{"id":5,"slug":"5-mots-c-met-1-norleucine-nle-substitution-isosteric-replacement-of-t","title":"MOTS-c Met1→Nle substitution to block N-terminal oxidation and improve metabolic stability","status":"PROMISING","fold_verdict":"PROMISING","discard_reason":null,"peptide":{"name":"MOTS-c","class":"LONGEVITY","sequence":"MRWQEMGYIFYPRKLR","modified_sequence":"(Nle)RWQEMGYIFYPRKLR","modification_description":"Met-1 → Norleucine (Nle) substitution; isosteric replacement of the oxidation-prone N-terminal methionine with a non-oxidizable straight-chain analog"},"target":{"protein":"5'-AMP-activated protein kinase catalytic subunit alpha-2","uniprot_id":"P54646","chembl_id":"CHEMBL2492","gene_symbol":"PRKAA2"},"rationale":{"hypothesis":"Replacing the N-terminal Met-1 of MOTS-c with norleucine will preserve the hydrophobic N-cap geometry needed for AMPK-pathway engagement while eliminating a major oxidation liability. We predict the modified peptide retains a near-identical predicted backbone and side-chain orientation compared to wild-type MOTS-c, supporting that the substitution is structurally silent at the binding interface.","rationale":"Methionine residues, especially solvent-exposed N-terminal ones, are highly susceptible to oxidation to methionine sulfoxide, which is a known degradation route limiting peptide shelf-life and in vivo half-life. Norleucine is a classical isosteric, non-oxidizable Met mimetic with nearly identical van der Waals volume and lipophilicity, widely used to stabilize peptide therapeutics (e.g., in oxytocin and GLP-1 analogs) without disrupting hydrophobic contacts. Because Met-1 sits at a flexible terminus rather than a known catalytic residue, the substitution is mechanistically conservative for downstream AMPK activation.","predicted_outcome":"Structure prediction should show backbone RMSD <1.0 Å versus wild-type MOTS-c, with the Nle side chain occupying the same hydrophobic envelope as Met-1; pLDDT at the N-terminus should be comparable or marginally improved, supporting a structurally silent, stability-enhancing modification.","mechanism_class":null,"biohacker_use":null},"confidence":{"plddt":0.6163824796676636,"ptm":0.57383793592453,"iptm":0.41800057888031006,"chai_agreement":null,"chai1_gated_decision":"RAN_BORDERLINE","binding_probability":null,"binding_pic50":null,"predicted_binding_change":null},"profile":{"aggregation_propensity":0.083,"stability_score":0.542,"bbb_penetration_score":0.149,"half_life_estimate":"moderate (~30 minutes – 2 hours)"},"narrative":{"tldr":"MOTS-c is a 16-amino acid mitochondrial-derived peptide that activates AMPK through folate cycle disruption, with established roles in metabolic homeostasis and longevity signaling. This fold explores replacing the oxidation-prone N-terminal methionine (Met-1) with norleucine (Nle), a classical isosteric, non-oxidizable methionine mimetic. Structure prediction returns a pLDDT of 0.62 and ipTM of 0.42 — moderate confidence consistent with a short, partially ordered peptide — and heuristic profiling suggests the substitution is structurally silent without introducing steric penalties. The signal is promising rather than conclusive: the chemical rationale is sound and precedented, but the absence of any MOTS-c SAR literature or resolved binding interface means validation remains entirely ahead of us.","detailed_analysis":"MOTS-c is a 16-amino acid peptide encoded within the 12S rRNA locus of mitochondrial DNA, first characterized in 2015 as a regulator of insulin sensitivity and skeletal muscle metabolism. Its canonical mechanism involves inhibition of the intracellular folate-methionine cycle, causing accumulation of AICAR — a naturally occurring AMPK activator — which in turn engages the AMPK catalytic alpha-2 subunit (PRKAA2) to drive downstream metabolic reprogramming. Beyond this pathway, MOTS-c is now understood to be a pleiotropic mitonuclear signaling molecule: under metabolic stress it translocates to the nucleus and directly modulates adaptive gene expression, influencing GLUT4, NRF2, STAT3, and IL-10 among others. Circulating MOTS-c levels decline with age, and exogenous peptide administration has shown efficacy in preclinical models of diet-induced obesity, insulin resistance, gestational diabetes, and even ovarian cancer suppression via LARS1 interaction. It sits squarely within the longevity peptide class.\n\nThe modification under investigation in this distillation is a single-residue substitution at position 1: methionine is replaced by norleucine (Nle), a non-proteinogenic amino acid carrying a straight four-carbon alkyl side chain in place of methionine's thioether. The oxidation hypothesis is chemically rigorous — N-terminal methionines are among the most oxidation-vulnerable residues in any peptide, readily forming methionine sulfoxide under physiological oxidative conditions or during formulation storage. Norleucine has been widely deployed as a pharmaceutical stabilization strategy in peptide drug development, including in GLP-1 and oxytocin analogs, precisely because it is isosteric and isosteric with methionine in terms of van der Waals volume and hydrophobicity, while being chemically inert to oxidation. The rationale for applying it to MOTS-c is therefore well-grounded in precedent.\n\nStructure prediction was performed using AlphaFold-class modeling targeting the AMPK alpha-2 catalytic subunit (PRKAA2). The predicted complex returned a pLDDT of 0.616 and a pTM of 0.574, with an interface-specific ipTM of 0.418. These values are characteristic of short peptides (16 residues) engaging a large protein partner — some disorder at termini is expected and does not by itself indicate a failed prediction. The caption from the structural agent describes the Nle-1 side chain occupying a hydrophobic envelope geometrically consistent with Met-1, with no steric clashes arising from the sulfur-to-methylene swap. This is exactly what an isosteric substitution should produce, and the model is consistent with the 'structurally silent' hypothesis.\n\nHeuristic sequence-based profiling adds complementary signal. The aggregation propensity score is low (0.083), which is favorable for a peptide therapeutic — this modification does not increase aggregation risk. The stability score is moderate (0.542), and the estimated half-life sits in the 30-minute to 2-hour range, which for a peptide of this class is a reasonable baseline that the Nle substitution is predicted to improve over the oxidation-labile native sequence. BBB penetration is low (0.149), consistent with MOTS-c's primarily peripheral and intracellular (skeletal muscle, liver) sites of action rather than any central nervous system target.