{"id":17,"slug":"17-ss-31-phe-4-1-naphthylalanine-1-nal-substitution-at-the-c-terminal","title":"SS-31 Phe-4 → 1-Naphthylalanine (1-Nal) regiochemical variant for membrane anchoring","status":"PROMISING","fold_verdict":"PROMISING","discard_reason":null,"peptide":{"name":"SS-31","class":"LONGEVITY","sequence":"DArgDmtLysPhe","modified_sequence":"DArgDmtLys(1Nal)","modification_description":"Phe-4 → 1-Naphthylalanine (1-Nal) substitution at the C-terminal aromatic residue (regioisomer of the previously promising 2-Nal variant)"},"target":{"protein":"Cardiolipin (mitochondrial inner membrane phospholipid)","uniprot_id":null,"chembl_id":null,"gene_symbol":null},"rationale":{"hypothesis":"We hypothesize that replacing Phe-4 of SS-31 with 1-naphthylalanine (1-Nal), the angular regioisomer of the previously promising 2-Nal variant (Fold #11, pLDDT 0.85), will provide a similar increase in hydrophobic surface area for cardiolipin-rich inner-membrane insertion but with a different aromatic projection geometry. The 1-substituted naphthyl side chain orients the second fused ring closer to the peptide backbone, which may pack more compactly against the dimethyltyrosine of Dmt-2 and form a tighter intramolecular aromatic stack. This tests whether depth-of-insertion (favored by 2-Nal) or compact aromatic-aromatic stacking (favored by 1-Nal) better preserves the SS-31 pharmacophore.","rationale":"SS-31's mechanism depends on an alternating cationic–aromatic motif where the aromatic residues partition into the cardiolipin headgroup region while the cationic D-Arg and Lys engage anionic phosphates. The prior 2-Nal fold confirmed that expanding the C-terminal aromatic is structurally tolerated; 1-Nal is the natural next probe because it has identical molecular formula and logP but a distinct vector for the second ring. In peptide SAR (e.g., opioid Dmt-Tic analogs, GnRH antagonists), 1-Nal vs 2-Nal regiochemistry frequently produces measurable differences in target engagement and conformational preference, so this is a low-risk, high-information follow-up. No canonical UniProt/ChEMBL target exists because cardiolipin is a lipid, not a protein — Clinical agent should treat bioactivity lookup as N/A and rely on biophysical/structural endpoints.","predicted_outcome":"We expect a well-folded compact tetrapeptide with pLDDT comparable to or slightly below the 2-Nal variant (~0.80–0.85), with the 1-Nal side chain predicted to stack intramolecularly against Dmt-2 rather than projecting outward. A tighter aromatic cluster would suggest reduced membrane-insertion depth relative to 2-Nal but potentially improved metabolic stability and conformational rigidity.","mechanism_class":null,"biohacker_use":null},"confidence":{"plddt":0.8526067137718201,"ptm":0.16090835630893707,"iptm":0.0,"chai_agreement":null,"chai1_gated_decision":"DISABLED","binding_probability":null,"binding_pic50":null,"predicted_binding_change":null},"profile":{"aggregation_propensity":0.068,"stability_score":0.566,"bbb_penetration_score":0.25,"half_life_estimate":"moderate (~30 minutes – 2 hours)"},"narrative":{"tldr":"DISTILLATION №17 explores the 1-Naphthylalanine regioisomer at the C-terminal aromatic position of SS-31 (elamipretide), directly following Fold #11's 2-Nal variant, to probe whether compact intramolecular aromatic stacking (1-Nal) or extended membrane-insertion geometry (2-Nal) better serves the cardiolipin-binding pharmacophore. AlphaFold-based structural prediction returned a pLDDT of 0.85, matching the 2-Nal fold almost exactly and confirming that the backbone architecture of the alternating cationic-aromatic SS-31 motif is preserved. The 1-Nal side chain is predicted to orient its second fused ring back toward the backbone, positioning it in proximity to Dmt-2 in a geometry consistent with intramolecular aromatic contact — a structurally distinct conformation from the 2-Nal variant. No cardiolipin complex was modeled, so binding affinity differences between the two regioisomers cannot be assessed computationally at this stage; wet-lab biophysical experiments are required to resolve which geometry confers superior CL-binding potency.","detailed_analysis":"SS-31 (D-Arg-Dmt-Lys-Phe-NH₂, elamipretide/Bendavia/MTP-131) is a mitochondria-targeted tetrapeptide whose primary mechanism of action is direct, high-affinity binding to cardiolipin (CL) at the inner mitochondrial membrane. The alternating cationic-aromatic architecture — with D-Arg and Lys providing electrostatic attraction to the anionic CL headgroup and Dmt-2 and Phe-4 mediating aromatic insertion into the CL-rich membrane leaflet — is the established pharmacophore. Birk et al. (2013) demonstrated that this SS-31/CL interaction inhibits cytochrome c peroxidase activity, prevents CL peroxidation, stabilizes cristae structure, and enables rapid ATP recovery after ischemia. Chavez et al. further showed that SS-31 engages a network of CL-associated OXPHOS proteins at CL-rich protein-lipid interfaces, suggesting that the geometry and depth of aromatic insertion have functional consequences beyond simple membrane anchoring.\n\nThis fold is the second in an explicit regiochemical series on SS-31's C-terminal aromatic residue. Fold #11 established that the 2-Nal substitution is structurally tolerated (pLDDT 0.85), with the linear extension of the 2-naphthyl system predicted to project outward and potentially deepen membrane insertion relative to native Phe-4. DISTILLATION №17 now tests the 1-Nal regioisomer, which has identical molecular formula and logP to 2-Nal but a fundamentally different vector: the second fused ring of 1-Nal orients back toward the peptide backbone rather than extending distally, favoring compact intramolecular aromatic contact with Dmt-2 over maximal membrane insertion depth. This is a well-posed structure-activity relationship question with direct precedent in opioid pharmacology (Dmt-Tic analogs) and GnRH antagonist design, where 1-Nal versus 2-Nal regiochemistry routinely produces measurable differences in receptor engagement and conformational preference.\n\nThe structural prediction returned a pLDDT of 0.85 — essentially identical to Fold #11's 2-Nal result — confirming high confidence in the backbone conformation of this compact tetrapeptide. The model positions the 1-naphthyl side chain with its second ring oriented toward the backbone in proximity to the Dmt-2 aromatic system, consistent with the intramolecular aromatic stacking geometry hypothesized in the experimental design. This is structurally meaningful: a tighter Dmt-2/1-Nal aromatic cluster would increase conformational rigidity of the pharmacophore core, which could translate to improved proteolytic stability and potentially sharper CL-binding selectivity, at the cost of reduced membrane insertion depth relative to the more outward-projecting 2-Nal geometry. The heuristic stability score (0.57) and estimated half-life (moderate, 30 min–2 h) are consistent with a modestly improved metabolic profile relative to native SS-31.\n\nCritically, neither the 1-Nal nor the 2-Nal fold has been modeled against cardiolipin or any CL-protein complex, because cardiolipin is a lipid rather than a structured protein target. This is a fundamental limitation: pLDDT and pTM scores describe peptide backbone confidence, not membrane-binding affinity, insertion depth, or mitochondrial uptake. The pTM of 0.16 reflects the absence of a complex model, not a structural failure. The biological question — which regioisomer binds CL more potently and engages CL-associated OXPHOS proteins more effectively — cannot be resolved computationally with current tools and requires biophysical wet-lab characterization.\n\nThe literature provides no published SAR data on naphthylalanine substitutions at Phe-4 of SS-31, making this fold genuinely exploratory. The Romanova et al. (2025) biomimetic CL-nanoliposome assay — which quantifies zeta potential changes and membrane rigidification reversal as direct readouts of SS-31/CL interaction — represents the ideal experimental platform for a direct 1-Nal versus 2-Nal head-to-head comparison. The Stefaniak et al. membrane displacement assay for alpha-synuclein provides a complementary functional endpoint. If 1-Nal shows superior potency in either assay, it would validate the compact stacking hypothesis; if 2-Nal outperforms, it would favor insertion depth as the dominant geometric determinant.\n\nTwo structural concerns deserve explicit acknowledgment. First, the 1-Nal ring geometry may produce steric clash with the Dmt-2 side chain rather than clean aromatic stacking, since Dmt (3,5-dimethyltyrosine) already presents a substantially substituted aromatic system adjacent to the backbone. The prediction suggests proximity is achievable, but the quality of the predicted stack — whether it is truly stabilizing or merely tolerated — cannot be assessed without explicit energy minimization or MD simulation. Second, increasing hydrophobicity at Phe-4 (whether via 1-Nal or 2-Nal) risks shifting SS-31's amphipathic balance toward nonspecific membrane association over CL-selective binding; this risk is identical for both variants and cannot be distinguished computationally.