pharmaceutical composition

专利摘要:
POLYPEPTIDE ISOLATED, PHARMACEUTICAL COMPOSITION, AND, METHOD OF TREATING DISEASE OR CONDITION MOTS3 is a new polypeptide. Methods of treating diseases such as diabetes, obesity, fatty liver and cancer using MOTS3 and respective pharmaceutical compositions are disclosed in this document.
公开号:BR112015023500B1
申请号:R112015023500-0
申请日:2014-03-14
公开日:2021-01-12
发明作者:Pinchas Cohen；Changhan Lee；Laura J. Cobb
申请人:The Regents Of The University Of California；
IPC主号:

专利说明:

CROSS REFERENCE TO RELATED ORDERS
[001] The present appeal claims the priority of U.S. Provisional Application No. 61 / 801,474, filed on March 15, 2013, which is incorporated into this document by reference in full for all purposes. GOVERNMENT RIGHTS
[002] This invention was made with government support under Concession No. 1R01AG034430 and Concession No. 1R01GM090311, granted by the National Institutes of Health. The Government has certain rights in the invention. FIELD OF THE INVENTION
[003] This invention relates to a new mitochondrial-derived peptide (MOTS3). This new peptide can be used in methods of treating diseases such as diabetes, obesity, fatty liver and cancer. FUNDAMENTALS
[004] Mitochondria, central to metabolic processes, is involved in energy production, programmed cell death and generation of reactive oxygen species (ROS) and is strongly implicated in various stages of serious illnesses including cancer, diabetes, neurodegenerative diseases and aging ; yet its role in pathogenesis remains largely unclear. Traditionally, mitochondria have been thought of as "end-function" organelles, receiving and processing large amounts of cellular signals to regulate energy production and cell death. However, retrograde signaling, in which mitochondria communicate back to the cell, is a poorly understood biological process.
[005] Calcium, cytochrome C and ROS have been considered a retrograde mitochondrial molecule. Notably, Durieux et al proposed that signals from mitochondria can regulate the life span of C. elegans in a non-autonomous cell way, but the nature of such signals has not been identified in worms (Durieux et al, Cell 144, 79 ( January 7, 2011) The identity of such mitochondrial-derived signals may have been discovered in 2001, when a 24-amino acid peptide, now known as humanin (HN), was cloned from a cDNA library constructed from the surviving fraction of an Alzheimer's patient's brain and was mapped to the mitochondrial 16S rRNA locus (Hashimoto et al, Proc Natl Acad Sci USA 98, 6336 (May 22, 2001). Since then, HN has been shown to be a cytoprotective and anti-apoptotic factor , partly due to its role as a Bax antagonist that prevents apoptosis in various cancer cells (Guo et al, Nature 423, 456 (May 22, 2003) as an IGFBP-3 partner that antagonizes the apoptotic actions of IGFBP- 3 in cancer cells (Ikonen et al, Proc Natl Acad Sci USA 100, 13042 (October 28, 2003). More recent work indicates that HN is a broad-spectrum survival factor (Nishimoto et al, Trends Mol Med 10, 102 (March 2004)), however, its exact mechanism of action is still unclear.
[006] Mitochondria-derived humanin shares 92-95% identity with several nuclear-encoded cDNAs, which represent domains within larger hypothetical genes, whose expression has not been validated (Tajima et al, NeurosciLett 324, 227 (May 24, 2002 )). HN transcripts of mitochondrial origin are present in the kidney, testis, brain and gastrointestinal tract. Noteworthy is that humanin is highly conserved among species (between 90-100% homology), including lower organisms. The peptide was demonstrated in the brain and testis and we demonstrated that it is present in concentrations of 1-10 ng / ml in plasma, CSF and seminal fluid. New mutants and HN analogs with more potent actions have been described, including HNG (S14G), HNG-F6A (non-IGFBP-3 binding, which we recently protected as a possible treatment for type 2 diabetes under a joint AECOM / UCLA patent that has been disclosed) and colivelin (hybrid peptide containing partial sequences of HN and ADNF9). Humanin and its analogs and derivatives are showing therapeutic potential for a variety of diseases, including kidney failure, diabetes and Alzheimer's disease.
[007] This report is the first description of a new open reading frame (ORF) in rRNA 12S. The name of this new ORF in the 12S rRNA is Mitochondrial ORF in the 12 S 3 rRNA (MOTS3). Similar to HN (Hashimoto Y. et al, Proc Natl Acad Sci USA 98, 6336 (May 22, 2001)), MOTS3 transcripts are polyadenylated and suggest an in-gene gene structure that is well conserved in the whole species.
[008] MOTS3 is detected in the liver, heart, testicles, with the same molecular weight, but with a slightly higher molecular weight in the brain of mice and rats. Its main biological function is metabolic regulation with a strong influence on mitochondrial respiration and the use of glucose in mice and cell culture. MOTS3 has also been tested in several ways in vitro and in vivo to affect mitochondrial respiration, glucose utilization, insulin regulation and cell proliferation / survival.
[009] Furthermore, MOTS3 is a naturally occurring non-toxic peptide derived from mitochondria that has a role in general metabolic regulation. It is the first of its kind that provides regulation of blood glucose and strong body weight, as well as the activation of AMPK, which is a major target of drugs for diabetes and cancer, via the mTOR pathway. That is, it is in a totally new category of drugs, that is, signal peptides derived from mtDNA that have only recently been described. In this sense, MOTS3 and respective pharmaceutical formulations can be used to treat various age-related diseases with many metabolic implications such as cancer, diabetes, obesity and neurodegenerative diseases are not sufficient to cure the disease.
[0010] Accordingly, this invention discloses new MOTS3 ORFs and methods of use to treat the disease. BRIEF SUMMARY OF THE INVENTION
[0011] In certain embodiments, this invention comprises an isolated polypeptide with 70% sequence identity to SEQ ID NO: 1. In certain embodiments, the isolated polypeptide comprises an 80% sequence identity for SEQ ID NO: 1. In certain embodiments, the isolated polypeptide comprises a 90% sequence identity for SEQ ID NO: 1. In certain embodiments, the isolated polypeptide comprises the sequence of SEQ ID NO: 1.