\n\nFrom the literature, a critical uncertainty shadows all structural interpretations: no co-crystal structure or cryo-EM structure of MOTS-c bound to any target exists. The AMPK pathway engagement appears to be mediated through upstream metabolic intermediates (AICAR) rather than direct AMPK binding, and MOTS-c's interactions with LARS1 and nuclear targets are protein-protein interactions whose structural determinants are unresolved. This means neither the importance of Met-1 to any binding interface nor its dispensability has been empirically tested. The 'N-cap geometry' rationale, while chemically intuitive, is inferential rather than evidence-based. There is also a secondary concern: Met-1 in a mitochondrially-encoded peptide may be a recognition element for methionine aminopeptidase or N-terminal acetyltransferase processing — substitution with Nle would block such post-translational events, potentially altering intracellular trafficking or stability through a different mechanism than oxidation.\n\nWhat elevates this fold to PROMISING rather than DISCARDED is the combination of: (1) strong chemical precedent for Nle as a Met replacement in therapeutic peptides, (2) a structurally reasonable prediction with no red flags such as steric clashes or dramatic backbone deviation, (3) a genuine and unaddressed oxidative stability liability in the native sequence, and (4) a biologically important target with preclinical proof-of-concept. What prevents a REFINED verdict is the absence of Chai-1 independent confirmation, the low ipTM (0.42) reflecting real uncertainty at the interface level, and the complete lack of MOTS-c-specific SAR data to anchor any confidence in functional tolerance of this substitution.\n\nThis distillation establishes a chemically motivated, structurally plausible baseline for MOTS-c oxidative stabilization. The most valuable next experiments would be parallel synthesis of native and Nle-1 MOTS-c with direct oxidative stress challenge assays, followed by cell-based AMPK activation readouts (phospho-ACC, phospho-AMPK) to confirm the modification is pharmacologically neutral. As the lab accumulates folds across the MOTS-c series, this Nle-1 variant provides a clean reference point for future modifications targeting other residues — particularly the internal Met-6 (position 6, sequence MRWQEMG…), which represents a second oxidation liability not addressed here.","executive_summary":"MOTS-c Met-1→Nle: pLDDT 0.62, ipTM 0.42 — moderate confidence, no steric red flags. Isosteric substitution predicted to eliminate N-terminal oxidation liability. Strong chemical precedent; no MOTS-c SAR data exists to confirm. Promising, not yet refined.","tweet_draft":"DISTILLATION №16 — promising.\nMOTS-c, Met-1 → Norleucine substitution.\nTarget: AMPK-α2 (PRKAA2).\npLDDT 0.62 | ipTM 0.42.\nObjective: block N-terminal oxidation, preserve structure.\nHeuristic stability: moderate ✓ | aggregation risk: low ✓\nIn silico only. No SAR data exists for this peptide.\nFull report: alembic.bio","research_brief_markdown":"# FOLD №16 — MOTS-c Met1→Nle Substitution\n**Verdict: PROMISING** | Class: Longevity | Target: AMPK α2 (PRKAA2)\n\n---\n\n## Mechanism of Action\n\nMOTS-c is a 16-amino acid mitochondrial-derived peptide (MDP) encoded within the 12S rRNA locus of mitochondrial DNA. Its primary mechanism operates intracellularly: MOTS-c inhibits the folate-methionine cycle in skeletal muscle and other metabolic tissues, causing accumulation of AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) — a naturally occurring AMP-mimetic. AICAR in turn activates 5'-AMP-activated protein kinase, specifically the catalytic alpha-2 subunit (PRKAA2/AMPK-α2), triggering downstream glucose uptake, fatty acid oxidation, and mitochondrial biogenesis. Beyond this canonical pathway, MOTS-c translocates to the nucleus under metabolic stress, directly modulating adaptive gene expression including NRF2, GLUT4, STAT3, and IL-10 — consistent with its identity as a pleiotropic mitonuclear communication molecule. More recently, interaction with LARS1 (leucyl-tRNA synthetase) has been reported in oncological contexts, expanding its known protein-protein interaction landscape. Circulating MOTS-c declines with age, and exogenous administration is bioactive in preclinical models of obesity, insulin resistance, and metabolic aging.\n\n---\n\n## Performance Applications\n\nMOTS-c has demonstrated preclinical efficacy across several contexts directly relevant to metabolic performance and longevity:\n\n- **Insulin sensitivity & glucose metabolism**: AMPK-α2 activation improves skeletal muscle glucose uptake and GLUT4 translocation, with demonstrated reversal of age-dependent and diet-induced insulin resistance in rodent models.\n- **Obesity resistance**: Exogenous MOTS-c reduces adiposity and improves lipid metabolism in diet-induced obesity models.\n- **Metabolic aging**: Declining MOTS-c levels are associated with the metabolic phenotype of aging; restoration of circulating levels is a proposed longevity intervention rationale.\n- **Gestational metabolic health**: Efficacy in gestational diabetes models extends potential applications.\n- **Proposed emerging contexts**: ME/CFS (AMPK/NRF2 rationale, preprint-stage evidence) and atrial fibrillation (alongside humanin, preprint-stage). Both remain speculative and low-evidence at this time.\n\n---\n\n## Modification Rationale\n\nThe native MOTS-c sequence (MRWQEMGYIFYPRKLR) opens with methionine at position 1. N-terminal methionines are among the most oxidation-vulnerable residues in peptide chemistry: they are solvent-exposed, subject to reactive oxygen species in biological fluids, and readily oxidized to methionine sulfoxide — a modification that can reduce peptide potency, increase proteolytic susceptibility, and limit shelf-life in formulation.\n\nNorleucine (Nle) is the classical pharmaceutical solution. It carries a straight four-carbon alkyl side chain (n-butyl) in place of methionine's thioether, producing:\n- **Near-identical van der Waals volume and hydrophobicity** — the substitution is isosteric in all practically relevant senses.\n- **No oxidizable sulfur atom** — complete elimination of the Met oxidation liability.\n- **Backbone geometry unchanged** — Nle is an alpha-amino acid with standard L-configuration and no unusual conformational preferences.\n\nThis substitution strategy is well-precedented in therapeutic peptide development (GLP-1 analogs, oxytocin analogs, and others). Application to MOTS-c is novel, and the field contains no prior SAR data for any MOTS-c analog — this fold represents the first in silico exploration of this modification.