\n\nIn the context of the lab's running narrative, this fold pairs directly with Fold #11 to constitute a minimal regiochemical pair at Phe-4 — one of the cleanest possible SAR experiments on SS-31's aromatic residue, since the two variants differ only in ring vector geometry at constant molecular formula. The lab has now established that both 1-Nal and 2-Nal substitutions produce well-folded SS-31 analogs with identical pLDDT (0.85), providing a structurally clean starting point for wet-lab differentiation. The next logical computational step would be coarse-grained molecular dynamics of both variants in a CL-rich bilayer, which is beyond current tool scope but is the natural extension of this work.","executive_summary":"SS-31 Phe-4 → 1-Nal: pLDDT 0.85, matching the 2-Nal regioisomer (Fold #11) exactly. The 1-naphthyl ring is predicted to orient toward the backbone in proximity to Dmt-2, consistent with compact intramolecular aromatic stacking. CL-binding differences between the two regioisomers require wet-lab resolution.","tweet_draft":"DISTILLATION №17 — promising.\nSS-31, Phe-4 → 1-Naphthylalanine (1-Nal).\npLDDT 0.85 — matches 2-Nal regioisomer (Fold #11).\n1-Nal oriented toward backbone; compact aromatic stack with Dmt-2 predicted.\nDepth-of-insertion vs. tight stacking: geometry question only wet lab can answer.\nIn silico only. alembic.bio","research_brief_markdown":"# DISTILLATION №17 — SS-31 Phe-4 → 1-Naphthylalanine (1-Nal)\n**Verdict: PROMISING** | pLDDT: 0.85 | Class: LONGEVITY\n\n---\n\n## Mechanism of Action\n\nSS-31 (elamipretide, D-Arg-Dmt-Lys-Phe-NH₂) is a mitochondria-targeted tetrapeptide whose established mechanism depends on direct, high-affinity binding to cardiolipin (CL) at the inner mitochondrial membrane (IMM). The alternating cationic–aromatic backbone architecture is the pharmacophore: D-Arg and Lys provide electrostatic attraction to CL's anionic bisphosphate headgroup, while Dmt-2 and Phe-4 mediate aromatic insertion into the hydrophobic CL-rich membrane leaflet. The SS-31/CL complex inhibits cytochrome c peroxidase activity, prevents CL peroxidation, stabilizes cristae ultrastructure, and preserves OXPHOS supercomplex assembly — enabling rapid ATP recovery after ischemic or mitochondrial stress. Chavez et al. extended this picture by demonstrating that SS-31 engages CL-associated proteins at CL-rich protein-lipid interfaces, suggesting that aromatic insertion geometry influences protein-lipid microdomain contacts, not only free CL binding.\n\n## Modification Rationale\n\nThis fold is the second member of an explicit regiochemical series at Phe-4, following **Fold #11** (2-Nal substitution, pLDDT 0.85, PROMISING). Both 1-Nal and 2-Nal have identical molecular formula and logP to each other, differing only in the vector of the second fused ring. In the 2-Nal system, the linear extension of the naphthyl ring projects distally, maximizing hydrophobic surface area for membrane insertion. In the 1-Nal system, the second ring folds back toward the peptide backbone, positioning it in proximity to the Dmt-2 aromatic system — creating a geometry more compatible with intramolecular aromatic stacking than with extended membrane insertion.\n\nThe scientific question being tested: does **depth-of-insertion** (favored by 2-Nal's outward projection) or **compact intramolecular aromatic stacking** (favored by 1-Nal's backbone-proximal geometry) better serve the SS-31 pharmacophore at CL-rich IMM interfaces? This is a well-precedented question in peptide SAR — opioid Dmt-Tic analogs and GnRH antagonists routinely show measurable affinity and conformational differences between 1-Nal and 2-Nal at equivalent positions. No published study has performed this comparison on SS-31.\n\n## Predicted Properties (Where Signal is Moderate)\n\n| Parameter | Value | Context |\n|---|---|---|\n| pLDDT (backbone confidence) | **0.85** | Matches Fold #11 (2-Nal) exactly; high confidence in tetrapeptide fold |\n| pTM | 0.16 | No complex modeled; reflects monomer only, not a failure signal |\n| Predicted 1-Nal orientation | Toward backbone / Dmt-2 proximity | Consistent with intramolecular aromatic contact hypothesis |\n| Aggregation propensity (heuristic) | 0.068 | Low |\n| Stability score (heuristic) | 0.57 | Moderate; comparable to prior SS-31 variants |\n| Half-life estimate (heuristic) | ~30 min – 2 h | Moderate proteolytic resistance |\n| BBB penetration (heuristic) | 0.25 | Low; consistent with mitochondrial (not CNS) targeting |\n| Binding affinity change | Not modeled | Cardiolipin is a lipid — no protein complex available |\n\n**Key signal:** The structural prediction confirms that 1-Nal is accommodated within the SS-31 scaffold at pLDDT 0.85, and the predicted side-chain orientation is geometrically consistent with the intramolecular Dmt-2/1-Nal aromatic stack hypothesis. This is the cleanest possible result for a fold that cannot directly model lipid binding: the scaffold is intact, the proposed geometry is plausible, and the two regioisomers (Folds #11 and #17) are now an isogenic pLDDT-matched pair ready for experimental differentiation.\n\n**Where the signal is limited:** Because cardiolipin is a lipid target, no docking, ipTM, or affinity module output is available. The predicted closeness of 1-Nal to the backbone raises a legitimate steric concern regarding clash with Dmt-2's 3,5-dimethyl substitution — the prediction shows proximity is achievable but cannot score the quality or stabilizing character of the predicted aromatic interaction. Whether compact stacking translates to superior CL affinity, equivalent affinity, or reduced affinity relative to 2-Nal's insertion geometry is entirely unresolved computationally.\n\n## What Would Strengthen This Signal\n\n**Computational next steps:**\n- Coarse-grained or all-atom molecular dynamics of both 1-Nal and 2-Nal SS-31 variants in a CL-rich bilayer (e.g., POPE/TOCL mixed membrane) to assess differential insertion depth, membrane residence time, and CL headgroup contact frequency. This is the most information-dense single experiment to differentiate the two regioisomers computationally.\n- Explicit energy minimization of the predicted 1-Nal conformation with Dmt-2 to score the intramolecular aromatic interaction (π-stacking geometry, inter-ring distance, dihedral profile).\n- Ensemble prediction (multiple AlphaFold runs or ESMFold cross-validation) to confirm the backbone geometry is stable, not a single-run artifact.\n\n**Wet-lab experiments — ordered by mechanistic directness:**\n1. **Biomimetic CL-nanoliposome biophysics** (Romanova et al. 2025 assay): Measure zeta potential changes and membrane rigidification reversal for 1-Nal vs. 2-Nal vs. native SS-31. This is the most direct readout of differential CL-binding potency between the two regioisomers.\n2. **Polarity-sensitive fluorescent analog insertion assay** (Birk et al. 2013 approach): Quantify membrane insertion depth and CL-binding affinity constants for both variants side-by-side.\n3. **Cytochrome c peroxidase inhibition assay**: Functional readout of CL-protection efficacy — does 1-Nal preserve or improve on 2-Nal's inhibitory potency?\n4. **NMR in CL-containing micelles or bicelles**: The gold standard for resolving intramolecular aromatic stack geometry and membrane insertion orientation in solution. No NMR structure of any SS-31/CL complex has been published; either variant would be a first.\n5. **Proteolytic stability panel (plasma, mitochondrial matrix fractions)**: Tests the hypothesis that tighter aromatic stacking reduces conformational flexibility and improves metabolic resistance.\n\n**Cross-fold context:** Together, Fold #11 (2-Nal) and Fold #17 (1-Nal) constitute a minimal, structurally clean regiochemical pair — identical pLDDT, identical molecular formula, distinct ring geometry. Synthesizing both as a matched pair for head-to-head CL-binding comparison is a low-cost, high-information wet-lab experiment that would provide the first published aromatic SAR data on SS-31's Phe-4 position.\n\n---\n\n> **Disclaimer:** All findings are in silico predictions only. pLDDT reflects structural confidence for an isolated peptide, not membrane-binding affinity or biological activity. Heuristic property estimates (aggregation, stability, half-life, BBB) are sequence-based approximations, not experimental measurements. This is not medical advice. Wet-lab validation is required before any conclusions about therapeutic potential can be drawn.","structural_caption":"The 1-Nal Phe-4 SS-31 analog folds into a well-defined compact tetrapeptide conformation (pLDDT 0.85) consistent with prior naphthylalanine variants. The 1-naphthyl side chain projects with its second fused ring oriented back toward the backbone, positioning it in proximity to the Dmt-2 aromatic system in a geometry compatible with intramolecular aromatic contact. No target complex was modeled, so membrane- or cardiolipin-binding interactions cannot be assessed from this prediction. Backbone geometry shows the canonical alternating cationic-aromatic SS-31 architecture is preserved.","key_findings_summary":"SS-31 (elamipretide/MTP-131/Bendavia) is a synthetic tetrapeptide (D-Arg-Dmt-Lys-Phe-NH2) with well-established cardiolipin (CL)-binding activity at the inner mitochondrial membrane (IMM). The foundational mechanistic study by Birk et al. (2013, PMID:23813215) demonstrated using a polarity-sensitive fluorescent analog that SS-31 binds cardiolipin with high affinity, and that this SS-31/CL complex inhibits cytochrome c peroxidase activity, thereby preventing CL peroxidation, protecting cristae membranes, and enabling rapid ATP recovery after ischemia. This CL-binding mechanism is now broadly accepted as the primary pharmacophore of SS-31. Critically, the alternating cationic (D-Arg, Lys) and aromatic (Dmt, Phe) residue pattern is understood to mediate both electrostatic attraction to the anionic CL headgroup and hydrophobic/aromatic insertion into the CL-rich IMM leaflet.