[0012] In certain embodiments, this invention comprises an isolated antibody that specifically binds to a polypeptide described above. In certain embodiments, the antibody is a monoclonal antibody.
[0013] In certain embodiments, this invention comprises an isolated polypeptide with 70% sequence identity to SEQ ID NO: 1. In certain embodiments, the polypeptide comprises 80% or 90% sequence identity with SEQ ID NO: 1. In certain embodiments, the isolated polypeptide comprises the sequence of SEQ ID NO: 1.
[0014] In certain embodiments, this invention comprises a method of treating diabetes, the method comprising the step of administering to a subject in need of a therapeutically effective amount of a pharmaceutical composition comprising an isolated polypeptide with 70% sequence identity for SEQ ID NO: 1. In certain embodiments, the polypeptide comprises 80% or 90% sequence identity with SEQ ID NO: 1. In certain embodiments, the isolated polypeptide comprises the sequence of SEQ ID NO: 1. In certain modalities, diabetes is type I diabetes. In certain modalities, diabetes is type II diabetes. In specific modalities, the subject is a human being.
[0015] In certain embodiments, this invention comprises a method of treating obesity and / or fatty liver, the method comprising the step of administering to a subject in need of a therapeutically effective amount of a pharmaceutical composition comprising an isolated polypeptide with identity 70% sequence for SEQ ID NO: 1. In certain embodiments, the polypeptide comprises 80% or 90% sequence identity with SEQ ID NO: 1. In certain embodiments, the isolated polypeptide comprises the sequence of SEQ ID NO: 1. In certain embodiments, the subject is a mammal. In specific modalities, the subject is a human being.
[0016] In certain embodiments, this invention comprises a method of treating cancer, the method comprising the step of administering to a subject in need of a therapeutically effective amount of a pharmaceutical composition comprising an isolated polypeptide with 70% sequence identity for SEQ ID NO: 1. In certain embodiments, the polypeptide comprises 80% or 90% sequence identity with SEQ ID NO: 1. In certain embodiments, the isolated polypeptide comprises the sequence of SEQ ID NO: 1. In specific modalities, the subject is a human being. In certain modalities, cancer is a cancer selected from a group consisting of breast cancer, brain cancer, colon cancer, melanoma, leukemia (eg, AML), lymphoma, pancreatic cancer, prostate cancer, ovarian cancer , lung cancer and gastric cancer.
[0017] In some embodiments, this invention comprises a cDNA molecule encoding SEQ ID NO: 1. In some embodiments, this invention comprises a cDNA at least 80%, 90% or 100% identical to SEQ ID NO: 2.
[0018] In some embodiments, this invention comprises an expression vector, with a nucleic acid sequence encoding SEQ ID NO: 1. In some embodiments, this invention comprises an expression vector, comprising a cDNA of at least 80%, 90% or 100% identical to SEQ ID NO: 2.
[0019] In some embodiments, this invention provides a host cell transformed or transfected with an expression vector as described herein in the present disclosure. BRIEF DESCRIPTION OF THE FIGURES
[0020] Figure 1 shows that MOTS3 is a polyadenylated transcription of mitochondrial 12S (A) rRNA. MOTS 3 sequence is well conserved in several organisms (B). Rabbit anti-MOTS3 polyclonal antibody recognizes MOTS3 as well as overexpressed MOTS3 labeled with cloned GFP into a cell expression vector (C). MOTS3 is found in the testis, liver and heart of rats at similar molecular weights, but at slightly higher molecular weight in the brain (D). MOTS3 is not detected in Rho-0 cells that do not have mitochondrial DNA (E). CO I / II (cytochrome oxidase I / II) were used to verify the lack of mitochondrial DNA.
[0021] Figure 2 shows the effect of MOTS3 on mitochondrial activity under culture conditions of 10% and 1% FBS (A). Reduction of MTT by mitochondrial reductase enzymes and the rate of oxygen consumption (OCR) and the rate of extracellular acidification of (ECAR; glycolysis measures due to changes in pH in the medium made by lactate secretion) (B).
[0022] Figure 3 depicts the effect of exogenous MOTS3 treatment on mitochondrial respiration. Each drug was treated to test the specific metabolic parameter described at the top of the graph; that is, renewal of oligomycin-ATP, FCCP-maximal respiratory capacity, rotenone-non-ATP respiration.
[0023] Figure 4 depicts the effect of overexpression of MOTS3 by transfection on mitochondrial activity. Several versions of MOTS3 have been cloned and expressed in the cells.
[0024] Figure 5 depicts the effect of exogenous MOTS3.
[0025] Figure 6 shows the effect of treating exogenous MOTS3 on glucose absorption and glycolysis.
[0026] Figure 7 shows that MOTS3 (M3) significantly induces the activation of AMPK in relation to the control group (C) after 96 hours of treatment. This correlates with the marked decrease in glucose availability in the medium after 96 hours of treatment in Figure 6.
[0027] Figure 8 shows that MOTS3 slows cell proliferation and survival in a glucose medium, but not in a galactose medium, which forces cells to rely largely on mitochondrial function for survival.
[0028] Figure 9 depicts the effect of intracellular MOTS3 expression on cell proliferation. MOTS3 was cloned into expression vectors and transfected into cells.
[0029] Figure 10 depicts the effect of exogenous MOTS 3 on cell senescence. Senescence was determined by the levels of β-galactosidase associated with senescence (blue).
[0030] Figure 11 shows that MOTS3 reduces the liver's mitochondrial breathing capacity and body weight. 4 days of injections of MOTS 3 (i.p .; 0.5 and 5.0 mg / kg / day) in mice significantly reduced body weight, but did not change food intake. Liver mitochondrial breathing capacity was also reduced at both doses of MOTS3
[0031] Figure 12 illustrates mice treated with MOTS3 for 4 days showing reduced levels of glucose (A) and elevated levels of insulin (B).
