{"id":56,"slug":null,"title":"SS-31 Phe-4 → Tyr: H-bond donor at C-terminal aromatic for ABCB1 efflux pump engagement","status":"DISCARDED","fold_verdict":"DISCARDED","discard_reason":"target_not_predictable: no UniProt ID resolved — target identity unconfirmed","peptide":{"name":"SS-31","class":"LONGEVITY","sequence":"DArgDmtLysPhe","modified_sequence":"DArg-Dmt-Lys-Tyr","modification_description":"Phe-4 → Tyr substitution at the C-terminal aromatic residue, adding a para-hydroxyl H-bond donor to engage the substrate-binding pocket of P-glycoprotein (ABCB1)"},"target":{"protein":"Multidrug resistance protein 1 (P-glycoprotein, ABCB1)","uniprot_id":null,"chembl_id":null,"gene_symbol":"ABCB1"},"rationale":{"hypothesis":"We hypothesize that replacing Phe-4 of SS-31 with Tyr will create a measurable interface with P-glycoprotein (ABCB1), the principal efflux transporter that limits cationic-aromatic peptide bioavailability and CNS penetration. SS-31's alternating cation-aromatic motif (DArg-Dmt-Lys-Phe) closely matches known ABCB1 substrate pharmacophores (cationic + aromatic + H-bond acceptor), and adding a para-OH at position 4 should test whether this analog is recognized as a substrate — informing whether ABCB1 efflux is a hidden PK liability for SS-31 itself.","rationale":"ABCB1 is a well-characterized class C transporter with multiple cryo-EM structures (a tractable target for Boltz-2, unlike a lipid bilayer). Its promiscuous substrate pocket recognizes small cationic-aromatic molecules with H-bond donors/acceptors; Tyr's para-OH is a canonical ABCB1 contact (cf. verapamil, doxorubicin). Phe→Tyr is a minimal, isosteric substitution preserving SS-31's pharmacophore while adding one polar handle, so a confident docked pose vs. a non-binding pose is structurally informative about efflux liability. This diverges from the last 3 lab folds (Semax cyclization/CONFORMATION, Retatrutide lactam/CONFORMATION, Sermorelin terminal/DELIVERY) by switching to PHARMACOKINETICS focus and a plain single substitution category, and from this peptide's recent 2-Nal/1-Nal aromatic-expansion line by testing a polar (not bulkier hydrophobic) edit against a protein (not lipid) target — directly addressing the prior rejection.","predicted_outcome":"Boltz-2 should place the Tyr-4-modified SS-31 in the central ABCB1 substrate cavity with the Tyr-OH H-bonding to a polar residue (e.g., Tyr307/Gln725 in human ABCB1) and the DArg/Lys cations contacting acidic residues lining the pocket. A pLDDT > 0.7 with a plausible interface would suggest SS-31 analogs are ABCB1 substrates (PK liability); poor docking would support the working assumption that SS-31's mitochondrial targeting bypasses efflux.","mechanism_class":null,"biohacker_use":null},"confidence":{"plddt":null,"ptm":null,"iptm":null,"chai_agreement":null,"chai1_gated_decision":null,"binding_probability":null,"binding_pic50":null,"predicted_binding_change":null},"profile":{"aggregation_propensity":null,"stability_score":null,"bbb_penetration_score":null,"half_life_estimate":null},"narrative":{"tldr":null,"detailed_analysis":null,"executive_summary":null,"tweet_draft":null,"research_brief_markdown":null,"structural_caption":"Structure prediction was not attempted — the orchestrator's predictability gate refused this fold (see discard_reason).","key_findings_summary":"SS-31 (elamipretide) is a synthetic tetrapeptide with the sequence D-Arg-Dmt-Lys-Phe-NH2, characterized by an alternating cationic-aromatic motif that enables selective accumulation in the inner mitochondrial membrane through electrostatic and hydrophobic interactions with cardiolipin. The literature strongly establishes SS-31's mechanism as primarily cardiolipin-dependent: Chavez et al. (2020) used chemical cross-linking mass spectrometry to show that SS-31-interacting proteins in mitochondria are cardiolipin binders involved in oxidative phosphorylation and 2-oxoglutarate metabolism, with binding regions proximal to known cardiolipin-protein interfaces. This cardiolipin-centric pharmacology is the dominant mechanistic framework in the current literature, with no published studies directly examining SS-31's interaction with ABCB1/P-glycoprotein.\n\nThe pharmacokinetic profile of SS-31 is discussed briefly in the renal disease review (Zhu et al., 2022), which notes favorable mitochondrial uptake in vivo, but does not characterize efflux transporter interactions, blood-brain barrier penetration mechanisms, or CNS bioavailability in mechanistic terms. Several studies demonstrate CNS activity of SS-31 following intraperitoneal administration — including reversal of LPS-induced memory impairment (Zhao et al., 2019) and improvement of cognitive function in sepsis-associated encephalopathy (Zhong et al., 2023) — implying some degree of CNS access, but none of these studies measure brain concentrations directly, characterize transport mechanisms, or test P-glycoprotein involvement.