acute Kidney Injury:

• Ischemia-reperfusion injury research
• Cisplatin nephrotoxicity models
• Contrast-induced nephropathy studies
• Sepsis-associated AKI research
• Transplantation models

Chronic Kidney Disease:

• Progressive CKD model investigations
• Diabetic nephropathy research
• Glomerulosclerosis studies
• Tubulointerstitial fibrosis mechanisms
• Mitochondrial dysfunction in CKD progression

Renal tissue’s high mitochondrial content and energy demands make it particularly susceptible to mitochondrial dysfunction, creating valuable research models for SS-31 investigation.

Pulmonary and Hepatic Research

Additional organ system investigations include:

Pulmonary Research:

• Acute lung injury and ARDS models
• Pulmonary hypertension research
• Radiation-induced lung injury studies
• Fibrosis mechanism investigations

Hepatic Research:

• Non-alcoholic fatty liver disease (NAFLD) models
• Ischemia-reperfusion injury in liver
• Drug-induced hepatotoxicity research
• Liver fibrosis and cirrhosis studies

Molecular Mechanisms Under Investigation

SS-31 research examines multiple potential mechanisms:

Cardiolipin Stabilization:

• Prevents cardiolipin oxidation and degradation
• Maintains cardiolipin localization in inner membrane
• Preserves cardiolipin’s scaffolding functions
• Stabilizes electron transport chain supercomplexes
• Maintains cristae structure integrity

ROS Reduction:

• Decreases mitochondrial superoxide generation
• Reduces hydrogen peroxide production
• Prevents lipid peroxidation
• Decreases protein oxidation and carbonylation
• Protects mitochondrial DNA from oxidative damage

Bioenergetic Enhancement:

• Improves electron transport chain efficiency
• Enhances ATP synthesis capacity
• Optimizes coupling of respiration to ATP production
• Preserves mitochondrial membrane potential
• Maintains calcium handling capacity

Apoptosis Modulation:

• Prevents cytochrome c release from mitochondria
• Inhibits caspase activation pathways
• Reduces apoptotic cell death in injury models
• Maintains BAX/BAK regulation
• Preserves mitochondrial outer membrane integrity

Mitochondrial Dynamics:

• Influences fission-fusion balance
• Affects mitophagy and quality control
• Modulates mitochondrial biogenesis signaling
• Impacts mitochondrial network architecture

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
• Stability data available for 12+ months at -20°C
• Avoid repeated exposure to room temperature

Reconstitution Guidelines:

• Reconstitute with sterile water, saline, or phosphate buffered saline
• Add solvent slowly down vial side to minimize foaming
• Gentle swirling motion recommended (avoid vigorous shaking)
• Allow complete dissolution before use (typically 1-2 minutes)
• Final pH should be 6.0-8.0 for optimal stability
• Typical reconstitution concentrations: 1-10 mg/mL

Reconstituted Solution Storage:

• Short-term storage: 4°C for up to 7 days
• Long-term storage: -20°C in single-use aliquots
• Aliquot preparation prevents repeated freeze-thaw cycles
• Maximum recommended freeze-thaw cycles: 2-3
• Protect from light during storage

Working Solution Preparation:

• Dilute stock solution in appropriate experimental buffer or media
• Filter sterilize if required for cell culture (0.22 μm filter)
• Prepare working solutions fresh when possible
• Consider vehicle controls matching final solvent composition

Quality Assurance and Analytical Testing

Each SS-31 batch undergoes comprehensive analytical characterization:

Purity Analysis:

• High-Performance Liquid Chromatography (HPLC): ≥98% purity
• Analytical method: Reversed-phase HPLC with UV detection at 220nm and 280nm
• Multiple wavelength detection to confirm peak identity
• Integration of all peaks for accurate purity determination

Structural Verification:

• Electrospray Ionization Mass Spectrometry (ESI-MS): Confirms molecular weight 640.7 Da
• Amino acid analysis: Verifies sequence composition
• Peptide content determination: Quantifies actual peptide content by weight
• Chiral verification when applicable

Contaminant Testing:

• Bacterial endotoxin: <5 EU/mg (Limulus Amebocyte Lysate method)
• Heavy metals: Below detection limits per USP standards
• Residual solvents: TFA, acetonitrile within ICH guidelines
• Water content: Karl Fischer titration (<8%)
• Microbial contamination: Sterility testing per USP standards