[0032] Figure 13 describes MOTS-c, a new peptide encoded in the mitochondrial genome. a) MOTS-c is encoded within the 12S rRNA of the mitochondrial genome as an open reading frame bp 51 (ORF). Use of the mitochondrial genetic code produces start / stop codons in tandem, while the cytoplasmic genetic code produces a viable peptide. b) rapid 3 'amplification of the ends of the cDNA (3' RACE) shows that the MOTS-c and humanin transcripts are polyadenylated. c) comparison of MOTS-c encoded in mitochondrial DNA (mtDNA) and peptides encoded in nuclear DNA that were transferred from mtDNA (NUMT) through evolution. de) HeLa p0 cells, devoid of mitochondrial DNA through prolonged exposure to ethidium bromide (EtBr) in low doses, have undetectable levels of: d) 12S rRNA and MOTS-c transcripts by qRT-PCR, e) oxidase I and Mitochondria-encoded proteins (COI / II) cytochrome C, as well as MOTS-c, but normally express nuclear-encoded GAPDH detected by immunostaining. f) immunostaining of MOTS-c (green) and hsp60 (red) in HEK293 cells. Scale bar, 20μm. g-h) MOTS-c is detected in: g, various tissues in mice and rats and h, circulation (plasma) in humans and rodents. i-j) Fasting, a metabolic stress, alters the expression of endogenous MOTS-c in i tissues and j, plasma in mice. Data presented as mean ± SEM. Student's t-test. * P <0.05, ** P <0.01, *** P <0.001
[0033] Figure 14 describes that MOTS-c modulates gene expression and regulates metabolism via AICAR and AMPK signaling. a) Principal component analysis (PCA) in HEK293 cells after 4- and 72-hours of MOTS-c treatment (10 μM; N = 6). b) Parametric analysis of gene pool enrichment (PAGE) showed even more time-dependent global gene expression changes (N = 6). c) The Venn diagram representing positively regulated genes (red) and negatively regulated genes (blue) is shown by time point (N = 6). d) The effect of 72 hours of MOTS-c treatment on HEK293 cells (10 μM; N = 6) on various sets of genes related to metabolism and inflammation. e) The effect of MOTS-c on de novo purine biosynthesis. The pentose-phosphate (PPP) pathway and NAD + feed for the de novo purine biosynthesis pathway (N = 5). Enzymes altered by 4 - and 72-hour MOTS-c were determined by microarray analysis (figure 18b). GAR: glycinamide ribonucleotide, FGAM: N-formylglycinamide ribonucleotide, air: aminoimidazole ribonucleotide, NCAIR: N5-carboxyaminoimidazole ribonucleotide, SAICAR: N-succinocarboxamide-5-aminoimidazole ribonucleotide. The colors indicate changes by MOTS-c; Red: positive regulation, blue: negative regulation and gray: not measurable. f) HEK293 stably overexpressing MOTS-c have significantly increased AICAR levels (N = 5). g) Metabolic intermediates from the de novo purine biosynthesis pathway in HEK293 cells stably overexpressing MOTS-c are shown (N = 5). h) AICAR is a potent AMPK activator. MOTS-c activates AMPK and its oxidation downstream pathway controls fatty acid oxidation (ACC and CPT-1) and glucose absorption (GLUT-4). i) MOTS-c promotes the phosphorylation of AMPK and Akt in a time- and dose-dependent manner. Data presented as mean ± SEM. Student's t-test. * P <0.05, ** P <0.01, *** P <0.001
[0034] Figure 15 describes MOTS-c coordinating cellular, mitochondrial and fatty acid metabolism. Measurements of: a, glucose and b, lactate in cell culture medium after treatment with MOTS-c (10 μM; N = 6). c) MOTS-c-dependent glycolytic intermediate changes detected by metabolomics. d) Glycolytic flow in real time was determined by the extracellular acidification rate (ECAR) (N = 6). Glycolysis, glycolytic capacity and glycolytic reserve were estimated by challenging cells with glucose, oligomycin and 2-deoxyglucose, respectively. e-f) the rate of oxygen consumption in real time (OCR) was measured after MOTS-c treatment (N = 6). e) ATP renewal and maximum respiratory capacity were estimated by challenging cells with oligomycin and FCCP and f) respiratory capacity, proton leakage and coupling efficiency were calculated. g) TCA intermediates after MOTS-c treatment determined by metabolomics are shown (N = 5). h) the cell number was counted after MOTS-c treatment under glucose or galactose. Galactose forces mammalian cells to depend on mitochondrial metabolism (N = 6). Changes induced by MOTS-c induced in the intracellular i) transports of acyl-carnitine, j) essential fatty acids and k) CoA of e-oxidation intermediary miristoil determined by the metabolomics (N = 5). Data presented as mean ± SEM. Student's t-test. * P <0.05, ** P <0.01, *** P <0.001.
[0035] Figure 16 describes how MOTS-c regulates weight, metabolic homeostasis and insulin sensitivity in mice. a) Effect of acute treatment of 4 days of MOTS-c (5 mg / kg / day; IP; BID) on the circulating levels of the main adipokines, IL-6 and TNFa in the exocrated CD-1 male mice (N = 6) . b-f) 8-week-old male CD-1 mice fed a high-fat (60% calorie) diet or a paired control diet (N = 10). MOTS-c was injected intraperitoneally daily (0.5 mg / kg / day). b) body weight, c) blood glucose, d) insulin, e) liver H&E staining, f) skeletal muscle AMPK phosphorylation and GLUT4 levels. g) the effect of the acute treatment of MOTS-c (5 mg / kg / day; IP) for 7 days in an intraperitoneal glucose tolerance test (IPGTT) performed on C57BL / 6 male mice (N = 7). hj) euglycemic-hyperglycemic clamps were performed on C57BL / 6 mice fed a high-fat (60% calorie) diet and treated with MOTS-c (5 mg / kg / day; IP) for 7 days (N = 6-8). h) glucose infusion rate (GIR), reflecting insulin sensitivity from the whole body, i) insulin-stimulated glucose elimination rate (IS-RDA), mainly reflecting muscle insulin sensitivity and j) liver production glucose (HGP). k-l) MOTS-c levels in young (4 months) and aged (32 months) (N = 3-4) mice decline in k) skeletal muscle and l) circulation (serum). m) the effect of the acute treatment of MOTS-c (5 mg / kg / day; IP) for 7 days on insulin-stimulated absorption of 2-deoxyglucose (60 μU / ml) in soleus muscles of young mice (3 months) and older (12 months) C57BL / 6 males (N = 6). n) proposed model for the MOTS-c action. Data presented as mean ± SEM. T- Student test * P <0.05, ** P <0.01, *** P <0.001.