\n\nThe structural pharmacophore hypothesis underlying the research question is chemically plausible. The D-Arg and Lys residues provide the cationic character, Dmt (2',6'-dimethyltyrosine) and Phe provide the aromatic character, and this pattern resembles known ABCB1 substrate pharmacophores. The Stefaniak et al. (2024) preprint provides indirect structural relevance by demonstrating that SS-31 can competitively displace alpha-synuclein from negatively charged membranes in a dose-dependent manner, supporting the idea that the aromatic-cationic motif mediates membrane and interface binding beyond just cardiolipin — a property consistent with potential ABCB1 substrate behavior. However, this is an indirect mechanistic inference.\n\nNo published or preprint study in this literature set directly investigates SS-31 as an ABCB1 substrate, inhibitor, or efflux target, nor has the Phe-4→Tyr substitution been reported in any context. The proposed modification adds a para-hydroxyl group to the C-terminal aromatic residue, converting it from a non-polar phenyl to a phenolic group structurally analogous to tyrosine. This would alter H-bond donor capacity, logP, and potentially the interaction geometry with ABCB1's transmembrane substrate-binding pocket. The hypothesis is novel and untested in the available 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"}],"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}],"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}],"consensus_view":"The literature consensus is that SS-31's pharmacological activity is mediated primarily through electrostatic and hydrophobic interactions with cardiolipin in the inner mitochondrial membrane, with secondary interactions involving cardiolipin-binding proteins of the oxidative phosphorylation and 2-oxoglutarate metabolic pathways. SS-31 is considered a cell-penetrating, mitochondria-targeting peptide with demonstrated efficacy across renal, cardiac, hepatic, and CNS disease models. There is no published consensus — nor any data — on SS-31's interaction with ABCB1/P-glycoprotein. The pharmacokinetic literature for SS-31 is thin and does not characterize efflux transporter contributions to its biodistribution. The CNS activity observed in multiple rodent studies implies some level of blood-brain barrier penetration, but whether this is limited by ABCB1 efflux (and thus whether efficacy could be enhanced by ABCB1 inhibition or modification) is entirely unexplored. The Phe-4→Tyr substitution has not been reported in any published or preprint study.","knowledge_gaps":"The most critical gap is the complete absence of any study characterizing SS-31 as a substrate, inhibitor, or non-interactor of ABCB1 or any other ABC efflux transporter. No study has measured brain concentrations of SS-31 directly using pharmacokinetic methods (e.g., LC-MS/MS), nor compared brain penetration in ABCB1 knockout versus wild-type animals. The contribution of efflux transporters to SS-31's tissue distribution — including in the kidney (where ABCB1 is highly expressed in the proximal tubule), liver (canalicular ABCB1), and blood-brain barrier — is entirely uncharacterized. There are no structure-activity relationship (SAR) studies on the C-terminal Phe-4 residue of SS-31, meaning the contribution of this position to membrane binding, ABCB1 recognition, and mitochondrial targeting is unknown. The proposed Phe-4→Tyr analog has not been synthesized or tested in any published context. Additionally, no study has used bidirectional transport assays (e.g., Caco-2 or MDCKII-MDR1 monolayers) to assess SS-31's efflux ratio, which is the standard method for identifying ABCB1 substrates.","supporting_evidence":"The alternating cationic-aromatic motif of SS-31 (D-Arg¹-Dmt²-Lys³-Phe⁴) structurally overlaps with known ABCB1 substrate pharmacophores, which typically require a hydrophobic/aromatic core, positive charge, and H-bond acceptor/donor capacity — features all present in SS-31. The demonstrated CNS activity of SS-31 following peripheral IP administration (Zhao et al., 2019; Zhong et al., 2023) suggests meaningful brain penetration, but the degree to which this is limited by ABCB1 efflux is unknown — making the question of ABCB1 recognition practically significant. The Stefaniak et al. (2024) preprint shows SS-31 engages negatively charged membrane interfaces through its cationic-aromatic motif in a dose-dependent and competitive manner, consistent with a peptide capable of fitting hydrophobic and ionic binding pockets such as those found in ABCB1's transmembrane domains. Adding a para-OH at Phe-4 would increase H-bond donor capacity, a modification known to enhance ABCB1 substrate recognition for several small molecule series. The NOS2 off-target finding (Duan et al., 2025) supports the idea that SS-31 is not exclusively a lipid-binding agent and can engage protein binding sites, lending credibility to ABCB1 as a potential interactor.","challenging_evidence":"No study in this literature set provides any direct or even indirect evidence that SS-31 interacts with ABCB1. The dominant mechanistic framework positions SS-31 as a lipid-binding (cardiolipin) rather than protein-transporter-binding agent. SS-31's small