\n\nA secondary consideration: Met-1 in mitochondrially-encoded peptides may serve as a substrate for methionine aminopeptidase (MAP) or N-terminal acetyltransferase (NAT) processing. Nle substitution would block any such co-translational or post-translational N-terminal modification. Whether this affects the biology of exogenously administered synthetic MOTS-c is unknown, but it warrants awareness in downstream interpretation.\n\n---\n\n## Predicted Properties — Where Signal Is Moderate\n\n| Parameter | Value | Interpretation |\n|---|---|---|\n| pLDDT | 0.616 | Moderate — typical for short peptide, not a failure signal |\n| pTM | 0.574 | Moderate global fold confidence |\n| ipTM | 0.418 | Low-moderate interface confidence — real uncertainty at binding surface |\n| Chai-1 agreement | Not available | Single-model prediction — not independently confirmed |\n| Aggregation propensity | 0.083 | Low — favorable; Nle does not increase aggregation risk |\n| Stability score | 0.542 | Moderate — consistent with heuristic improvement over native Met |\n| BBB penetration | 0.149 | Low — expected; MOTS-c's targets are peripheral/intracellular |\n| Half-life estimate | ~30 min – 2 hr | Moderate; Nle substitution predicted to reduce oxidative degradation |\n\n**Structural note**: The predicted structure shows the Nle-1 side chain occupying a hydrophobic envelope geometrically consistent with native Met-1, with no steric clashes introduced by the sulfur→methylene swap. The backbone topology is consistent with a short, partially ordered peptide. This is exactly the profile expected of a structurally silent isosteric substitution — but at ipTM 0.42, the interface prediction should not be over-interpreted.\n\n**What the heuristics suggest, not guarantee**: modest improvement in oxidative half-life, no degradation of aggregation behavior, preservation of the hydrophobic N-terminal character. All heuristic estimates only — these are sequence-derived proxies, not wet-lab measurements.\n\n---\n\n## What Would Strengthen This Signal\n\n**Additional in silico work:**\n- **Ensemble prediction**: Run 5+ AlphaFold-Multimer seeds and report median ipTM with variance — a single-run ipTM of 0.42 is insufficient to characterize interface confidence.\n- **Chai-1 independent confirmation**: The absence of Chai-1 agreement data leaves this as a single-model prediction; Chai-1 consensus would significantly strengthen the structural verdict.\n- **Boltz-2 affinity module**: Predicted ΔΔG or binding affinity change would directly test the 'pharmacologically neutral' hypothesis.\n- **Native MOTS-c fold reference**: Running the unmodified sequence (MRWQEMGYIFYPRKLR) through the same pipeline would enable direct RMSD comparison of backbone and side-chain geometry at position 1, providing the 'structurally silent' confirmation currently inferred rather than demonstrated.\n- **Met-6 variant**: The internal Met at position 6 (MRWQE**M**GYIFYPRKLR) is a second oxidation liability not addressed by this fold. A double-substitution Nle-1/Nle-6 variant would offer more comprehensive oxidative protection and is a logical next candidate in this series.\n\n**Wet-lab experiments that would be definitive:**\n- **Parallel synthesis + oxidative challenge**: Synthesize native MOTS-c and (Nle-1)MOTS-c; expose both to H₂O₂ or accelerated oxidative stress conditions; quantify methionine sulfoxide formation by LC-MS. This directly tests the stability hypothesis.\n- **Cell-based AMPK activation assay**: Treat myotubes (e.g., C2C12) with equimolar native vs. Nle-1 MOTS-c; measure phospho-AMPK-α2 (Thr172) and phospho-ACC by Western blot. This tests pharmacological neutrality of the substitution.\n- **Cellular uptake comparison**: Fluorescently label both variants and compare uptake kinetics in skeletal muscle cells to detect any N-terminus-dependent membrane interaction differences.\n- **Plasma stability assay**: Incubate both variants in human plasma and measure intact peptide by LC-MS/MS over 0–4 hours to quantify in vitro half-life improvement.\n\n---\n\n*This is the first MOTS-c fold in the Alembic Labs series. Future folds exploring Met-6, C-terminal modifications, or cyclic variants will be cross-referenced against this Nle-1 baseline.*","structural_caption":"The predicted Nle-1 MOTS-c structure adopts a backbone topology consistent with a short, partially ordered peptide engaging its target with moderate interface confidence (ipTM 0.42). The N-terminal Nle side chain occupies a hydrophobic envelope geometrically comparable to Met-1, with no obvious steric clash introduced by the sulfur→methylene swap. Per-residue confidence is moderate across the peptide core but, as expected for short peptides, weaker at termini. Overall, the model is consistent with a structurally silent substitution, though confidence is insufficient to rule out subtle local rearrangements.","key_findings_summary":"MOTS-c is a 16-amino acid mitochondrial-derived peptide (MDP) encoded within the 12S rRNA region of mitochondrial DNA, first characterized by Lee et al. (2015, PMID:25738459) as a regulator of insulin sensitivity and metabolic homeostasis. Its primary mechanism of action involves inhibition of the folate cycle and tethered de novo purine biosynthesis in skeletal muscle, leading to accumulation of AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) and subsequent AMPK activation. This AICAR-AMPK axis is consistently cited across multiple reviews (PMID:36677050, PMID:36761202) as the canonical signaling pathway through which MOTS-c exerts its metabolic effects, including improvements in glucose uptake, insulin sensitivity, and obesity resistance in preclinical models.\n\nBeyond its metabolic role, MOTS-c has been shown to translocate to the nucleus under metabolic stress conditions (glucose restriction, oxidative stress), where it directly regulates adaptive nuclear gene expression (PMID:31378979). This mitonuclear communication function suggests MOTS-c has pleiotropic effects beyond simple AMPK activation, including modulation of GLUT4, STAT3, IL-10, and NRF2 expression (PMID:36677050, PMID:36761202). Its circulating levels decline with age, and exogenous MOTS-c administration has demonstrated efficacy in mouse models of age-dependent insulin resistance, diet-induced obesity, gestational diabetes (PMID:34798268), and even oncological contexts such as ovarian cancer suppression via LARS1 ubiquitination (PMID:39321430).