Key findings
- A polarity-sensitive fluorescent analogue of the peptide bound cardiolipin with high affinity, and the resulting complex inhibited cytochrome c peroxidase activity by protecting the heme iron. [1]
- Chemical cross-linking with mass spectrometry found that every mitochondrial protein interactor recovered was a known cardiolipin binder, split between oxidative phosphorylation proteins and 2-oxoglutarate metabolism proteins, with cross-linked residues often near cardiolipin contact regions. [2]
- A genome-scale CRISPR screen identified phospholipid scramblase 3 as required for the mitoprotective effect, and biochemical work showed the peptide binding a previously uncharacterized N-terminal domain of that protein and stimulating its scramblase activity. [3]
- NMR and molecular dynamics models of the membrane-bound state show that three related analogues form compact reverse turns while this peptide does not, and the four differ in equilibrium binding and in their effect on membrane surface charge. [4]
Identity and structure
- Membrane partner
- Cardiolipin, the anionic phospholipid of the inner mitochondrial membrane that is required for cristae formation [1]
- Candidate protein target
- Phospholipid scramblase 3, an inner mitochondrial membrane protein whose N-terminal domain the peptide binds directly [3]
- Conformation
- Unlike three analogues tested beside it, this sequence does not adopt a compact reverse turn in the membrane-bound state [4]
- Form as supplied
- Sterile lyophilized powder
Mechanism as studied
The original account is electrostatic and lipid centred. Binding to cardiolipin is reported to protect cristae membranes and to inhibit the peroxidase activity of cytochrome c, which otherwise catalyses cardiolipin peroxidation, and the structure-activity work supports modulation of membrane surface charge as a shared feature of the peptide class. [1][4]
Cross-linking places the peptide among cardiolipin-binding proteins rather than at a single receptor. The interactors fall into an oxidative phosphorylation group and a 2-oxoglutarate metabolism group, and many cross-linked residues sit close to the regions where those proteins contact cardiolipin. [2]
A genetic screen supplies a protein-level requirement that the lipid account does not. Loss of phospholipid scramblase 3 abolished the protective effect in cells and in conditional knockout mice without changing baseline kidney function, and the peptide bound that protein directly and stimulated its scramblase activity. [3]
Research findings
In vitro
- System
- Isolated mitochondria and cardiolipin preparations probed with a polarity-sensitive fluorescent analogue of the peptide rather than the unlabelled material, with a parallel rat renal ischemia arm
- Measured
- Binding affinity of the labelled analogue for cardiolipin, cytochrome c peroxidase activity of the peptide cardiolipin complex, and cristae membrane integrity and mitochondrial swelling after renal ischemia
- Reported
- The labelled analogue bound cardiolipin with high affinity. The complex inhibited cytochrome c peroxidase activity by protecting the heme iron. In the rat arm cristae membranes were preserved during renal ischemia and mitochondrial swelling was prevented, with faster ATP recovery on reperfusion. [1]
Structural
- System
- This peptide benchmarked against three alternative tetrapeptide analogues differing in aromatic side chain composition and sequence register, in cardiolipin-containing model membranes and in mammalian cell culture
- Measured
- Membrane-bound conformation by NMR and molecular dynamics, equilibrium binding to cardiolipin-containing membranes, membrane surface charge, cell permeation and mitochondrial targeting, and mitochondrial membrane potential, ATP content and survival under serum withdrawal
- Reported
- All analogues except this peptide formed compact reverse turn conformations when membrane bound. All four bound cardiolipin-containing membranes but differed in equilibrium binding behaviour and markedly in their effect on membrane surface charge. None required a particular side chain composition or register to permeate cells and reach mitochondria, yet they differed in restoring membrane potential, preserving ATP and supporting survival, with the tryptophan-containing analogue strongest. [4]
Analytical
- System
- Intact mitochondria treated with the peptide and analysed by chemical cross-linking coupled to mass spectrometry
- Measured
- Identity of cross-linked mitochondrial protein interactors, the functional grouping of those proteins and the position of cross-linked residues relative to known cardiolipin contact regions
- Reported
- All recovered interactors were known cardiolipin binders, falling into an oxidative phosphorylation group and a 2-oxoglutarate metabolism group. Many cross-linked residues lie proximal to the regions where those proteins interact with cardiolipin. [2]
In vitro
- System