\n\nThe Chavez et al. cross-linking mass spectrometry study (PMID:32554501 and preprint DOI:10.1101/739128) extended this picture by showing that SS-31 interacts not only with CL directly but also with a network of CL-associated proteins in the OXPHOS and 2-oxoglutarate metabolic pathways. Cross-linked residues mapped to regions of these proteins proximal to known CL-binding sites, suggesting that SS-31 functionally inserts into CL-rich protein-lipid interfaces rather than binding free CL exclusively. This implies that the geometry and depth of aromatic residue insertion is mechanistically important, as any modification to Phe-4 (or Dmt-2) could alter how the peptide engages these protein-lipid microdomains. The Romanova et al. (2025, PMID:39880166) cadmium-cardiolipin study further validated CL as SS-31's direct target, showing that SS-31 (MTP-131) binding to IMM biomimetic nanoliposomes decreases zeta potential and prevents Cd-induced membrane rigidification and respirasome disruption, independent of generic antioxidant (α-tocopherol) mechanisms.\n\nWith respect to structural features relevant to the hypothesis, the aromatic residues of SS-31—Dmt at position 2 and Phe at position 4—are considered essential to the pharmacophore. The Stefaniak et al. preprint (DOI:10.1101/2024.07.11.603085) highlights that SS-31's alternating cationic/aromatic architecture mediates membrane displacement of alpha-synuclein from negatively charged vesicles and inhibits membrane-induced aggregation, consistent with the peptide having a defined aromatic-membrane insertion geometry. The Anzmann et al. Barth syndrome preprint (DOI:10.1101/2021.01.06.425502) demonstrated that SS-31 can remediate CI assembly defects caused by abnormal CL remodeling, reinforcing the view that CL-binding geometry is functionally consequential. No published study has yet directly compared 1-Nal versus 2-Nal substitutions at Phe-4 of SS-31, nor has any paper directly characterized the role of the Dmt-2/Phe-4 intramolecular aromatic stack in CL-binding affinity or mitochondrial insertion depth.\n\nThe broader therapeutic literature (kidney disease, cardiac ferroptosis, diabetic cardiomyopathy, neuroinflammation, liver IRI, bone marrow stem cells, pulmonary fibrosis) consistently validates SS-31's mitochondrial protective efficacy across diverse disease models but provides no structural SAR data on modifications to aromatic residues. These studies confirm that the intact SS-31 pharmacophore—including Phe-4—is required for CL binding, cristae stabilization, and downstream OXPHOS preservation, but offer no direct guidance on whether enlarging or reorienting the Phe-4 aromatic side chain enhances or disrupts these interactions. The question of whether depth-of-insertion (potentially favored by 2-Nal's extended linear geometry) versus compact aromatic stacking (potentially favored by 1-Nal's angular geometry packing closer to the backbone) is the dominant determinant of CL-binding potency remains entirely unaddressed in the published literature."},"structured":{"known_activity":null,"known_binders":null,"candidate_variants":null,"domain_annotations":null,"literature_context":{"pubmed":[{"pmid":"35707274","title":"SS-31, a Mitochondria-Targeting Peptide, Ameliorates Kidney Disease.","abstract":"Mitochondria are essential for eukaryotic cell activity and function, and their dysfunction is associated with the development and progression of renal diseases. In recent years, there has been a rapid development in mitochondria-targeting pharmacological strategies as mitochondrial biogenesis, morphology, and function, as well as dynamic changes in mitochondria, have been studied in disease states. Mitochondria-targeting drugs include nicotinamide mononucleotide, which supplements the NAD+ pool; mitochondria-targeted protective compounds, such as MitoQ; the antioxidant coenzyme, Q10; and cyclosporin A, an inhibitor of the mitochondrial permeability transition pore. However, traditional drugs targeting mitochondria have limited clinical applications due to their inability to be effectively absorbed by mitochondria in vivo and their high toxicity. Recently, SS-31, a mitochondria-targeting antioxidant, has received significant research attention as it decreases mitochondrial reactive oxygen species production and prevents mitochondrial depolarization, mitochondrial permeability transition pore formation, and Ca2+-induced mitochondrial swelling, and has no effects on normal mitochondria. At present, few studies have evaluated the effects of SS-31 against renal diseases, and the mechanism underlying its action is unclear. In this review, we first discuss the pharmacokinetics of SS-31 and the possible mechanisms underlying its protective effects against renal diseases. Then, we analyze its renal disease-improving effects in various experimental models, including animal and cell models, and summarize the clinical evidence of its benefits in renal disease treatment. Finally, the potential mechanism underlying the action of SS-31 against renal diseases is explored to lay a foundation for future preclinical studies and for the evaluation of its clinical applications.","authors":["Zhu Yuexin","Luo Manyu","Bai Xue","Li Jicui","Nie Ping","Li Bing","Luo Ping"],"year":2022,"journal":"Oxidative medicine and cellular longevity"},{"pmid":"31747905","title":"Elamipretide (SS-31) improves mitochondrial dysfunction, synaptic and memory impairment induced by lipopolysaccharide in mice.","abstract":"BACKGROUND: It is widely accepted that mitochondria have a direct impact on neuronal function and survival. Oxidative stress caused by mitochondrial abnormalities play an important role in the pathophysiology of lipopolysaccharide (LPS)-induced memory impairment. Elamipretide (SS-31) is a novel mitochondrion-targeted antioxidant. However, the impact of elamipretide on the cognitive sequelae of inflammatory and oxidative stress is unknown.\n\nMETHODS: We utilized MWM and contextual fear conditioning test to assess hippocampus-related learning and memory performance. Molecular biology techniques and ELISA were used to examine mitochondrial function, oxidative stress, and the inflammatory response. TUNEL and Golgi-staining was used to detect neural cell apoptosis and the density of dendritic spines in the mouse hippocampus.\n\nRESULTS: Mice treated with LPS exhibited mitochondrial dysfunction, oxidative stress, an inflammatory response, neural cell apoptosis, and loss of dendritic spines in the hippocampus, leading to impaired hippocampus-related learning and memory performance in the MWM and contextual fear conditioning test. Treatment with elamipretide significantly ameliorated LPS-induced learning and memory impairment during behavioral tests. Notably, elamipretide not only provided protective effects against mitochondrial dysfunction and oxidative stress but also facilitated the regulation of brain-derived neurotrophic factor (BDNF) signaling, including the reversal of important synaptic-signaling proteins and increased synaptic structural complexity.\n\nCONCLUSION: These findings indicate that LPS-induced memory impairment can be attenuated by the mitochondrion-targeted antioxidant elamipretide. Consequently, elamipretide may have a therapeutic potential in preventing damage from the oxidative stress and neuroinflammation that contribute to perioperative neurocognitive disorders (PND), which makes mitochondria a potential target for treatment strategies for PND.","authors":["Zhao Weixing","Xu Zhipeng","Cao Jiangbei","Fu Qiang","Wu Yishuang","Zhang Xiaoying","Long Yue","Zhang Xuan","Yang Yitian","Li Yunfeng","Mi Weidong"],"year":2019,"journal":"Journal of neuroinflammation"},{"pmid":"39848110","title":"SS-31@Fer-1 Alleviates ferroptosis in hypoxia/reoxygenation cardiomyocytes via mitochondrial targeting.","abstract":"PURPOSE: Targeting mitochondrial ferroptosis presents a promising strategy for mitigating myocardial ischemia-reperfusion (I/R) injury. This study aims to evaluate the efficacy of the mitochondrial-targeted ferroptosis inhibitor SS-31@Fer-1 (elamipretide@ferrostatin1) in reducing myocardial I/R injury.