\n\nWith respect to the proposed Met-1 → Norleucine (Nle) substitution, the literature does not directly address this or any specific N-terminal modification of MOTS-c. The hypothesis rests on the well-established chemical rationale that norleucine is an isosteric, non-oxidizable analog of methionine sharing an equivalent straight-chain four-carbon side chain geometry. The N-terminal methionine of MOTS-c (sequence: MRWQEMGYIFYPRKLR) is recognized as an oxidation-prone residue, and its substitution with Nle is a standard pharmaceutical strategy to enhance oxidative stability. Since the MOTS-c mechanism operates primarily through intracellular metabolic pathway engagement (folate cycle disruption, AMPK activation) rather than through a well-characterized receptor-ligand binding interface that has been structurally resolved, the 'structurally silent' nature of the substitution at a binding interface remains unverified by the available literature.\n\nThe therapeutic development landscape for MOTS-c is currently preclinical and predominantly observational. Preprint evidence (Cheema et al., 2025) proposes MOTS-c for ME/CFS based on its AMPK and NRF2 activation properties, while another preprint (Liao et al., 2026) investigates MOTS-c alongside humanin for atrial fibrillation. No clinical trial data or pharmacokinetic/stability studies specifically addressing oxidation of native MOTS-c in formulation or in vivo are available in the indexed abstracts provided. The gap between the biological rationale for Nle substitution and empirical validation in the context of MOTS-c specifically is thus substantial."},"structured":{"known_activity":null,"known_binders":null,"candidate_variants":null,"domain_annotations":null,"literature_context":{"pubmed":[{"pmid":"36677050","title":"MOTS-c Functionally Prevents Metabolic Disorders.","abstract":"Mitochondrial-derived peptides are a family of peptides encoded by short open reading frames in the mitochondrial genome, which have regulatory effects on mitochondrial functions, gene expression, and metabolic homeostasis of the body. As a new member of the mitochondrial-derived peptide family, mitochondrial open reading frame of the 12S rRNA-c (MOTS-c) is regarding a peptide hormone that could reduce insulin resistance, prevent obesity, improve muscle function, promote bone metabolism, enhance immune regulation, and postpone aging. MOTS-c plays these physiological functions mainly through activating the AICAR-AMPK signaling pathways by disrupting the folate-methionine cycle in cells. Recent studies have shown that the above hormonal effect can be achieved through MOTS-c regulating the expression of genes such as GLUT4, STAT3, and IL-10. However, there is a lack of articles summarizing the genes and pathways involved in the physiological activity of MOTS-c. This article aims to summarize and interpret the interesting and updated findings of MOTS-c-associated genes and pathways involved in pathological metabolic processes. Finally, it is expected to develop novel diagnostic markers and treatment approaches with MOTS-c to prevent and treat metabolic disorders in the future.","authors":["Gao Yue","Wei Xinran","Wei Pingying","Lu Huijie","Zhong Luying","Tan Jie","Liu Hongbo","Liu Zheng"],"year":2023,"journal":"Metabolites"},{"pmid":"36761202","title":"MOTS-c: A promising mitochondrial-derived peptide for therapeutic exploitation.","abstract":"Mitochondrial ORF of the 12S rRNA Type-C (MOTS-c) is a mitochondrial-derived peptide composed of 16 amino acids encoded by the 12S rRNA region of the mitochondrial genome. The MOTS-c protein is transferred to the nucleus during metabolic stress and directs the expression of nuclear genes to promote cell balance. Different tissues co-expressed the protein with mitochondria, and plasma also contained the protein, but its level decreased with age. In addition, MOTS-c has been shown to improve glucose metabolism in skeletal muscle, which indicates its benefits for diseases such as diabetes, obesity, and aging. Nevertheless, MOTS-c has been used less frequently in disease treatment, and no effective method of applying MOTS-c in the clinic has been developed. Throughout this paper, we discussed the discovery and physiological function of mitochondrial-derived polypeptide MOTS-c, and the application of MOTS-c in the treatment of various diseases, such as aging, cardiovascular disease, insulin resistance, and inflammation. To provide additional ideas for future research and development, we tapped into the molecular mechanisms and therapeutic potentials of MOTS-c to improve diseases and combined the technology with synthetic biology in order to offer a new approach to its development and application.","authors":["Zheng Yuejun","Wei Zilin","Wang Tianhui"],"year":2023,"journal":"Frontiers in endocrinology"},{"pmid":"31378979","title":"MOTS-c: A Mitochondrial-Encoded Regulator of the Nucleus.","abstract":"Mitochondria are increasingly being recognized as information hubs that sense cellular changes and transmit messages to other cellular components, such as the nucleus, the endoplasmic reticulum (ER), the Golgi apparatus, and lysosomes. Nonetheless, the interaction between mitochondria and the nucleus is of special interest because they both host part of the cellular genome. Thus, the communication between genome-bearing organelles would likely include gene expression regulation. Multiple nuclear-encoded proteins have been known to regulate mitochondrial gene expression. On the contrary, no mitochondrial-encoded factors are known to actively regulate nuclear gene expression. MOTS-c (mitochondrial open reading frame of the 12S ribosomal RNA type-c) is a recently identified peptide encoded within the mitochondrial 12S ribosomal RNA gene that has metabolic functions. Notably, MOTS-c can translocate to the nucleus upon metabolic stress (e.g., glucose restriction and oxidative stress) and directly regulate adaptive nuclear gene expression to promote cellular homeostasis. It is hypothesized that cellular fitness requires the coevolved mitonuclear genomes to coordinate adaptive responses using gene-encoded factors that cross-regulate the opposite genome. This suggests that cellular gene expression requires the bipartite split genomes to operate as a unified system, rather than the nucleus being the sole master regulator.","authors":["Benayoun Bérénice A","Lee Changhan"],"year":2019,"journal":"BioEssays : news and reviews in molecular, cellular and developmental biology"},{"pmid":"25738459","title":"The mitochondrial-derived peptide MOTS-c promotes metabolic homeostasis and reduces obesity and insulin resistance.","abstract":"Mitochondria are known to be functional organelles, but their role as a signaling unit is increasingly being appreciated. The identification of a short open reading frame (sORF) in the mitochondrial DNA (mtDNA) that encodes a signaling peptide, humanin, suggests the possible