[0036] Figure 17 describes a highly conserved MOTS-c peptide sequence. b) putative post-translation modifications of MOTS-c. c) MOTS-c antibodies were generated and tested for specificity against MOTS-c by immunoblotting. Western blots showing MOTS-c detection of HEK293 cells transfected with pEV, PMOT-c, PMOT-c-EGFP, pEGFP. d) HEK293 cells were stained with anti-MOTS-c antibody (green) alone or in the presence of MOTS-c peptide (block). e) MOTS-c (green) and hsp60 immunostaining (red) in HEK293 cells. Cores were stained with Hoechst 33258 (blue). Scale bar, 20μm. f) Standard curve of an MOTS-c ELISA in-house, which was used to measure MOTS-c in plasma.
[0037] Figure 18 describes the effect of MOTS-c on the de novo purine biosynthesis pathway (N = 5). a) Treatment of MOTS-c (10 μM; 24 and 72 hours post-treatment) regulates the de novo purine biosynthesis pathway in HEK293 cells. b) MOTS-c treatment (10 μM) alters the gene expression of the pathways involved in de novo purine biosynthesis as determined by microarray and Ingenuity Pathway Analysis (IPA) (N = 6). See figure 14e for overlap with metabolomic analysis. Red: positive regulation, green: negative regulation. Student's t-test between groups within the same time point. * P <0.05, ** P <0.01, *** P <0.001
[0038] Figure 19 describes the effect of MOTS-c on components of the adenyl system and components of the adenine nicotinamide dinucleotide (NAD) cycle. a) ATP, ADP, AMP and cyclic AMP levels (cAMP) and b) levels of NAD + and NADH in HEK293 cells stably overexpressing MOTS-c. EV: HEK293 stably transfected with empty vector. Student's t-test. * P <0.05, ** P <0.01, *** P <0.001
[0039] Figure 20 describes the effect of MOTS-c on the components of a) purine metabolism and b) cofactor and vitamin metabolism in HEK293 cells stably overexpressing MOTS-c, determined by metabolomics (N = 5). EV: HEK293 stably transfected with empty vector.
[0040] Figure 21 describes the effect of MOTS-c on a) extracellular levels (culture medium) of glucose and lactate in HEK293 cells stably overexpressing MOTS-c (N = 6) and b) intracellular glucose absorption rate, determined by the fluorescent glucose analog 2- NBDG in HEK293 cells treated with MOTS-c (10 μM; 72 hours) (N = 3). Student's t-test. *** P <0.001
[0041] Figure 22 describes the effect of MOTS-c on the metabolism of various sugar substrates in HEK293 cells stably overexpressing MOTS-c, determined by metabolomics (N = 5). EV: HEK293 stably transfected with empty vector.
[0042] Figure 23 describes intermediate metabolites, determined by metabolomics (N = 5), of a) glycolysis and b) pentose phosphate pathway (PPP) after 24 or 72 hours of exogenous treatment with MOTS-c (10 μM) in HEK293 cells and c) PPP in HEK293 cells stably overexpressing MOTS-c. EV: HEK293 stably transfected with empty vector. Student's t-test between groups within the same time point. * P <0.05, ** P <0.01, *** P <0.001
[0043] Figure 24 describes the effect of exogenous MOTS-c treatment (10 μM; 24 or 72 hours) on the levels of tricarboxylic acid cycle (TCA) intermediates in HEK293 cells, determined by metabolomics (N = 5). Student's t-test between groups within the same time point. * P <0.05, ** P <0.01, *** P <0.001
[0044] Figure 25 describes the effect of several MOTS-c expression clones on the rate of oxygen consumption (OCR) in HEK293 cells (N = 12). MOTS-c-HA: MOTS-c marked with HA at the N-terminal; MOTS-c- IRES-GFP: MOTS-c cloned into an expression vector that independently expresses GFP through the internal ribosome entry site (IRES); MOTS-c-HA-IRES-GFP: MOTS-c-HA cloned into an expression vector with IRES-GFP. Student's t-test. * P <0.05, ** P <0.01, *** P <0.001
[0045] Figure 26 describes the cellular redox homeostasis that was determined by the state of the glutathione system. Glutathione in its reduced (GSH) and oxidized (GSSG) form, its ratio (GSH / GSSG) and total glutathione levels (GSH + GSSG) were determined in a) HEK293 cells stably overexpressing MOTS-c and b) MOTS-treated HEK293 cells -c exogenous (10 μM) for 24 or 72 hours (N = 5), EV: HEK293 stably transfected with empty vector. Student's t-test. * P <0.05, ** P <0.01
[0046] Figure 27 describes the effect of MOTS-c on lipid metabolism. a) MOTS-c regulates lipid metabolism in HEK293 cells stably overexpressing MOTS-c. Treatment of exogenous MOTS-c (10 μM; 24 or 72 hours) regulates the levels of b) members of the carnitine-transport system, c) essential fatty acids and d) e-oxidation myristoil-CoA. Student's t-test. * P <0.05, ** P <0.01, *** P <0.001
[0047] Figure 28 describes the levels of long-chain fatty acids in a) HEK293 cells stably overexpressing MOTS-c and b) HEK293 cells treated with exogenous MOTS-c (10 μM) for 24 or 72 hours, determined by metabolomics (N = 5). EV: HEK293 stably transfected with empty vector. Student's t-test. * P <0.05, ** P <0.01, *** P <0.001
[0048] Figure 29 describes the effect of acute MOTS-c (5 mg / kg / day; IP; BID) for 4 days in a) body weight change (%), b) food intake, c) glucose in blood and d) adipokines in male CD-1 mice (N = 6). and f) The effect of MOTS-c (0.5 mg / kg / day) on e) daily food intake and f) caloric / total food intake of mice fed a high-fat (60% calorie) diet for 8 weeks. Student's t-test between groups within the same time point. * P <0.05, ** P <0.01
[0049] Figure 30 describes microarray data in HEK293 cells treated with MOTS-c (10 μM) for 72 hours (N = 6). See Figure 14d.