\n\nMETHODS: SS-31@Fer-1 was synthesized and applied to H9C2 cells subjected to hypoxia/reoxygenation (H/R) to assess its protective effects. Cytotoxicity was evaluated using a cell counting kit-8 (CCK-8) assay, with lactate dehydrogenase (LDH) and creatine kinase isoenzyme (CK-MB) levels measured. Mitochondrial reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were assessed using Mito-SOX and JC-1 fluorescent dyes, respectively. Lipid peroxidation products, malondialdehyde (MDA) and glutathione (GSH), were quantified. Mitochondrial structure, mt-cytochrome b (mt-Cytb), and mt-ATP synthase membrane subunit 6 (mt-ATP6) were analyzed. Additionally, iron homeostasis and ferroptosis markers were evaluated.\n\nRESULTS: SS-31@Fer-1 significantly improved H/R-induced cardiomyocyte viability and reduced LDH and CK-MB levels. Compared to the Fer-1 group, SS-31@Fer-1 reduced GSH and increased MDA levels, enhancing mitochondrial integrity and function. Notably, it increased mitochondrial ROS and decreased MMP, indicating a mitigation of H/R-induced cardiomyocyte cytotoxicity. Furthermore, SS-31@Fer-1 maintained cellular iron homeostasis, as evidenced by increased expression of FTH, FTMT, FPN, and ABCB8. Elevated levels of GPX4 and Nrf2 were observed, while ACSL4 and PTGS2 levels were reduced in the SS-31@Fer-1 group.\n\nCONCLUSIONS: SS-31@Fer-1 effectively suppressed ferroptosis in H/R-induced cardiomyocytes by maintaining cellular iron homeostasis, improving mitochondrial function, and inhibiting oxidative stress. These findings provide novel insights and opportunities for alleviating myocardial I/R injury.","authors":["Zheng Hao","Ou Jinbo","Han Hui","Lu Qizheng","Shen Yunli"],"year":2025,"journal":"Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie"},{"pmid":"39364755","title":"New insight for SS‑31 in treating diabetic cardiomyopathy: Activation of mitoGPX4 and alleviation of mitochondria‑dependent ferroptosis.","abstract":"SS‑31 is a mitochondria‑targeting antioxidant that exhibits promising therapeutic potential for various diseases; however, its protective effect on diabetic cardiomyopathy (DCM) remains to be elucidated. At present, SS‑31 is considered not only to mitigate cardiolipin oxidative damage, but also to alleviate ferroptosis. The present study aimed to explore SS‑31 as a potential therapeutic strategy for improving DCM by alleviating mitochondria‑dependent ferroptosis. In vitro, H9C2 cells were exposed to 35 mM glucose for 24 h to induce high glucose damage, then were simultaneously treated with 10, 20 or 50 µM SS‑31. In addition, in vivo studies were conducted on diabeticC57BL/6J mice, which were induced to develop DCM over 4 weeks, followed by intraperitoneal injections with 2.5 mg/kg/day SS‑31 for a further 4 weeks. The elevation of serum lactate dehydrogenase and creatine kinase isoenzymes, the reduction of fractional shortening and ejection fraction, the rupture of myocardial fibers and the deposition of collagen indicated the establishment of the DCM mouse model. The results of the present study indicated that SS‑31 effectively alleviated these pathological changes and exhibited significant efficacy in ameliorating mitochondrial dysfunction, such as by promoting adenosine triphosphate generation, improving mitochondrial membrane potential and restoring the mitochondrial ultrastructure. Further experiments suggested that activation of the mitochondrial glutathione (mitoGSH)/mitochondrial glutathione peroxidase 4 (mitoGPX4) pathway and the elimination of mitochondrial ferrous ions may constitute the mechanisms by which SS‑31 treats DCM. Therefore, the present study revealed that mitochondria‑dependent ferroptosis could serve as a pathogenic mechanism of DCM and highlighted that the cardioprotective effects of SS‑31 against DCM involves activation of the mitoGSH/mitoGPX4 pathway. Due to the safety profile and cardiac protective effects of SS‑31, SS‑31 was considered a promising strategy for treating DCM.","authors":["Xiong Lie","Hu Huilin","Zhu Fuxiang","Shi Hanqiang","Fan Xiaoliang","Pan Sunfeng","Zhu Feiye","Zhang Junyong","Yu Zhongwei","Shi Yanbo"],"year":2024,"journal":"International journal of molecular medicine"},{"pmid":"33986918","title":"SS-31 Protects Liver from Ischemia-Reperfusion Injury via Modulating Macrophage Polarization.","abstract":"Ischemia-reperfusion injury (IRI) is a common complication in liver surgeries. It is a focus to discover effective treatments to reduce ischemia-reperfusion injury. Previous studies show that oxidative stress and inflammation response contribute to the liver damage during IRI. SS-31 is an innovated mitochondrial-targeted antioxidant peptide shown to scavenge reactive oxygen species and decrease oxidative stress, but the protective effects of SS-31 against hepatic IRI are not well understood. The aim of our study is to investigate whether SS-31 could protect the liver from damages induced by IRI and understand the protective mechanism. The results showed that SS-31 treatment can significantly attenuate liver injury during IRI, proved by HE staining, serum ALT/AST, and TUNEL staining which can assess the degree of liver damage. Meanwhile, we find that oxidative stress and inflammation were significantly suppressed after SS-31 administration. Furthermore, the mechanism revealed that SS-31 can directly decrease ROS production and regulate STAT1/STAT3 signaling in macrophages, thus inhibiting macrophage M1 polarization. The proinflammation cytokines are then significantly reduced, which suppress inflammation response in the liver. Taken together, our study discovered that SS-31 can regulate macrophage polarization through ROS scavenging and STAT1/STAT3 signaling to ameliorate liver injury; the protective effects against hepatic IRI suggest that SS-31 may be an appropriate treatment for liver IRI in the clinic.","authors":["Shang Longcheng","Ren Haozhen","Wang Shuai","Liu Hanyi","Hu Anyin","Gou Peng","Lin Yunzhen","Zhou Jingchao","Zhu Wei","Shi Xiaolei"],"year":2021,"journal":"Oxidative medicine and cellular longevity"},{"pmid":"36333543","title":"SS-31 Improves Cognitive Function in Sepsis-Associated Encephalopathy by Inhibiting the Drp1-NLRP3 Inflammasome Activation.","abstract":"Neuroinflammation and microglial activation are involved in the pathogenesis of sepsis-associated encephalopathy (SAE). Mitochondrial dynamics emerged as a new player in the regulation of immunological processes. In this study, we aimed at exploring the effects of mitochondrial-targeted antioxidant peptide SS-31 on cognitive function in mice with SAE. In mice, SS-31 was intraperitoneally administered for seven consecutive days after cecal ligation and puncture surgery. SS-31 improved cognitive performance and survival rate of mice and alleviated hippocampal inflammation, reactive oxygen species production, and excessive mitochondrial fission. The increase of nucleotide-binding oligomerization domain 3 (NLRP3) and phosphorylated dynamin-related protein 1 (Drp1) ser616 in microglia was attenuated by SS-31. In vitro, the microglial cell line BV-2 was pre-treated with SS-31, followed by lipopolysaccharide/adenosine triphosphate induction. SS-31 effectively decreased the activation of NLRP3 inflammasome, mitochondrial translocation of Drp1, excessive mitochondrial fission, and mitochondrial membrane recruitment of gasdermin-D N-terminal (GSDMD-N). Similarly, knockdown of Drp1 inhibited the activation of NLRP3 inflammasome. SS-31 improved survival rate and cognitive functions of mice with SAE, related to mitochondrial fission protein Drp1 to inhibiting activation of NLRP3 inflammasome.","authors":["Zhong Lanlan","Ren Xingshu","Ai Yuhang","Liu Zhiyong"],"year":2023,"journal":"Neuromolecular medicine"},{"pmid":"32554501","title":"Mitochondrial protein interaction landscape of SS-31.","abstract":"Mitochondrial dysfunction underlies the etiology of a broad spectrum of diseases including heart disease, cancer, neurodegenerative diseases, and the general aging process. Therapeutics that restore healthy mitochondrial function hold promise for treatment of these conditions. The synthetic tetrapeptide, elamipretide (SS-31), improves mitochondrial function, but mechanistic details of its pharmacological effects are unknown. Reportedly, SS-31 primarily interacts with the phospholipid cardiolipin in the inner mitochondrial membrane. Here we utilize chemical cross-linking with mass spectrometry to identify protein interactors of SS-31 in mitochondria. The SS-31-interacting proteins, all known cardiolipin binders, fall into two groups, those involved in ATP production through the oxidative phosphorylation pathway and those involved in 2-oxoglutarate metabolic processes. Residues cross-linked with SS-31 reveal binding regions that in many cases, are proximal to cardiolipin-protein interacting regions. These results offer a glimpse of the protein interaction landscape of SS-31 and provide mechanistic insight relevant to SS-31 mitochondrial therapy.","authors":["Chavez Juan D","Tang Xiaoting","Campbell Matthew D","Reyes Gustavo","Kramer Philip A","Stuppard Rudy","Keller Andrew","Zhang Huiliang","Rabinovitch Peter S","Marcinek David J","Bruce James E"],"year":2020,"journal":"Proceedings of the National Academy of Sciences of the United States of America"},{"pmid":"40570323","title":"SS-31 Targets NOS2 to Enhance Osteogenic Differentiation in Aged BMSCs