existence of additional sORFs in the mtDNA. Here we report a sORF within the mitochondrial 12S rRNA encoding a 16-amino-acid peptide named MOTS-c (mitochondrial open reading frame of the 12S rRNA-c) that regulates insulin sensitivity and metabolic homeostasis. Its primary target organ appears to be the skeletal muscle, and its cellular actions inhibit the folate cycle and its tethered de novo purine biosynthesis, leading to AMPK activation. MOTS-c treatment in mice prevented age-dependent and high-fat-diet-induced insulin resistance, as well as diet-induced obesity. These results suggest that mitochondria may actively regulate metabolic homeostasis at the cellular and organismal level via peptides encoded within their genome.","authors":["Lee Changhan","Zeng Jennifer","Drew Brian G","Sallam Tamer","Martin-Montalvo Alejandro","Wan Junxiang","Kim Su-Jeong","Mehta Hemal","Hevener Andrea L","de Cabo Rafael","Cohen Pinchas"],"year":2015,"journal":"Cell metabolism"},{"pmid":"37307934","title":"MOTS-c: A potential anti-pulmonary fibrosis factor derived by mitochondria.","abstract":"Pulmonary fibrosis (PF) is a serious lung disease characterized by diffuse alveolitis and disruption of alveolar structure, with a poor prognosis and unclear etiopathogenesis. While ageing, oxidative stress, metabolic disorders, and mitochondrial dysfunction have been proposed as potential contributors to the development of PF, effective treatments for this condition remain elusive. However, Mitochondrial open reading frame of the 12S rRNA-c (MOTS-c), a peptide encoded by the mitochondrial genome, has shown promising effects on glucose and lipid metabolism, cellular and mitochondrial homeostasis, as well as the reduction of systemic inflammatory responses, and is being investigated as a potential exercise mimetic. Additionally, dynamic expression changes of MOTS-c have been closely linked to ageing and ageing-related diseases, indicating its potential as an exercise mimetic. Therefore, the review aims to comprehensively analyze the available literature on the potential role of MOTS-c in improving PF development and to identify specific therapeutic targets for future treatment strategies.","authors":["Zhang Zewei","Chen Dongmei","Du Kaili","Huang Yaping","Li Xingzhe","Li Quwen","Lv Xiaoting"],"year":2023,"journal":"Mitochondrion"},{"pmid":"39321430","title":"Mitochondrial-Derived Peptide MOTS-c Suppresses Ovarian Cancer Progression by Attenuating USP7-Mediated LARS1 Deubiquitination.","abstract":"Mitochondrial-nuclear communication plays a vital role in maintaining cellular homeostasis. MOTS-c, a short peptide derived from the 12S rRNA of mitochondrial DNA, has been suggested as a retrograde mitochondrial signal. Although recent clinical studies have suggested a possible link between MOTS-c and human cancer, the role of MOTS-c in tumorigenesis has yet to be investigated. Here, MOTS-c levels are found to be reduced in both serum and tumor tissues from ovarian cancer (OC) patients, which are associated with poor patients' prognosis. Exogenous MOTS-c inhibits the proliferation, migration and invasion of OC cells, and induces cell cycle arrest and apoptosis. Mechanistically, MOTS-c interacts with LARS1 and promotes its ubiquitination and proteasomal degradation. In addition, USP7 was identified as a deubiquitinase of LARS1, and MOTS-c can attenuates USP7-mediated LARS1 deubiquitination by competing with USP7 for binding to LARS1. Besides, LARS1 was found to be increased and play an important oncogenic function in OC. More importantly, MOTS-c displays a marked anti-tumor effect on OC growth without systemic toxicity in vivo. In conclusion, this study reveals a crucial role of MOTS-c in OC and provides a possibility for MOTS-c as a therapeutic target for the treatment of this manlignacy.","authors":["Yin Yadong","Li Yujie","Ma Boyi","Ren Chenlu","Zhao Shuhua","Li Jia","Gong Yun","Yang Hong","Li Jibin"],"year":2024,"journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)"},{"pmid":"37200834","title":"Role of MOTS-c in the regulation of bone metabolism.","abstract":"MOTS-c, a mitochondrial-derived peptide (MDP), is an essential regulatory mediator of cell protection and energy metabolism and is involved in the development of specific diseases. Recent studies have revealed that MOTS-c promotes osteoblast proliferation, differentiation, and mineralization. Furthermore, it inhibits osteoclast production and mediates the regulation of bone metabolism and bone remodeling. Exercise effectively upregulates the expression of MOTS-c, but the specific mechanism of MOTS-c regulation in bone by exercise remains unclear. Therefore, this article reviewed the distribution and function of MOTS-c in the tissue, discussed the latest research developments in the regulation of osteoblasts and osteoclasts, and proposed potential molecular mechanisms for the effect of exercise on the regulation of bone metabolism. This review provides a theoretical reference for establishing methods to prevent and treat skeletal metabolic diseases.","authors":["Yi Xuejie","Hu Guangxuan","Yang Yang","Li Jing","Jin Junjie","Chang Bo"],"year":2023,"journal":"Frontiers in physiology"},{"pmid":"34798268","title":"The mitochondrial-derived peptide MOTS-c relieves hyperglycemia and insulin resistance in gestational diabetes mellitus.","abstract":"The most common complication during pregnancy, gestational diabetes mellitus (GDM), can cause adverse pregnancy outcomes and result in the mother and infant having a higher risk of developing type 2 diabetes after pregnancy. However, existing therapies for GDM remain scant, with the most common being lifestyle intervention and appropriate insulin treatment. MOTS-c, a mitochondrial-derived peptide, can target skeletal muscle and enhance glucose metabolism. Here, we demonstrate that MOTS-c can be an effective treatment for GDM. A GDM mouse model was established by short term high-fat diet combined with low-dose streptozotocin (STZ) treatment while MOTS-c was administrated daily during pregnancy. GDM symptoms such as blood glucose and insulin levels, glucose and insulin tolerance, as well as reproductive outcomes were investigated. MOTS-c significantly alleviated hyperglycemia, improved insulin sensitivity and glucose tolerance, and reduced birth weight and the death of offspring induced by GDM. Similar to a previous study, MOTS-c also could activate insulin sensitivity in the skeletal muscle of GDM mice and elevate glucose uptake in vitro. In addition, we found that MOTS-c protects pancreatic β-cell from STZ-mediated injury. Taken together, our findings demonstrate that MOTS-c could be a promising strategy for the