[0050] Figure 31 describes the total number of biochemicals (metabolites) in HEK293 cells with significant (p <0.05) and almost significant (0.05 <P <0.1) changes 24 and 72 hours after treatment of exogenous MOTS-c ( 10 μM) or MOTS-c stably overexpressing or empty vector (EV) (N = 5). Red: increased, blue: decreased. Two Sample Welch t-tests
[0051] Figure 32 describes the metabolic parameters and circulating factors of the euglycemic-hyperinsulinemic clamp study in Figure 16h-j. Student's t-test.
[0052] Figure 33 depicts fasting blood sugars from day 6. ZDF diabetic rats were followed until blood sugar was> 300 ng / ml, at which point treatment was started with one of two doses of MOTS-c or insulin, given IV at 8:00 am for 6 days. 6 rats per group were used. Fasting blood sugars were measured after 6 days. ANOVA and Student's t-tests were used for analysis and p-values.
[0053] Figure 34 describes the absolute change in blood glucose on day 6. In the same experiment described in figure 33, absolute changes in blood glucose are shown. Salt-treated ZDF rats exhibit a further 100 ng / ml increase in their blood sugar. Rats treated with daily insulin stabilize blood sugar at levels similar to the baseline, while rats with MOTS-c (1 mg per 400-gram rat) show a substantial reduction in their blood sugar.
[0054] Figure 35 describes how MOTS-c stimulates the glycolytic flow evidenced by increased lactate production and glucose absorption. Extracellular glucose (A) and lactate (B) in HEK293 cell culture medium stably overexpressing MOTS-c (N = 6)
[0055] Figure 36 describes how MOTS-c increases the flow of cellular glucose in vitro and acute treatment reduces glucose levels in mice fed a normal diet. To test insulin sensitivity, we treated mice with intraperitoneal injections of MOTS-c (5 mg / kg / day) for 7 days and then subjected them to a glucose tolerance test (GTT; glucose 1g / kg) in C57BL / mice 6 males (N = 7). We found significantly increased glucose clearance, indicative of improved insulin sensitivity.
[0056] Figure 37 describes tests of the effects of MOTS-c (0.5 mg / kg / day; PI) on metabolism in the context of a high-fat diet (HFD 60% of calories) (N = 10). Although treatment of MOTS-c had no effect on body weight when fed a normal diet, it remarkably prevented obesity in mice fed an HFD (A). (B-C) This difference in body weight was not attributed to food intake, since the caloric intake was identical between groups. (D-E) In addition, MOTS-c treatment prevented HFD-induced hyperinsulinemia, indicating improved glucose homeostasis. (F) MOTS-c also reduced HFD-induced liver steatosis determined by liver H&E staining.
[0057] Figure 38 describes tests for the effects of MOTS-c (5.0 mg / kg / day; PI) on the high-fat diet (HFD 60% of calories) in mice from a different genetic background, C57BL / 6 (N = 12). (A) Similar to CD-1 mice, MOTS-c treatment prevented obesity in C57BL / 6 mice fed an HFD. (B) Reduced weight gain can be partly attributed to reduced levels of visceral fat. (C) This difference in body weight was not due to lower food intake. In addition, treatment of MOTS-c prevented HFD-induced hyperinsulinemia, indicating improved glucose homeostasis (D-E). ND: normal diet, HFD: high fat diet.
[0058] Figure 39 shows the effect of MOTS-c on the metabolism of the whole body, male ex-crossed CD-1 mice fed a normal diet were treated with MOTS-c (5 mg / kg / day; Bid, 4 days) or vehicle control (N = 6). (4 days) MOTS-c treatment reduced body weight, food levels and blood glucose. Also, baseline levels of circulating IL-6 and TNFa (A), implicated in the pathogenesis of obesity and insulin resistance, were significantly reduced by the treatment of MOTS-c.
[0059] Figure 40 shows cancer cells that are known to have unbalanced metabolism to support their unrestrained growth. MOTS-c is a regulator of metabolic homeostasis and affects the proliferative capacity of the malignant cell. Breast cancer cells (4Q1) stably overexpressing MOTS-c show reduced proliferation rate (A) and (B) and reduced respiration. In addition, the treatment of MOTS-c (C) delayed breast cancer proliferation (4T1) in a mouse model of subcutaneous allograft; (D.) MOTS-c treatment was non-toxic, as reflected by maintaining stable body weight.
[0060] Figure 41 shows that similar to breast cancer, MOTS-c also slows the growth of prostate cancer cells. (A) MOTS-c treatment delayed prostate cancer cell proliferation (22Rv1) in vitro. (B-C) Furthermore, the treatment of MOTS-c delayed the proliferation of prostate cancer (22Rv1) in a mouse model of subcutaneous allograft; (D) MOTS-c treatment was non-toxic, as reflected by maintaining stable body weight.
[0061] Figure 42 describes that MOTS-c treatment slows the proliferation of 4 different prostate cancer cell lines in vitro in a dose-dependent manner. MTT reduction was used to assess cell proliferation and relative quantity (RQ) for the control is presented. The cells were treated for 48-72 hours under ideal growth conditions. DEFINITIONS
[0062] For your convenience, the meanings of certain terms and phrases used in the specification, examples and appended claims, are provided below.
[0063] As used here, the term "diabetes" refers to the broad class of disorders characterized by impaired insulin production and glucose tolerance. Diabetes includes type 1 and type 2 diabetes (also called juvenile and adult onset, respectively), gestational diabetes, pre-diabetes, insulin resistance, metabolic syndrome and impaired glucose tolerance. Common symptoms include frequent urination, excessive thirst, extreme hunger, abnormal weight loss, increased fatigue, irritability and blurred vision. Diagnosis of these individual disorders is described in more detail below.
[0064] Type I diabetes is also known as insulin dependent diabetes mellitus (IDDM), type 1 diabetes and juvenile diabetes. The terms are used interchangeably in this document. Treatment and diagnosis of the disease is described in more detail below
[0065] "Pancreatic beta cells," "beta islet cells" and similar terms refer to a population of endocrine cells in the pancreas found on islets of Langerhans. Beta islet cells produce and secrete insulin and amylin into the bloodstream.