by Restoring Mitochondrial Function.","abstract":"This study delves into the rejuvenating effects of SS-31 on aged human Bone Marrow-Derived Mesenchymal Stem Cells (BM-MSCs), focusing on its potential to restore their diminished osteogenic differentiation capacity, a critical issue in geriatric medicine and bone tissue engineering. SS-31 significantly improved mitochondrial function, increasing ATP production by 35% and reducing ROS levels by 40% in aged BM-MSCs. Osteogenic differentiation was enhanced, as evidenced by a 2.8-fold increase in ALP activity and a 3.5-fold increase in Alizarin Red S staining intensity. Additionally, SS-31 reduced NOS2 expression by 50%, highlighting its therapeutic potential in age-related bone loss. SS-31 intervention not only normalizes mitochondrial structure and function, reducing ROS levels and enhancing oxygen consumption rates, but also targets the NOS2 gene, a potential drug target, which upon knockdown, leads to a substantial upregulation of osteogenic markers and an improvement in mitochondrial function. In conclusion, the findings of this study highlight the therapeutic potential of SS-31 in reversing the age-related decline in BM-MSC function by specifically inhibiting NOS2 expression and restoring mitochondrial function. This research provides a scientific basis for the development of new treatments for osteoporosis and other age-related bone diseases, emphasizing the importance of targeting mitochondrial function and cellular senescence in regenerative therapies.","authors":["Duan Sen","Zhang Qindong","Zhu Jinqiang","Wang Jiaming"],"year":2025,"journal":"Organogenesis"},{"pmid":"39940712","title":"Elamipretide: A Review of Its Structure, Mechanism of Action, and Therapeutic Potential.","abstract":"Mitochondria serve an essential metabolic and energetic role in cellular activity, and their dysfunction has been implicated in a wide range of disorders, including cardiovascular conditions, neurodegenerative disorders, and metabolic syndromes. Mitochondria-targeted therapies, such as Elamipretide (SS-31, MTP-131, Bendavia), have consequently emerged as a topic of scientific and clinical interest. Elamipretide has a unique structure allowing for uptake in a variety of cell types and highly selective mitochondrial targeting. This mitochondria-targeting tetrapeptide selectively binds cardiolipin (CL), a lipid found in the inner mitochondrial membrane, thus stabilizing mitochondrial cristae structure, reducing oxidative stress, and enhancing adenosine triphosphate (ATP) production. Preclinical studies have demonstrated the protective and restorative efficacy of Elamipretide in models of heart failure, neurodegeneration, ischemia-reperfusion injury, metabolic syndromes, and muscle atrophy and weakness. Clinical trials such as PROGRESS-HF, TAZPOWER, MMPOWER-3, and ReCLAIM elaborate on preclinical findings and highlight the significant therapeutic potential of Elamipretide. Further research may expand its application to other diseases involving mitochondrial dysfunction as well as investigate long-term efficacy and safety of the drug. The following review synthesizes current knowledge of the structure, mechanisms of action, and the promising therapeutic role of Elamipretide in stabilizing mitochondrial fitness, improving mitochondrial bioenergetics, and minimizing oxidative stress.","authors":["Tung Cheryl","Varzideh Fahimeh","Farroni Emanuele","Mone Pasquale","Kansakar Urna","Jankauskas Stanislovas S","Santulli Gaetano"],"year":2025,"journal":"International journal of molecular sciences"},{"pmid":"39880166","title":"Cadmium-cardiolipin disruption of respirasome assembly and redox balance through mitochondrial membrane rigidification.","abstract":"The environmental pollutant cadmium (Cd) poses a threat to human health through the consumption of contaminated foodstuffs culminating in chronic nephrotoxicity. Mitochondrial dysfunction and excessive reactive oxygen species (ROS) are key to Cd cellular toxicity. Cd-lipid interactions have been less considered. We hypothesized Cd binding to the inner mitochondrial membrane (IMM) phospholipid cardiolipin (CL) and membrane rigidification underlies defective electron transfer by disrupted respiratory supercomplexes (SCs). In Cd-treated rat kidney cortex (rKC) mitoplasts, laurdan (lipid-water interface), and diphenylhexatriene (hydrophobic core) revealed increased and decreased membrane fluidity, respectively. Laurdan-loaded pure CL or IMM biomimetic (40 mol % POPC, 35 mol % DOPE, 20 mol % TOCL, 5 mol % SAPI) nanoliposomes were rigidified by 25 μM Cd, which was confirmed in live-cell imaging of laurdan or di-4-ANEPPDHQ loaded human proximal convoluted tubule (HPCT) cells. Blue native gel electrophoresis evidenced ∼30% loss of I+III2+IVn SC formation after 5 μM Cd for 6 h in HPCTs, which was reversed by CL-binding drug MTP-131/SS-31/elamipretide (0.1 μM), yet α-tocopherol-insensitive. Moreover, MTP-131 attenuated Cd-induced H2O2 (∼30%) and cytochrome c release (∼25%), but not osmotic swelling, in rKC mitochondria as well as Cd-induced ROS (∼25%) in HPCTs. MTP-131 binding to IMM biomimetic nanoliposomes decreased zeta potential, prevented Cd-induced liposome size increase, and membrane rigidification reported by laurdan. Heterologous CRLS1 expression reversed Cd (5 μM, 24 h) cytotoxicity (∼25%) by MTT assay, Cd (5 μM, 3 h)-induced ROS and mitochondrial membrane rigidification by Cd (1 μM, 1 h) in HPCT cells. In summary, we report a novel mechanism for Cd toxicity in which Cd-CL interactions cause IMM rigidification, thereby disrupting correct SC assembly and increasing ROS.","authors":["Romanova Nadiya","Sule Kevin","Issler Travis","Hebrok Daniel","Persicke Marcus","Thévenod Frank","Prenner Elmar J","Lee Wing-Kee"],"year":2025,"journal":"Journal of lipid research"},{"pmid":"23813215","title":"The mitochondrial-targeted compound SS-31 re-energizes ischemic mitochondria by interacting with cardiolipin.","abstract":"Ischemia causes AKI as a result of ATP depletion, and rapid recovery of ATP on reperfusion is important to minimize tissue damage. ATP recovery is often delayed, however, because ischemia destroys the mitochondrial cristae membranes required for mitochondrial ATP synthesis. The mitochondria-targeted compound SS-31 accelerates ATP recovery after ischemia and reduces AKI, but its mechanism of action remains unclear. Here, we used a polarity-sensitive fluorescent analog of SS-31 to demonstrate that SS-31 binds with high affinity to cardiolipin, an anionic phospholipid expressed on the inner mitochondrial membrane that is required for cristae formation. In addition, the SS-31/cardiolipin complex inhibited cytochrome c peroxidase activity, which catalyzes cardiolipin peroxidation and results in mitochondrial damage during ischemia, by protecting its heme iron. Pretreatment of rats with SS-31 protected cristae membranes during renal ischemia and prevented mitochondrial swelling. Prompt recovery of ATP on reperfusion led to rapid repair of ATP-dependent processes, such as restoration of the actin cytoskeleton and cell polarity. Rapid recovery of ATP also inhibited apoptosis, protected tubular barrier function, and mitigated renal dysfunction. In conclusion, SS-31, which is currently in clinical trials for ischemia-reperfusion injury, protects mitochondrial cristae by interacting with cardiolipin on the inner mitochondrial membrane.","authors":["Birk Alexander V","Liu Shaoyi","Soong Yi","Mills William","Singh Pradeep","Warren J David","Seshan Surya V","Pardee Joel D","Szeto Hazel H"],"year":2013,"journal":"Journal of the American Society of Nephrology : JASN"}],"biorxiv":[{"pmid":"","doi":"10.1101/2024.11.28.625848","title":"SS-31 protects against bleomycin-induced lung injury and fibrosis","abstract":"<h4>Objective</h4> The aim of this research was to investigate if the mitochondria-targeting peptide SS-31 could serve as a protective measure against bleomycin-induced pulmonary fibrosis in mice. <h4>Method</h4> Mice were split into four groups named CON group, SS-31 group, BLM group, and the BLM+ SS-31 group. SS-31 was administered daily from the day prior to the experiment for the control and model groups. Mice were euthanized after 28 days of the experiment, following which blood, bronchoalveolar lavage fluid, and lung tissue were collected for analysis. <h4>Results</h4> The study demonstrated that SS-31 could potentially mitigate the reduction in mice. It was observed through HE and Masson staining, immunohistochemistry, hydroxyproline detection, and fibrosis index measurement via Western blot that SS-31 could alleviate pulmonary fibrosis caused by BLM. Electron microscopy and ATP detection further suggested that SS-31 might help protect mitochondrial structure and function. It was also found that SS-31 could reduce reactive oxygen species and myeloperoxidase, thereby alleviating the reduction of antioxidant factor MPO and SOD, as well as diminishing the inflammatory factors TNF-α, IL-1 β, and IL-6. <h4>Conclusion</h4> The mitochondria-targeting drug SS-31 exhibited potential in mitigating bleomycin-induced pulmonary fibrosis, improving mitochondrial structural and functional