treatment of GDM.","authors":["Yin Yadong","Pan Yihui","He Jin","Zhong Hong","Wu Yangyang","Ji Chenbo","Liu Lan","Cui Xianwei"],"year":2022,"journal":"Pharmacological research"}],"biorxiv":[{"pmid":"","doi":"10.20944/preprints202604.0328.v1","title":"Humanin and MOTS-c Attenuate Atrial Fibrillation by Suppressing Fibrosis and Mitochondrial Dysfunction","abstract":"A single paragraph of about 200 words maximum. For research articles, abstracts should give a pertinent overview of the work. We strongly encourage authors to use the following style of structured abstracts, but without headings: (1) Background: Place the question addressed in a broad context and highlight the purpose of the study; (2) Methods: briefly describe the main methods or treatments applied; (3) Results: summarize the article’s main findings; (4) Conclusions: indicate the main conclusions or interpretations. The abstract should be an objective representation of the article and it must not contain results that are not presented and substantiated in the main text and should not exaggerate the main conclusions.","authors":["Liao Y","Xu J","Jiao Y","Sun X","Gao M","Ding Y","Cai D","Shen Y","Zhou X","Han W."],"year":2026,"journal":"PPR","source":"PPR","preprint":true},{"pmid":"","doi":"10.20944/preprints202507.0058.v2","title":"Redefining Mitochondrial Therapy for ME/CFS: The Case for MOTS-c","abstract":"Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating, multi-system disease characterized by profound fatigue, post-exertional malaise (PEM), and a constellation of immune, neurological, and autonomic symptoms. Despite its global prevalence, the pathophysiology of ME/CFS remains elusive, and there are no FDA-approved treatments targeting the underlying mechanisms. Symptom-based pharmacologic management is often complicated by hypersensitivity reactions and mitochondrial toxicity. Non-pharmacologic interventions, such as energy conservation, autonomic regulation, and nutritional strategies, are frequently employed to mitigate symptom burden. Emerging research points to mitochondrial dysfunction as a core contributor to ME/CFS pathology, marked by impaired ATP production, oxidative stress, and bioenergetic failure. Mitochondrial-derived peptides (MDPs), particularly MOTS-c, offer a novel therapeutic avenue by enhancing mitochondrial biogenesis, reducing oxidative damage, and modulating inflammatory responses through AMPK and NRF2 activation. Preclinical evidence suggests that MOTS-c improves glucose metabolism, increases mitochondrial density, and enhances fatigue resistance. However, safety and efficacy data in humans are lacking. Future investigations are needed to evaluate MOTS-c&#039;s potential as a disease-modifying therapy in ME/CFS.","authors":["Cheema AK","Tehrani L","Patel S","Rozenfeld I","Renesca V","Fornos A","Kempuraj D","Klimas NG."],"year":2025,"journal":"PPR","source":"PPR","preprint":true},{"pmid":"","doi":"10.20944/preprints202507.0058.v1","title":"Redefining Mitochondrial Therapy for ME/CFS: The Case for MOTS-c","abstract":"Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating, multi-system disease characterized by profound fatigue, post-exertional malaise (PEM), and a constellation of immune, neurological, and autonomic symptoms. Despite its global prevalence, the pathophysiology of ME/CFS remains elusive, and there are no FDA-approved treatments targeting the underlying mechanisms. Symptom-based pharmacologic management is often complicated by hypersensitivity reactions and mitochondrial toxicity. Non-pharmacologic interventions, such as energy conservation, autonomic regulation, and nutritional strategies, are frequently employed to mitigate symptom burden. Emerging research points to mitochondrial dysfunction as a core contributor to ME/CFS pathology, marked by impaired ATP production, oxidative stress, and bioenergetic failure. Mitochondrial-derived peptides (MDPs), particularly MOTS-c, offer a novel therapeutic avenue by enhancing mitochondrial biogenesis, reducing oxidative damage, and modulating inflammatory responses through AMPK and NRF2 activation. Preclinical evidence suggests that MOTS-c improves glucose metabolism, increases mitochondrial density, and enhances fatigue resistance. However, safety and efficacy data in humans are lacking. Future investigations are needed to evaluate MOTS-c&#039;s potential as a disease-modifying therapy in ME/CFS.","authors":["Cheema AK","Tehrani L","Patel S","Rozenfeld I","Renesca V","Fornos A","Kempuraj D","Klimas NG."],"year":2025,"journal":"PPR","source":"PPR","preprint":true}],"preprints":[{"pmid":"","doi":"10.20944/preprints202604.0328.v1","title":"Humanin and MOTS-c Attenuate Atrial Fibrillation by Suppressing Fibrosis and Mitochondrial Dysfunction","abstract":"A single paragraph of about 200 words maximum. For research articles, abstracts should give a pertinent overview of the work. We strongly encourage authors to use the following style of structured abstracts, but without headings: (1) Background: Place the question addressed in a broad context and highlight the purpose of the study; (2) Methods: briefly describe the main methods or treatments applied; (3) Results: summarize the article’s main findings; (4) Conclusions: indicate the main conclusions or interpretations. The abstract should be an objective representation of the article and it must not contain results that are not presented and substantiated in the main text and should not exaggerate the main conclusions.","authors":["Liao Y","Xu J","Jiao Y","Sun X","Gao M","Ding Y","Cai D","Shen Y","Zhou X","Han W."],"year":2026,"journal":"PPR","source":"PPR","preprint":true},{"pmid":"","doi":"10.20944/preprints202507.0058.v2","title":"Redefining Mitochondrial Therapy for ME/CFS: The Case for MOTS-c","abstract":"Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating, multi-system disease characterized by profound fatigue, post-exertional malaise (PEM), and a constellation of immune, neurological, and autonomic symptoms. Despite its global prevalence, the pathophysiology of ME/CFS remains elusive, and there are no FDA-approved treatments targeting the underlying mechanisms. Symptom-based pharmacologic management is often complicated by hypersensitivity reactions and mitochondrial toxicity. Non-pharmacologic interventions, such as energy conservation, autonomic regulation, and nutritional strategies, are frequently employed to mitigate symptom burden. Emerging research points to mitochondrial dysfunction as a core contributor to ME/CFS pathology, marked by impaired ATP production, oxidative stress, and bioenergetic failure. Mitochondrial-derived peptides (MDPs), particularly MOTS-c, offer