[0066] As used here, "improving cell survival" refers to an increase in the number of cells that survive a given condition, compared to a control, for example, the number of cells that would survive under the same conditions , in the absence of treatment. Conditions can be in vitro, in vivo, ex vivo or in situ. Improved cell survival can be expressed as a comparative value, for example, twice as many cells survive if cell survival is improved twice. Improved cell survival can result from a decrease in apoptosis, an increase in cell life, or an improvement in cell function and condition. In some embodiments, cell survival is improved by 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%, compared to control levels. In some modalities, cell survival is by two, three, four, five or ten times the levels of control. Alternatively, improved cell survival can be expressed as a decrease in the percentage of apoptosis. In some modalities, for example, apoptosis is reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90 or even 100%, compared to a control sample.
[0067] The term "preventing a disorder", as used here, is not intended as an absolute term. Instead, prevention, for example, of type 1 diabetes, refers to delayed onset, reduced frequency of symptoms, or reduced severity of symptoms associated with the disorder. Prevention, therefore, refers to a wide range of prophylactic measures that will be understood by those skilled in the art. In some circumstances, the frequency and severity of symptoms is reduced to non-pathological levels, for example, so that the individual does not need traditional insulin replacement therapy. In some circumstances, the symptoms of an individual receiving the compositions of the invention are only 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 or 1% frequent or severe as symptoms experienced by an individual not treated with the disorder.
[0068] Likewise, the term "treating a disorder" is not intended to be an absolute term. In some aspects, the compositions of the invention seek to reduce the loss of insulin-producing cells that leads to diabetic symptoms. In some circumstances, treatment with the composition leads to a better prognosis or a reduction in the frequency or severity of symptoms.
[0069] As used here, the term "an individual in need of treatment or prevention" refers to an individual who has been diagnosed with type 1 diabetes, type 2 diabetes, gestational diabetes, pre-diabetes, insulin resistance, metabolic syndrome , or impaired glucose tolerance or who is at risk of developing any of these disorders. Individuals in need of treatment also include those who have suffered an injury, illness or surgical intervention affecting the pancreas, or individuals who are otherwise impaired in their ability to take insulin. Such individuals can be any mammal for example, human, dog, cat, horse, pig, sheep, cattle, rat, mouse, rabbit or primate.
[0070] As used in this document, the term "percentage of sequence identity" is determined by comparing two sequences ideally aligned in a comparison window, in which the part of the polynucleotide sequence in the comparison window can include additions or deletions (i.e., gaps) compared to the reference sequence (e.g., a polypeptide of the invention), which do not comprise, additions or deletions, for the optimal alignment of the two sequences. The percentage is calculated by determining the number of positions in which the identical nucleic acid base or amino acid residue occurs in both sequences to produce the number of corresponding positions, dividing the number of corresponding positions by the total number of positions in the comparison window and multiply the result by 100 to yield the percentage of sequence identity.
[0071] As used herein, the terms "identical" or percentage of "identity," in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same. The sequences are "substantially identical" if two sequences have a certain percentage of amino acid or nucleotide residues that are the same (ie, 60% identity, optionally, 65%, 70%, 75%, 80%, 85%, 90% or 95% identity in a specified region, or, when unspecified, over the entire sequence), when compared and aligned for maximum match in a comparison window, region designated as a measurement using one of the following comparison algorithms of sequence or manual alignment and visual inspection, or in the whole sequence where not indicated. The invention provides polypeptides or polynucleotides that encode polypeptides that are substantially identical, or comprise sequences substantially identical to the polypeptides exemplified here (for example, humanin). This definition also refers to the complement of a nucleotide test sequence.
[0072] A "prediabetic individual" refers to an adult with a fasting blood glucose level greater than 110 mg / dl but less than 126 mg / dl or a 2-hour PG reading greater than 140 mg / dl, but less than 200 mg / dl. A "diabetic individual" refers to an adult with a fasting blood glucose level greater than 126 mg / dl or a 2-hour PG reading greater than 200 mg / dl. DETAILED DESCRIPTION OF THE INVENTION Introduction
[0073] A decade ago, the first mitochondria-derived peptide (MDP), humanin (HN), was cloned from a cDNA library built from the unaffected fraction of the brain of an Alzheimer's patient and was mapped to the rRNA locus Mitochondrial 16s. HN has been demonstrated as a cytoprotective and anti-apoptotic factor (Bachar, AR, et al., (2010). Humanin is expressed in human vascular walls and has a cytoprotective effect against oxidized LDL-induced oxidative stress. Cardiovascular research 88, 360366; Guo, B., et al., (2003). Humanin peptide suppresses apoptosis by interfering with Bax activation. Nature 423, 456-461; Hashimoto, Y., et al ... (2001). A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer's disease genes and Abeta. Proc Natl Acad Sci USA 98, 6336-6341; Hoang, PT, et al., (2010). The neurosurvival factor Humanin inhibits beta-cell apoptosis via signal transducer and activator of transcription 3 activation and delays and ameliorates diabetes in nonobese diabetic mice (Metabolism: clinical and experimental 59, 343-349; and Ikonen, M., et al. 2003). Interaction between the Alzheimer’s survival peptide humanin and insulin-like growth factor-binding protein 3 regulates cell survival and apoptosis. Proc Natl Acad Sci USA 100, 13042-13047). There are now more than 130 publications describing the various aspects of HN biology.
[0074] Stated in previous observations, an additional MDP encoded within the 12S rRNA and named MOTS3 (Mitochondrial Open-reading-frame within the Twelve S rRNA) was discovered in silicon and is disclosed in this document (table 1). Table 1. Sequence and location of MOTS3 (SEQ ID NO: 1 and SEQ ID NO: 2).

[0075] As reported here, MOTS3 shifts the cellular metabolic state by modulating mitochondrial function / metabolism and glucose utilization. This has a lot of potential to be applied to various diseases in which mitochondria play an important role in the pathogenesis or maintenance / progression of the disease, such as cancer, diabetes, fatty liver, neurodegenerative diseases, hypertension and aging.
[0076] In addition, mitochondrial dysfunction plays a central role in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease (AD) as well as hypertension.
[0077] In cancer, mitochondria are central organelles for the production of biosynthetic precursor to supply their unrestrained proliferation of signature, which can be counterbalanced by suppression of MOTS3-dependent mitochondrial function and reduced cell proliferation (Ward and Thompson, (2012) Metabolic reprogramming: a cancer hallmark even warburg did not anticipate (Cancer cell 21, 297-308).