damage, stabilizing the balance between oxidative and antioxidant systems, reducing inflammatory factor expression, and improving apoptosis in lung tissue.","authors":["Gu Q","Wang Y","Zhang H","Yang W","Meng X","Zhao M."],"year":2024,"journal":"PPR","source":"PPR","preprint":true},{"pmid":"","doi":"10.1101/2024.07.11.603085","title":"Therapeutic Peptide SS-31 Modulates Membrane Binding and Aggregation of Alpha-Synuclein and Restores Impaired Mitochondrial Function","abstract":"Membrane binding and aggregation properties of alpha-synuclein are closely associated with Parkinson's disease and a class of related syndromes named as synucleinopathy. This study explored the potential of SS-31 (Elamipretide), a therapeutic tetrapeptide with alternating cationic and aromatic residues and known properties of mitochondrial inner membrane binding and oxidative stress reduction, in modulating alpha-synuclein interaction with the lipid membranes and mitigating impairment of mitochondrial function induced by alpha-synuclein oligomers. It was demonstrated by both fluorescence correlation spectroscopy and fluorescence anisotropy that SS-31 displaces both wild-type and N-terminus acetylated alpha-synuclein from negatively charged small unilamellar vesicles in a dose-dependent manner. Thioflavin-T assay and transmission electron microscopy (TEM) showed that SS-31 inhibits membrane-induced alpha-synuclein aggregation and alters the morphology of alpha-synuclein fibrils. Moreover, Seahorse Mito Stress Test indicated that SS-31 restores impaired mitochondrial function in alpha-synuclein oligomer-treated neuroblastoma cells. Finally, confocal imaging revealed that SS-31 hinders cellular uptake of alpha-synuclein oligomers, possibly by modifying cell membrane electrostatics. These findings underscore the multifaceted protective role of SS-31 against mitochondrial dysfunction caused by alpha-synuclein aggregation. Consequently, SS-31 emerges as a promising therapeutic candidate to attenuate neurodegeneration pertinent to alpha-synuclein misfolding and aggregation. There is a good potential for further refinement of such peptide against many diseases linked to mitochondrial dysfunction and oxidative stress.","authors":["Stefaniak E","Cui B","Sun K","Yan X","Teng X","Ying L."],"year":2024,"journal":"PPR","source":"PPR","preprint":true},{"pmid":"","doi":"10.22541/au.168372042.22287207/v1","title":"Protective effects of SS-31 on Post-Contrast Acute Kidney Injury in Diabetes Mice","abstract":"Post-contrast acute kidney injury (PC-AKI) has emerged as the third leading cause of iatrogenic acute renal failure. Diabetes mellitus not only represents an independent risk factor for PC-AKI but also remains a major cause of chronic kidney disease (CKD). SS-31, an antioxidant peptide targeting mitochondria, is a potential preventive drug for PC-AKI. In this study, we established a PC-AKI model by injecting iodixanol in type 1 diabetic mice. Blood and tissue samples were collected to confirm that iodine injection caused excessive reactive oxygen species (ROS) in the kidney, activated the NLRP3 inflammasome pathway, and subsequently aggravated the development of PC-AKI. We also confirmed that SS-31 can reduce acute kidney injury induced by iodine contrast agent in diabetes by protecting mitochondrial function and inhibiting the ROS-NLRP3 signaling pathway.","authors":["hu j","wu z","Li Y","Yao X","Shi D","zhang h","Ren K","guo q."],"year":2023,"journal":"PPR","source":"PPR","preprint":true},{"pmid":"","doi":"10.1101/2021.01.06.425502","title":"Barth syndrome cellular models have dysregulated respiratory chain complex I and mitochondrial quality control due to abnormal cardiolipin","abstract":"Barth syndrome (BTHS) is an X-linked genetic condition caused by defects in TAZ , which encodes a transacylase involved in the remodeling of the inner mitochondrial membrane phospholipid, cardiolipin (CL). As such, CL has been implicated in numerous mitochondrial functions, and the role of defective CL in the clinical pathology of BTHS is under intense investigation. We used untargeted proteomics, shotgun lipidomics, gene expression analysis, and targeted metabolomics to identify novel areas of mitochondrial dysfunction in a new model of TAZ deficiency in HEK293 cells. Functional annotation analysis of proteomics data revealed abnormal regulation of mitochondrial respiratory chain complex I (CI), driven by the reduced abundance of 6 CI associated proteins in TAZ-deficient HEK293 cells: MT-ND3, NDUFA5, NDUFAB1, NDUFB2, NDUFB4, and NDUFAF1. This resulted in reduced assembly and function of CI in TAZ-deficient HEK293 cells as well as BTHS patient derived lymphoblast cells. We also identified increased abundance of PARL, a rhomboid protein involved in the regulation of mitophagy and apoptosis, and abnormal downstream processing of PGAM5, another mediator of mitochondrial quality control, in TAZ-deficient cells. Lastly, we modulated CL via the phospholipase inhibitor bromoenol lactone and the CL targeted SS-peptide, SS-31, and showed that each is able to remediate abnormalities in CI abundance as well as PGAM5 processing. Thus, mitochondrial respiratory chain CI and PARL/PGAM5 regulated mitochondrial quality control, both of whose functions localize to the inner mitochondrial membrane, are dysregulated due to TAZ deficiency and are partially remediated via modulation of CL.","authors":["Anzmann AF","Sniezek OL","Pado A","Busa V","Vaz FM","Kreimer SD","Cole RN","Le A","Kirsch BJ","Claypool SM","Vernon HJ."],"year":2021,"journal":"PPR","source":"PPR","preprint":true},{"pmid":"","doi":"10.1101/739128","title":"Mitochondrial protein interaction landscape of SS-31","abstract":"Mitochondrial dysfunction underlies the etiology of a broad spectrum of diseases including heart disease, cancer, neurodegenerative diseases, and the general aging process. Therapeutics that restore healthy mitochondrial function hold promise for treatment of these conditions. The synthetic tetrapeptide, elamipretide (SS-31), improves mitochondrial function, but mechanistic details of its pharmacological effects are unknown. Reportedly, SS-31 primarily interacts with the phospholipid cardiolipin in the inner mitochondrial membrane. Here we utilize chemical cross-linking with mass spectrometry to identify protein interactors of SS-31 in mitochondria. The SS-31-interacting proteins, all known cardiolipin binders, fall into two groups, those involved in ATP production through the oxidative phosphorylation pathway and those involved in 2-oxoglutarate metabolic processes. Residues cross-linked with SS-31 reveal binding regions that in many cases, are proximal to cardiolipin-protein interacting regions. These results offer the first glimpse of the protein interaction landscape of SS-31 and provide new mechanistic insight relevant to SS-31 mitochondrial therapy. <h4>Significance Statement</h4> SS-31 is a synthetic peptide that improves mitochondrial function and is currently undergoing clinical trials for treatments of heart failure, primary mitochondrial myopathy, and other mitochondrial diseases. SS-31 interacts with cardiolipin which is abundant in the inner mitochondrial membrane, but mechanistic details of its pharmacological effects are unknown. Here we apply a novel chemical cross-linking/mass spectrometry method to provide the first direct evidence for specific interactions between SS-31 and mitochondrial proteins. The identified SS-31 interactors are functional components in ATP production and 2-oxoglutarate metabolism and signaling, consistent with improved mitochondrial function resultant from SS-31 treatment. These results offer the first glimpse of the protein interaction landscape of SS-31 and provide new mechanistic insight relevant to SS-31 mitochondrial therapy.","authors":["Chavez JD","Tang X","Campbell MD","Reyes G","Kramer PA","Stuppard R","Keller A","Marcinek DJ","Bruce JE."],"year":2019,"journal":"PPR","source":"PPR","preprint":true}],"preprints":[{"pmid":"","doi":"10.1101/2024.11.28.625848","title":"SS-31 protects against bleomycin-induced lung injury and fibrosis","abstract":"<h4>Objective</h4> The aim of this research was to investigate if the mitochondria-targeting peptide SS-31 could serve as a protective measure against bleomycin-induced pulmonary fibrosis in mice. <h4>Method</h4> Mice were split into four groups named CON group, SS-31 group, BLM group, and the BLM+ SS-31 group. SS-31 was administered daily from the day prior to the experiment for the control and model groups. Mice were euthanized after 28 days of the experiment, following which blood, bronchoalveolar lavage fluid, and lung tissue were collected for analysis. <h4>Results</h4> The study demonstrated that SS-31 could potentially mitigate the reduction in mice. It was observed through HE and Masson staining, immunohistochemistry, hydroxyproline detection, and fibrosis index measurement via Western blot that SS-31 could alleviate pulmonary fibrosis caused by BLM. Electron microscopy and ATP detection further suggested that SS-31 might help protect mitochondrial structure and function. It was also found that SS-31 could reduce reactive oxygen species and