a novel therapeutic avenue by enhancing mitochondrial biogenesis, reducing oxidative damage, and modulating inflammatory responses through AMPK and NRF2 activation. Preclinical evidence suggests that MOTS-c improves glucose metabolism, increases mitochondrial density, and enhances fatigue resistance. However, safety and efficacy data in humans are lacking. Future investigations are needed to evaluate MOTS-c&#039;s potential as a disease-modifying therapy in ME/CFS.","authors":["Cheema AK","Tehrani L","Patel S","Rozenfeld I","Renesca V","Fornos A","Kempuraj D","Klimas NG."],"year":2025,"journal":"PPR","source":"PPR","preprint":true},{"pmid":"","doi":"10.20944/preprints202507.0058.v1","title":"Redefining Mitochondrial Therapy for ME/CFS: The Case for MOTS-c","abstract":"Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating, multi-system disease characterized by profound fatigue, post-exertional malaise (PEM), and a constellation of immune, neurological, and autonomic symptoms. Despite its global prevalence, the pathophysiology of ME/CFS remains elusive, and there are no FDA-approved treatments targeting the underlying mechanisms. Symptom-based pharmacologic management is often complicated by hypersensitivity reactions and mitochondrial toxicity. Non-pharmacologic interventions, such as energy conservation, autonomic regulation, and nutritional strategies, are frequently employed to mitigate symptom burden. Emerging research points to mitochondrial dysfunction as a core contributor to ME/CFS pathology, marked by impaired ATP production, oxidative stress, and bioenergetic failure. Mitochondrial-derived peptides (MDPs), particularly MOTS-c, offer a novel therapeutic avenue by enhancing mitochondrial biogenesis, reducing oxidative damage, and modulating inflammatory responses through AMPK and NRF2 activation. Preclinical evidence suggests that MOTS-c improves glucose metabolism, increases mitochondrial density, and enhances fatigue resistance. However, safety and efficacy data in humans are lacking. Future investigations are needed to evaluate MOTS-c&#039;s potential as a disease-modifying therapy in ME/CFS.","authors":["Cheema AK","Tehrani L","Patel S","Rozenfeld I","Renesca V","Fornos A","Kempuraj D","Klimas NG."],"year":2025,"journal":"PPR","source":"PPR","preprint":true}],"consensus_view":"The literature consensus firmly establishes that MOTS-c exerts its principal metabolic effects through inhibition of the intracellular folate-methionine cycle, leading to AICAR accumulation and downstream AMPK activation in skeletal muscle and other tissues. This mechanism is consistently reproduced across independent reviews and primary studies. The consensus also supports MOTS-c as a pleiotropic signaling peptide with nuclear translocation capacity. However, there is no consensus—or even discussion—in the existing literature regarding the structural requirements of the N-terminus of MOTS-c for AMPK pathway engagement, nor any prior reports of MOTS-c analogs bearing amino acid substitutions. The field has not yet resolved whether MOTS-c operates through a defined receptor or direct enzymatic inhibition; this mechanistic ambiguity limits confident structural predictions about which residues are 'interface-critical.' The Met-1 → Nle substitution is chemically reasonable and precedented in the broader peptide therapeutics field as an oxidative stabilization strategy, but is unsupported by MOTS-c-specific structural or SAR (structure-activity relationship) data.","knowledge_gaps":"Several critical knowledge gaps are directly relevant to the hypothesis. First, no structure of MOTS-c in complex with any binding partner (AMPK subunits, folate cycle enzymes, LARS1, or nuclear targets) has been reported, making it impossible to confirm from the literature whether Met-1 participates in a defined binding interface or is structurally dispensable. Second, no SAR studies or analog series for MOTS-c have been published, meaning the functional tolerance of any single-residue substitution—including at position 1—is entirely unknown empirically. Third, the oxidative stability of native MOTS-c in biological fluids or pharmaceutical formulations has not been characterized in the published literature, so the magnitude of the oxidation liability being addressed is unquantified. Fourth, the cellular uptake mechanism of exogenous MOTS-c is not well defined; if Met-1 participates in interactions with membrane transporters or endocytosis machinery, a substitution could affect bioavailability independently of target engagement. Fifth, the ME/CFS and other emerging therapeutic preprints lack safety and pharmacokinetic data, highlighting that even native MOTS-c clinical pharmacology is not established.","supporting_evidence":"The chemical rationale supporting the hypothesis is sound: norleucine (n-butyl side chain) is isoelectronic and nearly isosteric with methionine (thioether side chain), differing only by substitution of sulfur with a methylene group, and lacks the oxidation-susceptible sulfur atom. This substitution is well-precedented in peptide chemistry for improving oxidative stability without altering backbone conformation or gross hydrophobic character. The N-terminal position of MOTS-c (Met-1) is inherently exposed to solvent and reactive oxygen species, making it a genuine liability in oxidative environments. The MOTS-c mechanism as described by Lee et al. (2015) is intracellular and pathway-mediated rather than dependent on a structurally resolved extracellular receptor interface, which is mildly consistent with the possibility that the N-terminus serves a structural/stability role rather than a precise pharmacophore role. Multiple papers confirm that exogenous synthetic MOTS-c is bioactive when administered in vivo, indicating the peptide survives cellular processing adequately, and a more stable analog might improve upon this.","challenging_evidence":"Several considerations from the literature complicate or challenge the hypothesis. The MOTS-c sequence begins Met-Arg-Trp (MRW), and it is notable that Met-1 is directly followed by a positively charged Arg and a bulky Trp residue; the precise contribution of Met-1 to any local structural motif or N-cap geometry has not been characterized, so the claim that Nle preserves 'N-cap geometry' is inferential rather than evidence-based. The discovery that MOTS-c interacts with LARS1 (a leucyl-tRNA synthetase) through a protein-protein interaction (PMID:39321430) and translocates to the nucleus (PMID:31378979) suggests that MOTS-c engages multiple macromolecular partners through potentially different structural determinants; any of these could involve the N-terminal region. Furthermore, methionine at position 1 in a mitochondrially encoded peptide may carry biological significance beyond oxidation liability—N-terminal methionine is a recognition element for methionine aminopeptidase processing and N-terminal acetylation, modifications that could influence the peptide's intracellular fate. Substitution with Nle would block any such processing. The literature also contains no direct evidence that MOTS-c oxidation (e.g., Met sulfoxide formation) reduces its biological activity, meaning the oxidation 'liability' is assumed by analogy rather than demonstrated for this specific peptide. Finally, the preprint evidence (Cheema et al., 2025; Liao et al., 2026) is low-quality: one provides no results (uninformative structured abstract placeholder), and both lack peer review, reducing their evidentiary weight."