[0078] Metabolic diseases such as obesity and hepatic steatosis can also be treated with MOTS3 as it is effective in regulating body weight and liver mitochondrial breathing under the same amount of food consumption (Figure 11). In diabetes, drugs that activate 5 'adenosine monophosphate-activated kinase (AMP) -activated (AMPK), such as Metformin and AICAR, have been well documented regulating glucose homeostasis and are currently used in clinics (Zhang et al, (2009 ) AMPK: an emerging drug target for diabetes and the metabolic syndrome. Cell metabolism 9, 407-416). Likewise, MOTS3 is also a strong activator of AMPK (Figure 6) as well as a significant regulator of glucose levels (Figure 12A) and therefore could be used to treat diabetes. Diabetes
[0079] Type II diabetes (non-insulin-dependent diabetes) is a common metabolic disorder that is increasing rapidly particularly in the developed world. It can be characterized by insulin resistance, insulin deficiency and hyperglycemia. Factors that are related to type II diabetes include hypertension, obesity and high cholesterol.
[0080] Type II diabetes may not be diagnosed for many years, as the symptoms can be sporadic and are certainly milder than those associated with type I diabetes. However, high blood sugar levels in patients with untreated type II diabetes can lead to functional impairment of the kidneys, eyes and cardiovascular systems.
[0081] In addition, type II diabetes can be caused by the failure of beta cells to compensate for insulin resistance. High-calorie diets and insufficient muscle work seem to be important environmental factors involved in the pathogenesis of obesity and type II diabetes. Environmental factors appear to act through two main targets. One is the processing of glucose, fatty acids and other metabolites, as regulated by insulin and other hormones in most tissues, and the other is the function of the beta cell.
[0082] Obesity has become a public health problem. Health conditions caused or made worse by obesity include hypertension, diabetes mellitus, sleep apnea, obesity hypoventilation, back and joint problems, cardiovascular disease, non-alcoholic fatty liver disease and gastroesophageal reflux disease.
[0083] The body mass index (BMI) (calculated as weight in kilograms divided by the square of height in meters) is the most commonly accepted measurement for overweight and / or obesity. A BMI greater than 25 is considered overweight, while obesity is defined as a BMI of 30 or more, with a BMI of 35 or more considered to be severe comorbidity and a BMI of 40 or more considered to be morbid obesity.
[0084] Type I diabetes is an autoimmune disease characterized by the progressive destruction of pancreatic beta cells after islet infiltration by lymphocytes. This results in insulin deficiency.
[0085] Apoptosis is the primary mode of beta cell death during the development of type-1 diabetes (O'Brien et al. (1997) Diabetes 46: 750-57). Il-1, TNF-α and IFN-y are released by T cells and macrophages during this autoimmune response and are important mediators of beta cell destruction (Eizirik and Mandrup-Poulsen (2001) Diabetologia44: 2115- 2133).
[0086] Protein-3 binding to insulin-like growth factor (IGFBP-3) induces apoptosis in a way unrelated to its IGF binding (Rajah et al. (1997) J Biol Chem. 272: 12181- 88). Pro-inflammatory cytokines Th1 increase intranuclear aggregation of endogenous IGFBP-3 and addition of exogenous IGFBP-3 to beta cells induces apoptosis (Shim et al. (2004) Growth Norm IGF Res. 14: 216-25). IGFBP-3 is one of a series of peptides that includes insulin, leptin, adiponectin and resistin, which has been shown to act on the central nervous system to regulate glucose metabolism (Musa et al. (2007) J Clin Invest. 117: 1670 -78; Obici et al. (2002) Nat Med 8: 1376-82). Loss of beta cells by apoptosis also occurs after islet grafting (Paraskevas et al. (1999) FEBS Lett. 455: 2038); Tobiasch et al. (2001) J Investig Med. 49: 566-71). Recent studies have shown that isolated human islets express the pro-apoptotic protein Bax at a higher level than the anti-apoptotic protein Bcl-2 (Thomas et al. (2002) Transplantation 74: 1489).
[0087] More than 1 million people in the United States have type I diabetes. According to the American Diabetes Association, the disease causes thousands of deaths every year and costs more than $ 20 billion annually. An effective therapeutic or preventive agent is available for type I diabetes.
[0088] There is a need to develop methods to treat and prevent type I and type II diabetes. Therefore, in certain embodiments of the present invention, MOTS3 and respective pharmaceutical compositions can be used to treat and prevent type I and type II diabetes. Cancer
[0089] Cancer is a serious threat to modern society. Worldwide, more than 10 million people are diagnosed with cancer every year and it is estimated that this number will grow to 15 million new cases every year by 2020. Cancer causes 6 million deaths every year or 12% of deaths worldwide.
[0090] Malignant cancerous tumors, due to their unique characteristics, pose serious challenges for modern medicine. Its characteristics include uncontrollable cell proliferation, resulting in unregulated growth of malignant tissue, an ability to invade local and even remote tissues, lack of differentiation, lack of detectable symptoms and, more significantly, the lack of effective therapy and prevention.
[0091] Cancer can develop in any tissue of any organ at any age. The etiology of cancer is not clearly defined, but mechanisms such as genetic susceptibility, rupture of chromosomal disorders, viruses, environmental factors and immunological disorders have been associated with malignant cell growth and transformation. Cancer covers a large category of medical conditions, which affect millions of individuals worldwide. Cancer cells can appear in almost any organ or tissue in the body. Cancer develops when cells in a part of the body start to grow or differentiate out of control. All types of cancer begin with out-of-control growth of abnormal cells.
[0092] There are many types of cancer, including, but not limited to brain cancer, colon cancer, melanoma, leukemia (eg, AML), lymphoma, pancreatic cancer, prostate cancer, ovarian cancer, lung cancer and cancer gastric.
[0093] Currently, some of the main treatments available are surgery, radiotherapy and chemotherapy. Surgery is often a drastic measure and can have serious consequences. For example, all treatments for ovarian cancer can result in infertility. Some treatments for cervical cancer and bladder cancer can cause infertility and / or sexual dysfunction. Surgical procedures to treat pancreatic cancer can result in partial or total removal of the pancreas and can lead to significant risks for the patient. Breast cancer surgery invariably involves removing part or all of the breast. Some surgical procedures for prostate cancer carry the risk of urinary incontinence and impotence. Procedures for lung cancer patients often have significant postoperative pain, since the ribs must be cut to access and remove the cancerous lung tissue. In addition, patients who have lung cancer and another lung disease, such as emphysema or chronic bronchitis, typically experience an increase in their shortness of breath after surgery.