myeloperoxidase, thereby alleviating the reduction of antioxidant factor MPO and SOD, as well as diminishing the inflammatory factors TNF-α, IL-1 β, and IL-6. <h4>Conclusion</h4> The mitochondria-targeting drug SS-31 exhibited potential in mitigating bleomycin-induced pulmonary fibrosis, improving mitochondrial structural and functional damage, stabilizing the balance between oxidative and antioxidant systems, reducing inflammatory factor expression, and improving apoptosis in lung tissue.","authors":["Gu Q","Wang Y","Zhang H","Yang W","Meng X","Zhao M."],"year":2024,"journal":"PPR","source":"PPR","preprint":true},{"pmid":"","doi":"10.1101/2024.07.11.603085","title":"Therapeutic Peptide SS-31 Modulates Membrane Binding and Aggregation of Alpha-Synuclein and Restores Impaired Mitochondrial Function","abstract":"Membrane binding and aggregation properties of alpha-synuclein are closely associated with Parkinson's disease and a class of related syndromes named as synucleinopathy. This study explored the potential of SS-31 (Elamipretide), a therapeutic tetrapeptide with alternating cationic and aromatic residues and known properties of mitochondrial inner membrane binding and oxidative stress reduction, in modulating alpha-synuclein interaction with the lipid membranes and mitigating impairment of mitochondrial function induced by alpha-synuclein oligomers. It was demonstrated by both fluorescence correlation spectroscopy and fluorescence anisotropy that SS-31 displaces both wild-type and N-terminus acetylated alpha-synuclein from negatively charged small unilamellar vesicles in a dose-dependent manner. Thioflavin-T assay and transmission electron microscopy (TEM) showed that SS-31 inhibits membrane-induced alpha-synuclein aggregation and alters the morphology of alpha-synuclein fibrils. Moreover, Seahorse Mito Stress Test indicated that SS-31 restores impaired mitochondrial function in alpha-synuclein oligomer-treated neuroblastoma cells. Finally, confocal imaging revealed that SS-31 hinders cellular uptake of alpha-synuclein oligomers, possibly by modifying cell membrane electrostatics. These findings underscore the multifaceted protective role of SS-31 against mitochondrial dysfunction caused by alpha-synuclein aggregation. Consequently, SS-31 emerges as a promising therapeutic candidate to attenuate neurodegeneration pertinent to alpha-synuclein misfolding and aggregation. There is a good potential for further refinement of such peptide against many diseases linked to mitochondrial dysfunction and oxidative stress.","authors":["Stefaniak E","Cui B","Sun K","Yan X","Teng X","Ying L."],"year":2024,"journal":"PPR","source":"PPR","preprint":true},{"pmid":"","doi":"10.22541/au.168372042.22287207/v1","title":"Protective effects of SS-31 on Post-Contrast Acute Kidney Injury in Diabetes Mice","abstract":"Post-contrast acute kidney injury (PC-AKI) has emerged as the third leading cause of iatrogenic acute renal failure. Diabetes mellitus not only represents an independent risk factor for PC-AKI but also remains a major cause of chronic kidney disease (CKD). SS-31, an antioxidant peptide targeting mitochondria, is a potential preventive drug for PC-AKI. In this study, we established a PC-AKI model by injecting iodixanol in type 1 diabetic mice. Blood and tissue samples were collected to confirm that iodine injection caused excessive reactive oxygen species (ROS) in the kidney, activated the NLRP3 inflammasome pathway, and subsequently aggravated the development of PC-AKI. We also confirmed that SS-31 can reduce acute kidney injury induced by iodine contrast agent in diabetes by protecting mitochondrial function and inhibiting the ROS-NLRP3 signaling pathway.","authors":["hu j","wu z","Li Y","Yao X","Shi D","zhang h","Ren K","guo q."],"year":2023,"journal":"PPR","source":"PPR","preprint":true},{"pmid":"","doi":"10.1101/2021.01.06.425502","title":"Barth syndrome cellular models have dysregulated respiratory chain complex I and mitochondrial quality control due to abnormal cardiolipin","abstract":"Barth syndrome (BTHS) is an X-linked genetic condition caused by defects in TAZ , which encodes a transacylase involved in the remodeling of the inner mitochondrial membrane phospholipid, cardiolipin (CL). As such, CL has been implicated in numerous mitochondrial functions, and the role of defective CL in the clinical pathology of BTHS is under intense investigation. We used untargeted proteomics, shotgun lipidomics, gene expression analysis, and targeted metabolomics to identify novel areas of mitochondrial dysfunction in a new model of TAZ deficiency in HEK293 cells. Functional annotation analysis of proteomics data revealed abnormal regulation of mitochondrial respiratory chain complex I (CI), driven by the reduced abundance of 6 CI associated proteins in TAZ-deficient HEK293 cells: MT-ND3, NDUFA5, NDUFAB1, NDUFB2, NDUFB4, and NDUFAF1. This resulted in reduced assembly and function of CI in TAZ-deficient HEK293 cells as well as BTHS patient derived lymphoblast cells. We also identified increased abundance of PARL, a rhomboid protein involved in the regulation of mitophagy and apoptosis, and abnormal downstream processing of PGAM5, another mediator of mitochondrial quality control, in TAZ-deficient cells. Lastly, we modulated CL via the phospholipase inhibitor bromoenol lactone and the CL targeted SS-peptide, SS-31, and showed that each is able to remediate abnormalities in CI abundance as well as PGAM5 processing. Thus, mitochondrial respiratory chain CI and PARL/PGAM5 regulated mitochondrial quality control, both of whose functions localize to the inner mitochondrial membrane, are dysregulated due to TAZ deficiency and are partially remediated via modulation of CL.","authors":["Anzmann AF","Sniezek OL","Pado A","Busa V","Vaz FM","Kreimer SD","Cole RN","Le A","Kirsch BJ","Claypool SM","Vernon HJ."],"year":2021,"journal":"PPR","source":"PPR","preprint":true},{"pmid":"","doi":"10.1101/739128","title":"Mitochondrial protein interaction landscape of SS-31","abstract":"Mitochondrial dysfunction underlies the etiology of a broad spectrum of diseases including heart disease, cancer, neurodegenerative diseases, and the general aging process. Therapeutics that restore healthy mitochondrial function hold promise for treatment of these conditions. The synthetic tetrapeptide, elamipretide (SS-31), improves mitochondrial function, but mechanistic details of its pharmacological effects are unknown. Reportedly, SS-31 primarily interacts with the phospholipid cardiolipin in the inner mitochondrial membrane. Here we utilize chemical cross-linking with mass spectrometry to identify protein interactors of SS-31 in mitochondria. The SS-31-interacting proteins, all known cardiolipin binders, fall into two groups, those involved in ATP production through the oxidative phosphorylation pathway and those involved in 2-oxoglutarate metabolic processes. Residues cross-linked with SS-31 reveal binding regions that in many cases, are proximal to cardiolipin-protein interacting regions. These results offer the first glimpse of the protein interaction landscape of SS-31 and provide new mechanistic insight relevant to SS-31 mitochondrial therapy. <h4>Significance Statement</h4> SS-31 is a synthetic peptide that improves mitochondrial function and is currently undergoing clinical trials for treatments of heart failure, primary mitochondrial myopathy, and other mitochondrial diseases. SS-31 interacts with cardiolipin which is abundant in the inner mitochondrial membrane, but mechanistic details of its pharmacological effects are unknown. Here we apply a novel chemical cross-linking/mass spectrometry method to provide the first direct evidence for specific interactions between SS-31 and mitochondrial proteins. The identified SS-31 interactors are functional components in ATP production and 2-oxoglutarate metabolism and signaling, consistent with improved mitochondrial function resultant from SS-31 treatment. These results offer the first glimpse of the protein interaction landscape of SS-31 and provide new mechanistic insight relevant to SS-31 mitochondrial therapy.","authors":["Chavez JD","Tang X","Campbell MD","Reyes G","Kramer PA","Stuppard R","Keller A","Marcinek DJ","Bruce JE."],"year":2019,"journal":"PPR","source":"PPR","preprint":true}],"consensus_view":"The literature strongly consensus that SS-31's primary mechanism is direct, high-affinity cardiolipin binding at the IMM, mediated by the alternating cationic-aromatic architecture of its tetrapeptide sequence. The aromatic residues—Dmt-2 and Phe-4—are understood to be critical for membrane insertion and intramolecular electronic interactions (particularly the Dmt iminium/aromatic system). Cardiolipin binding stabilizes cristae structure, inhibits CL peroxidation by cytochrome c, and preserves OXPHOS supercomplex assembly. This is well-established across multiple independent mechanistic studies and validated in diverse disease models. However, there is no published SAR literature on naphthylalanine (1-Nal or 2-Nal) substitutions at Phe-4, and the specific contributions of Phe-4 ring geometry to CL-binding affinity, membrane insertion depth, and