},"caveats":["In silico prediction only — requires wet-lab synthesis and biological validation before any conclusions can be drawn about real-world activity or stability.","Single-run prediction (not ensembled) — ipTM 0.42 is based on one model seed; ensemble prediction across multiple seeds is needed to characterize confidence variance.","Predicted properties may not reflect real-world biological behavior — heuristic scores (aggregation propensity, stability, half-life, BBB penetration) are sequence-derived proxies, not measured values.","This is research, not medical advice — MOTS-c has no approved clinical indications; all described applications are preclinical or investigational.","No MOTS-c SAR data exists in the literature — functional tolerance of any substitution at any position, including Met-1, is entirely uncharacterized empirically.","No resolved structure of MOTS-c bound to any target — the 'structurally silent at the binding interface' conclusion is inferred from isosteric chemistry, not confirmed by a co-crystal or cryo-EM reference structure.","Nle substitution blocks potential N-terminal methionine processing (MAP/NAT) — biological consequences of this for exogenous synthetic peptide are unknown.","Chai-1 independent structural confirmation was not available for this fold — the structural verdict rests on a single prediction platform.","The oxidation liability of native MOTS-c has not been empirically quantified in the literature — the magnitude of the stability problem being solved is assumed by analogy, not demonstrated for this specific peptide."],"works_cited":[{"pmid_or_doi":"25738459","title":"The mitochondrial-derived peptide MOTS-c promotes metabolic homeostasis and reduces obesity and insulin resistance","year":2015,"relevance":"Foundational paper establishing MOTS-c's mechanism of AMPK activation via folate cycle disruption and AICAR accumulation, directly defining the pathway our hypothesis seeks to preserve through the Nle substitution."},{"pmid_or_doi":"36677050","title":"MOTS-c Functionally Prevents Metabolic Disorders","year":2023,"relevance":"Comprehensive review summarizing AICAR-AMPK as the primary signaling axis of MOTS-c, and cataloguing downstream gene targets (GLUT4, STAT3, IL-10) relevant to understanding what functional activity must be retained in the modified peptide."},{"pmid_or_doi":"36761202","title":"MOTS-c: A promising mitochondrial-derived peptide for therapeutic exploitation","year":2023,"relevance":"Reviews MOTS-c therapeutic applications and mechanism, including AMPK and NRF2 activation, providing context for why preserving AMPK engagement in a more stable analog is therapeutically meaningful."},{"pmid_or_doi":"31378979","title":"MOTS-c: A Mitochondrial-Encoded Regulator of the Nucleus","year":2019,"relevance":"Establishes that MOTS-c translocates to the nucleus under stress to regulate gene expression, indicating its mechanism involves intracellular trafficking that could be sensitive to N-terminal structural changes affecting cellular uptake or processing."},{"pmid_or_doi":"34798268","title":"The mitochondrial-derived peptide MOTS-c relieves hyperglycemia and insulin resistance in gestational diabetes mellitus","year":2022,"relevance":"Demonstrates exogenous MOTS-c administration efficacy in vivo, establishing a translational model against which a Nle-substituted analog's activity could be benchmarked."},{"pmid_or_doi":"39321430","title":"Mitochondrial-Derived Peptide MOTS-c Suppresses Ovarian Cancer Progression by Attenuating USP7-Mediated LARS1 Deubiquitination","year":2024,"relevance":"Reveals a protein-protein interaction mechanism (MOTS-c binding LARS1) distinct from the AMPK pathway, indicating that N-terminal structure may matter for multiple functional interfaces, not solely AMPK engagement."},{"pmid_or_doi":"37307934","title":"MOTS-c: A potential anti-pulmonary fibrosis factor derived by mitochondria","year":2023,"relevance":"Discusses oxidative stress as a pathological context where MOTS-c is deployed, supporting the rationale that an oxidation-stable MOTS-c analog would be advantageous for therapeutic development."},{"pmid_or_doi":"37200834","title":"Role of MOTS-c in the regulation of bone metabolism","year":2023,"relevance":"Illustrates the breadth of MOTS-c's tissue-level effects and reinforces that AMPK-dependent signaling is consistently required across multiple physiological contexts, underscoring the importance of preserving this pathway in the modified peptide."},{"pmid_or_doi":"10.20944/preprints202507.0058.v2","title":"Redefining Mitochondrial Therapy for ME/CFS: The Case for MOTS-c","year":2025,"relevance":"Preprint proposing MOTS-c for clinical use in ME/CFS via AMPK/NRF2 mechanisms; the absence of human safety data cited here underscores the translational gap that a more stable Nle analog could help address."},{"pmid_or_doi":"10.20944/preprints202604.0328.v1","title":"Humanin and MOTS-c Attenuate Atrial Fibrillation by Suppressing Fibrosis and Mitochondrial Dysfunction","year":2026,"relevance":"Preprint exploring MOTS-c in a cardiovascular disease context; the uninformative abstract limits utility, but indicates expanding therapeutic interest that would benefit from a more shelf-stable formulation."}]},"onchain":{"hash":"5k2kreEFCTkEyb1aeVVcAGVz61P5k8UmbBJsNQETgMVPm77Tp8t5ExzyzLPCFBE343Eb8LwDauxENGxwx3vQxjuw","signature":"5k2kreEFCTkEyb1aeVVcAGVz61P5k8UmbBJsNQETgMVPm77Tp8t5ExzyzLPCFBE343Eb8LwDauxENGxwx3vQxjuw","data_hash":"04910ae440fe2d5a96245f43221c94684cf53adc94b34ef20fea3f2cee45f5c4","logged_at":"2026-05-02T13:27:42.935475+00:00","explorer_url":"https://solscan.io/tx/5k2kreEFCTkEyb1aeVVcAGVz61P5k8UmbBJsNQETgMVPm77Tp8t5ExzyzLPCFBE343Eb8LwDauxENGxwx3vQxjuw"},"ipfs_hash":null,"created_at":"2026-05-02T12:52:47.979416+00:00","updated_at":"2026-05-02T13:27:42.938480+00:00"}