- A genome-scale CRISPR screen in a human kidney cell model of cisplatin-associated mitochondrial dysfunction, with knockout lines, siRNA knockdown, primary murine tubular epithelial cells and tubular epithelial conditional knockout mice for validation
- Measured
- Screen hits required for the mitoprotective effect, validation of the hit by gene ablation in cells and mice, and direct biochemical interaction of the peptide with the identified protein
- Reported
- Phospholipid scramblase 3 was essential for the protective effect. Ablating it did not change kidney function at baseline or the severity of the injury models, but it completely abolished the protective effect of the peptide. The peptide bound a previously uncharacterized N-terminal domain of the protein and stimulated its scramblase activity. [3]
Ex vivo
- System
- Permeabilized ventricular fibers and mitochondria from rats after cardiac ischemia reperfusion, plus biomimetic cardiolipin-containing membranes
- Measured
- Respirometry activity of complexes I, II and IV, three dimensional cristae ultrastructure by serial block face scanning electron microscopy, cardiolipin concentration by mass spectrometry, and biophysical properties of the model membranes
- Reported
- The ischemia reperfusion decrements in complex I, II and IV activity were alleviated and the fragmentation of cristae networks was reduced. The peptide did not prevent the fall in cardiolipin concentration after ischemia reperfusion. In model membranes it aggregated cardiolipin and changed the biophysical properties of the bilayer. [5]
In vitro
- System
- INS1 beta-cell cultures under control conditions and after short-term nutrient excess
- Measured
- Mitochondrial morphology and membrane potential, engulfment of mitochondria into autophagosomes, lysosomal function, p62 and LC3II accumulation and insulin secretory function
- Reported
- Engulfment of mitochondria into autophagosomes increased in control cells without widespread change in mitochondrial morphology or membrane potential. Under nutrient excess the peptide prevented mitochondrial fragmentation and the defect in engulfment, but it did not prevent the lysosomal defects, the p62 and LC3II accumulation or the change in secretory function. [6]
Handling for in-vitro work
- Labelled analogue in cited work
- The cardiolipin binding measurement used a polarity-sensitive fluorescent analogue, so the reported affinity describes the labelled construct [1]
- Membrane system in cited work
- Binding and surface charge measurements used cardiolipin-containing model membranes rather than intact mitochondria [4][5]
- Storage
- Lyophilized at -20 °C, dark and dry; reconstituted aliquots kept cold and used promptly
Open questions
- The cited work offers two accounts of the target, a lipid interaction with cardiolipin and a direct protein interaction with phospholipid scramblase 3, and does not reconcile them into one mechanism.
- The affinity figure for cardiolipin comes from a fluorescent analogue, so the unlabelled material has no independently measured affinity in the cited set.
- This sequence is the one analogue in the structure-activity study that does not form a compact reverse turn when membrane bound, and the cited work does not explain why it remains active without that fold.
- In the cardiac model the peptide preserved cristae structure without preventing the loss of cardiolipin itself, leaving the relationship between lipid amount and cristae integrity open.
A clinical trial literature on this tetrapeptide exists and concerns finished formulations; it is out of scope for a research material profile and none of it is summarized here.
Lot records
Check the record for the exact material you order. A published paper and a batch certificate answer different questions.
- RV-24-0013-2 ↗SS-31 · 99.60% HPLC2026-09-22
- RV-24-0013-1 ↗SS-31 · 99.60% HPLC2026-09-16
References
- Birk AV, Liu S, Soong Y, et al. The mitochondrial-targeted compound SS-31 re-energizes ischemic mitochondria by interacting with cardiolipin. Journal of the American Society of Nephrology : JASN. 2013.
- Chavez JD, Tang X, Campbell MD, et al. Mitochondrial protein interaction landscape of SS-31. Proceedings of the National Academy of Sciences of the United States of America. 2020.
- Silvaroli JA, Bisunke B, Kim JY, et al. Genome-Wide CRISPR Screen Identifies Phospholipid Scramblase 3 as the Biological Target of Mitoprotective Drug SS-31. Journal of the American Society of Nephrology : JASN. 2024.
- Mitchell W, Tamucci JD, Ng EL, et al. Structure-activity relationships of mitochondria-targeted tetrapeptide pharmacological compounds. eLife. 2022.
- Allen ME, Pennington ER, Perry JB, et al. The cardiolipin-binding peptide elamipretide mitigates fragmentation of cristae networks following cardiac ischemia reperfusion in rats. Communications biology. 2020.
- Petcherski A, Trudeau KM, Wolf DM, et al. Elamipretide Promotes Mitophagosome Formation and Prevents Its Reduction Induced by Nutrient Excess in INS1 β-cells. Journal of molecular biology. 2018.
Publication records fetched from PubMed on 2026-09-20. Profile text reviewed 2026-09-20.