intramolecular Dmt-2/Phe-4 aromatic stacking are not characterized in any available publication.","knowledge_gaps":"No published study has performed systematic aromatic residue substitution at Phe-4 of SS-31 to interrogate the role of ring geometry, size, or regiochemistry in CL binding. The specific question of whether 1-Nal versus 2-Nal at position 4 differentially affects (a) membrane insertion depth into CL-rich IMM leaflets, (b) intramolecular aromatic stacking with Dmt-2, (c) CL-binding affinity constants, (d) cytochrome c peroxidase inhibition potency, or (e) mitochondrial uptake/accumulation is entirely uncharacterized. The structural basis for SS-31's selectivity for CL over other anionic phospholipids is also not fully resolved. Additionally, while the Dmt-2/Phe-4 aromatic interaction is implied by the alternating architecture, no NMR or crystallographic study of the SS-31/CL complex has been published to directly validate the hypothesized intramolecular aromatic stack geometry that our 1-Nal variant tests.","supporting_evidence":"The Birk et al. (2013) polarity-sensitive fluorescence study establishes that SS-31 inserts into the hydrophobic environment of CL-containing membranes, suggesting aromatic residue properties directly influence insertion depth and affinity—consistent with the premise that naphthylalanine's larger hydrophobic surface could enhance membrane contact. The Chavez cross-linking data showing SS-31 contacts are proximal to CL-protein interfaces supports the notion that aromatic geometry matters for engaging the CL-rich protein-lipid microenvironment. The Romanova et al. (2025) biomimetic liposome data, showing SS-31 decreases zeta potential of CL-containing vesicles and reverses membrane rigidification, provides a tractable biophysical assay in which 1-Nal versus 2-Nal potency differences could be directly measured. The conservation of CL-binding function when SS-31 is used as a targeting moiety in SS-31@Fer-1 conjugates (PMID:39848110) implies the peptide tolerates some structural modification at its periphery, lending cautious support to the idea that a Phe-4 naphthyl variant might retain the core pharmacophore.","challenging_evidence":"No published SAR study supports the specific benefit of naphthylalanine at Phe-4; the hypothesis rests on structural inference without direct experimental validation. The compact 1-Nal geometry—with the second fused ring oriented toward the backbone—may sterically clash with the Dmt-2 side chain rather than forming the envisioned tighter stack, since Dmt already occupies significant space adjacent to the backbone. The Stefaniak et al. preprint shows SS-31's membrane displacement activity is dose-dependent and tied to its overall charge/aromatic balance; increasing hydrophobicity at Phe-4 could reduce aqueous solubility or alter the peptide's amphipathic balance in ways that diminish CL-selective binding relative to nonspecific membrane association. Several therapeutic studies show that the intact SS-31 sequence is highly efficacious, raising a high bar for modifications to demonstrate improvement. The Anzmann et al. Barth syndrome preprint suggests CL quantity/quality rather than SS-31 binding geometry is the primary determinant of CI rescue, which could mean that geometric differences between 1-Nal and 2-Nal are functionally negligible at the cellular level even if measurable biophysically. Finally, all preprint data (Stefaniak, Anzmann, Chavez 2019, Gu et al., hu et al.) should be treated as preliminary pending peer review."},"caveats":["in silico prediction only — requires wet lab validation","single-run prediction (not ensembled)","predicted properties may not reflect real-world biological behavior","this is research, not medical advice","cardiolipin is a lipid target — no docking, complex pTM/ipTM, or affinity module output is available; pLDDT reflects backbone confidence only, not binding affinity","heuristic property estimates (aggregation propensity 0.068, stability 0.57, half-life ~30 min–2 h, BBB 0.25) are sequence-based approximations only","the predicted proximity of 1-Nal to Dmt-2 may reflect steric accommodation rather than a stabilizing aromatic interaction; energy minimization is needed to score this geometry","no NMR or crystallographic data exists for any SS-31/CL complex in the published literature; the intramolecular aromatic stack hypothesis is structurally plausible but experimentally unvalidated","increased hydrophobicity at Phe-4 (shared by both 1-Nal and 2-Nal variants) may shift amphipathic balance toward nonspecific membrane association — cannot be distinguished computationally"],"works_cited":[{"pmid_or_doi":"23813215","title":"The mitochondrial-targeted compound SS-31 re-energizes ischemic mitochondria by interacting with cardiolipin","year":2013,"relevance":"Foundational study establishing SS-31's direct high-affinity cardiolipin binding and the mechanistic link between CL interaction, cristae protection, and ATP recovery; defines the pharmacophore that Phe-4 modifications must preserve."},{"pmid_or_doi":"32554501","title":"Mitochondrial protein interaction landscape of SS-31","year":2020,"relevance":"Cross-linking MS shows SS-31 engages CL-associated OXPHOS proteins at CL-binding interfaces, implying that aromatic residue geometry (including Phe-4) influences insertion into protein-lipid microdomains relevant to our 1-Nal vs 2-Nal comparison."},{"pmid_or_doi":"10.1101/739128","title":"Mitochondrial protein interaction landscape of SS-31","year":2019,"relevance":"Preprint version of the Chavez cross-linking study; provides detailed binding residue data suggesting SS-31's aromatic contacts are proximal to CL-protein interaction regions, underscoring the structural importance of Phe-4 orientation."},{"pmid_or_doi":"39880166","title":"Cadmium-cardiolipin disruption of respirasome assembly and redox balance through mitochondrial membrane rigidification","year":2025,"relevance":"Demonstrates SS-31 binding to IMM biomimetic CL-containing liposomes reverses membrane rigidification and restores respirasome assembly, providing a direct biophysical readout that could distinguish 1-Nal from 2-Nal variants in membrane-insertion assays."},{"pmid_or_doi":"10.1101/2024.07.11.603085","title":"Therapeutic Peptide SS-31 Modulates Membrane Binding and Aggregation of Alpha-Synuclein and Restores Impaired Mitochondrial Function","year":2024,"relevance":"Preprint showing SS-31's alternating aromatic/cationic architecture mediates displacement from negatively charged membranes in a dose-dependent manner, supporting the importance of aromatic residue geometry for membrane interaction that our 1-Nal substitution tests."},{"pmid_or_doi":"10.1101/2021.01.06.425502","title":"Barth syndrome cellular models have dysregulated respiratory chain complex I and mitochondrial quality control due to abnormal cardiolipin","year":2021,"relevance":"Preprint demonstrating SS-31 remediates CI assembly defects caused by aberrant CL, confirming that CL-binding geometry is functionally relevant to OXPHOS restoration—the ultimate test for our modified variants."},{"pmid_or_doi":"39940712","title":"Elamipretide: A Review of Its Structure, Mechanism of Action, and Therapeutic Potential","year":2025,"relevance":"Comprehensive review synthesizing SS-31's structure-mechanism relationships and clinical trial data, confirming CL-binding as the central pharmacophore and the aromatic residues as critical structural elements."},{"pmid_or_doi":"39364755","title":"New insight for SS-31 in treating diabetic cardiomyopathy: Activation of mitoGPX4 and alleviation of mitochondria-dependent ferroptosis","year":2024,"relevance":"Confirms SS-31 mitigates cardiolipin oxidative damage in a cardiac disease model, establishing in vivo efficacy benchmarks against which modified variants must be compared."},{"pmid_or_doi":"35707274","title":"SS-31, a Mitochondria-Targeting Peptide, Ameliorates Kidney Disease","year":2022,"relevance":"Reviews SS-31 pharmacokinetics and renal protective mechanisms, noting its selective mitochondrial accumulation; relevant context for whether bulkier aromatic substitutions (1-Nal) might alter mitochondrial uptake kinetics."},{"pmid_or_doi":"39848110","title":"SS-31@Fer-1 Alleviates ferroptosis in hypoxia/reoxygenation cardiomyocytes via mitochondrial targeting","year":2025,"relevance":"Demonstrates SS-31 retains mitochondrial targeting and CL-related function when used as a targeting moiety in conjugates, suggesting the CL-binding domain tolerates some structural elaboration—relevant background for naphthylalanine substitution."}]},"onchain":{"hash":"478hoNENhwDBhQJtZZmULxfG4PBDicmvMDYpwxaHnhQ4qkUYTtySegJkFSEGjjvTceLocYBRUXXjfAt7UPSYFx7o","signature":"478hoNENhwDBhQJtZZmULxfG4PBDicmvMDYpwxaHnhQ4qkUYTtySegJkFSEGjjvTceLocYBRUXXjfAt7UPSYFx7o","data_hash":"9734a17edce37156ad12da4dd9264f90b03ebfaafcaece75a2b74488111987b9","logged_at":"2026-05-03T00:35:02.938915+00:00","explorer_url":"https://solscan.io/tx/478hoNENhwDBhQJtZZmULxfG4PBDicmvMDYpwxaHnhQ4qkUYTtySegJkFSEGjjvTceLocYBRUXXjfAt7UPSYFx7o"},"ipfs_hash":null,"created_at":"2026-05-03T00:32:04.601253+00:00","updated_at":"2026-05-03T00:35:02.943282+00:00"}