Documentation:

• Certificate of Analysis (COA) provided with each batch
• Includes analytical chromatograms and spectra
• Third-party analytical verification available upon request
• Stability data documented for recommended storage conditions
• Batch-specific QC results traceable by lot number
• Complete chain of custody documentation

Research Considerations

Experimental Design Factors:

Researchers should consider several factors when designing SS-31 experiments:

1. Concentration Selection: Published research reports effective concentrations ranging from nanomolar (in vitro cell culture) to micromolar (some assay systems). Dose-response characterization recommended for new experimental systems.

2. Temporal Considerations: SS-31’s plasma half-life of 2-4 hours informs dosing schedules. Tissue accumulation and residence time exceed plasma presence. Timing of tissue collection and functional measurements should account for pharmacokinetic profile.

3. Route Considerations: Intravenous, intraperitoneal, and subcutaneous routes show efficacy in preclinical models. Route selection influences onset time and tissue distribution patterns. Oral administration investigated in some research models.

4. Model Selection: Choose appropriate cell culture systems (primary cells, cell lines), isolated mitochondria, isolated perfused organs, or in vivo animal models based on specific research questions.

5. Control Groups: Include appropriate vehicle controls, sham-operated controls (surgical models), and positive controls where applicable. Consider using structurally similar but inactive peptides as negative controls.

6. Outcome Measures: Mitochondrial function assessment methods include respirometry (Seahorse, Oroboros), ATP measurement, membrane potential (TMRM, JC-1), ROS detection, electron microscopy for structural analysis, and cardiolipin analysis.

Mechanism Investigation:

SS-31’s mechanisms involve cardiolipin interactions, ROS reduction, bioenergetic enhancement, and structural protection. Experimental approaches to mechanism investigation include:

• Cardiolipin Binding Studies: Liposome binding assays, surface plasmon resonance, isothermal titration calorimetry
• Mitochondrial Function: High-resolution respirometry, ATP synthesis rates, membrane potential measurements
• ROS Detection: Fluorescent probes (MitoSOX, H2DCFDA), electron paramagnetic resonance, lipid peroxidation assays
• Structural Analysis: Transmission electron microscopy, super-resolution microscopy, cristae quantification
• Molecular Pathways: Western blotting for apoptotic proteins, gene expression analysis, proteomic approaches

Multiple complementary approaches provide comprehensive mechanism characterization.

Clinical Translation Context

Clinical Development Background:

SS-31 (Elamipretide) has progressed through clinical trials for several conditions, providing context for research applications:

• Phase 2 trials completed in heart failure with preserved ejection fraction (HFpEF)
• Phase 2 trials in primary mitochondrial myopathy (Barth syndrome, mitochondrial diseases)
• Phase 1 safety and pharmacokinetic studies completed
• Clinical program name: Elamipretide (formerly Bendavia)
• Development company: Stealth BioTherapeutics

Research use of SS-31 builds on this clinical development foundation while investigating fundamental mechanisms, expanding disease model applications, and characterizing biological pathways.

Compliance and Safety Information

Regulatory Status:
SS-31 (Elamipretide) is provided as a research chemical for in-vitro laboratory studies and preclinical research only. Clinical trials are conducted under separate regulatory frameworks. This research-grade product has not been approved by the FDA for human therapeutic use, dietary supplementation, or medical applications.

Intended Use:

• In-vitro cell culture studies and isolated mitochondria research
• In-vivo preclinical research in approved animal models
• Laboratory investigation of mitochondrial biology and dysfunction
• Academic and institutional research applications
• Mechanism of action characterization

NOT Intended For:

• Human consumption or administration
• Therapeutic treatment or diagnosis
• Dietary supplementation
• Veterinary therapeutic applications without appropriate oversight
• Self-administration or personal experimentation

Safety Protocols:
Researchers should follow standard laboratory safety practices when handling SS-31:

• Use appropriate personal protective equipment (lab coat, gloves, safety glasses)
• Handle in well-ventilated areas
• Follow institutional biosafety guidelines and IACUC protocols
• Dispose of waste according to local regulations for biological/chemical waste
• Consult material safety data sheet (MSDS) for additional safety information
• Maintain laboratory records and inventory logs

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