size (MW ~639 Da as free base) and all D-amino acid/modified residue composition may reduce susceptibility to ABCB1-mediated efflux, as many ABCB1 substrates are larger, more flexible molecules with multiple rotatable bonds. The use of D-amino acids (D-Arg at position 1) and the non-natural Dmt residue may reduce protease susceptibility in a way that also reduces ABCB1 recognition, since ABCB1 substrate binding can depend on backbone conformation accessible only in L-amino acid peptides. The observed CNS efficacy in multiple rodent studies, while measured without quantitative brain distribution data, could be interpreted as evidence against significant ABCB1-mediated efflux limiting brain exposure — though this interpretation is speculative without proper PK studies. Adding a para-OH (Phe→Tyr) increases polarity and reduces logP, which could decrease passive membrane permeability and thus reduce overall CNS penetration independent of ABCB1 status, confounding interpretation of any observed change in brain exposure. The preprint evidence (Stefaniak et al., 2024; Gu et al., 2024; Hu et al., 2023) should be weighted lower than peer-reviewed publications given the absence of full peer review."},"caveats":null,"works_cited":[{"pmid_or_doi":"32554501","title":"Mitochondrial protein interaction landscape of SS-31","year":2020,"relevance":"Defines SS-31's primary mitochondrial binding partners as cardiolipin-associated proteins, establishing the structural basis for its pharmacology and providing context for why ABCB1 interactions have not been studied — the field has focused entirely on mitochondrial targets."},{"pmid_or_doi":"35707274","title":"SS-31, a Mitochondria-Targeting Peptide, Ameliorates Kidney Disease","year":2022,"relevance":"Reviews SS-31 pharmacokinetics in vivo and discusses systemic distribution, but does not address efflux transporter interactions, making it informative for what is absent in the PK literature regarding ABCB1."},{"pmid_or_doi":"31747905","title":"Elamipretide (SS-31) improves mitochondrial dysfunction, synaptic and memory impairment induced by lipopolysaccharide in mice","year":2019,"relevance":"Demonstrates CNS activity of SS-31 after peripheral (IP) administration, implying CNS penetration occurs, but does not investigate the transport mechanism or whether ABCB1 efflux limits brain exposure."},{"pmid_or_doi":"36333543","title":"SS-31 Improves Cognitive Function in Sepsis-Associated Encephalopathy by Inhibiting the Drp1-NLRP3 Inflammasome Activation","year":2023,"relevance":"Provides additional evidence for CNS penetration of SS-31 following IP dosing in mice, reinforcing the relevance of understanding blood-brain barrier transport mechanisms including potential ABCB1 efflux."},{"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 demonstrating SS-31's ability to displace proteins from negatively charged membranes via its cationic-aromatic motif, supporting the structural argument that SS-31 can engage amphipathic binding pockets — a property relevant to ABCB1 substrate pharmacophore considerations."},{"pmid_or_doi":"39848110","title":"SS-31@Fer-1 Alleviates ferroptosis in hypoxia/reoxygenation cardiomyocytes via mitochondrial targeting","year":2025,"relevance":"Demonstrates SS-31 used as a mitochondrial targeting moiety in a conjugate drug, highlighting its cell-penetrating properties and the importance of understanding how modifications to SS-31's aromatic residues affect biodistribution."},{"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":"Reports systemic IP dosing of SS-31 at 2.5 mg/kg/day with therapeutic efficacy, indicating acceptable bioavailability in rodents, but provides no mechanistic data on transporter-mediated efflux that could limit exposure."},{"pmid_or_doi":"33986918","title":"SS-31 Protects Liver from Ischemia-Reperfusion Injury via Modulating Macrophage Polarization","year":2021,"relevance":"Demonstrates hepatic distribution of SS-31, relevant because the liver expresses high levels of ABCB1 and other efflux transporters — yet transporter-mediated clearance is not addressed."},{"pmid_or_doi":"40570323","title":"SS-31 Targets NOS2 to Enhance Osteogenic Differentiation in Aged BMSCs by Restoring Mitochondrial Function","year":2025,"relevance":"Identifies NOS2 as an off-target interaction of SS-31, suggesting SS-31 may have protein binding interactions beyond cardiolipin, supporting the plausibility that the peptide could engage other protein targets including ABCB1."},{"pmid_or_doi":"10.22541/au.168372042.22287207/v1","title":"Protective effects of SS-31 on Post-Contrast Acute Kidney Injury in Diabetes Mice","year":2023,"relevance":"Preprint reporting SS-31 efficacy in a renal model where renal ABCB1 expression is relevant to drug clearance, yet transporter interactions are not discussed — highlighting the gap in PK mechanistic understanding."}]},"onchain":{"hash":null,"signature":null,"data_hash":null,"logged_at":null,"explorer_url":null},"ipfs_hash":null,"created_at":"2026-05-04T08:42:35.125180+00:00","updated_at":"2026-05-04T08:44:15.117881+00:00"}