[0094] Radiotherapy has the advantage of killing cancer cells, but it also damages non-cancerous tissue at the same time. Chemotherapy involves the administration of various anticancer drugs to a patient, but it is often accompanied by adverse side effects.
[0095] Thus, there is still a need for methods that can treat and prevent cancer. These methods can provide the basis for pharmaceutical compositions useful in the prevention and treatment of cancer in humans and other mammals. Therefore, in certain embodiments of the present invention, MOTS3 and respective pharmaceutical compositions can be used to treat and prevent cancer. Specifically, as examples of non-limitation, MOTS3 and respective pharmaceutical compositions can be used to treat and prevent brain cancer, colon cancer, melanoma, leukemia (eg, AML), lymphoma, pancreatic cancer, prostate cancer, cancer of ovary, lung cancer and gastric cancer. Fatty Liver and Obesity
[0096] Fatty liver, also known as hepatic steatosis (FLD), is a reversible condition where vacuoles of triglyceride fats accumulate in the liver. The process of fat accumulation in liver cells is known as steatosis.
[0097] Obesity is a general term to describe having too much body fat. Obesity occurs when a subject consumes more calories than he / she can burn, leading to fat deposits. Obesity is caused by eating more calories than a guy needs and not getting enough exercise. Two of the most common methods for assessing obesity are body mass index (BMI) and waist circumference. BMI is calculated using height and weight. A high BMI, that is, obesity, increases the risk of type II diabetes, heart disease and stroke.
[0098] Thus, there is still a need for methods that can treat and prevent obesity and fatty liver. Therefore, in certain embodiments of the present invention, MOTS3 and respective pharmaceutical compositions can be used to treat and prevent obesity and fatty liver. Polypeptide Expression and Purification
[0099] Naturally occurring, synthetic or recombinant polypeptides of the invention can be purified for use in compositions and functional assays. The naturally occurring polypeptides of the invention can be purified from any source. Recombinant polypeptides can be purified from any suitable expression system (for example, mammals, insects, yeast or bacterial cell culture).
[00100] The peptides of the present invention (i.e. MOTS3 and MOTS3 analogs) can include modified peptides and synthetic peptide analogs. In some embodiments the peptide is a 70%, 75%, 80%, 85%, 90%, 95%, 97% or 100% peptide identical to SEQ ID NO: 1. In some embodiments the peptide is a peptide comprising SEQ ID NO: 1. In some embodiments, the peptide is SEQ ID NO: 1. Peptides as described herein can be modified to improve formulation and storage properties, or to protect labile peptide bonds by incorporating non-peptide structures. Peptides of the present invention can be prepared using methods known in the art. For example, peptides can be produced by chemical synthesis, for example, using solid phase techniques and / or automated peptide synthesizers. In certain cases, peptides can be synthesized using solid phase strategies in an automated multiple peptide synthesizer (Abimed AMS 422) using 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry. The peptides can be purified by inverted phase HPLC and lyophilized.
[00101] For recombinant approaches, the present invention includes isolated nucleic acids encoding the polypeptides disclosed herein, expression vectors comprising the nucleic acids and host cells comprising the expression vectors. More particularly, the invention provides isolated nucleic acids encoding MOTS3 peptides and MOTS3 peptide analogs having MOTS3 activities, the peptides including, but not limited to, SEQ ID NO: 1. In some embodiments, this invention comprises a cDNA molecule that encodes a peptide at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or 100% identical to SEQ ID NO: 1. In some embodiments, this invention comprises a cDNA molecule encoding SEQ ID NO: 1. In some embodiments, this invention comprises a cDNA at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or 100% identical to SEQ ID NO: 2.
[00102] When recombinant proteins are expressed by the transformed bacterium in large quantities, usually after induction of the promoter, although expression may be constitutive, the proteins can form insoluble aggregates. There are several protocols that are suitable for the purification of protein inclusion bodies. For example, purification of aggregated proteins (hereinafter referred to as inclusion bodies) typically involves the extraction, separation and / or purification of inclusion bodies by disrupting bacterial cells normally, but not limited to, by incubating in a buffer of approximately 100 -150 μ g / ml lysozyme and 0.1% Nonidet P40, a non-ionic detergent. The cell suspension can be ground using a Polytron grinder (Brinkman Instruments, Westbury, N.Y.) Alternatively, the cells can be sonicated on ice. Alternative methods of lysis of bacteria are described in Ausubel et al and Sambrooket al, both above and will be evident to those skilled in the art.
[00103] The cell suspension is usually centrifuged and the pellet containing the inclusion bodies resuspended in the buffer that does not dissolve but washes the inclusion bodies, for example, 20 mM Tris-HCl (pH 7.2), 1 mM EDTA , 150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. It may be necessary to repeat the washing step to remove as much cell debris as possible. The remaining pellet of inclusion bodies can be resuspended in an appropriate buffer (eg, 20 mM sodium phosphate, pH 6.8, 150 mM NaCl). Other suitable buffers will be evident to those skilled in the art.
[00104] After the washing step, the inclusion bodies are solubilized by the addition of a solvent that is a strong hydrogen acceptor and a strong hydrogen donor (or a combination of solvents each having one of these properties). The proteins that form the inclusion bodies can then be renatured by dilution or dialysis with a compatible buffer. Suitable solvents include, but are not limited to, urea (from about 4 M to about 8 M), formamide (at least about 80%, volume / volume basis) and guanidine hydrochloride (from about 4 M to about 8 M). Some solvents that are capable of solubilizing aggregate-forming proteins, such as SDS (sodium dodecyl sulfate) and 70% formic acid, are unsuitable for use in this procedure due to the possibility of irreversible denaturation of the proteins, accompanied by a lack of immunogenicity and / or activity. Although guanidine hydrochloride and similar agents are denaturants, this denaturation is not irreversible and renaturalization can occur after removal (by dialysis, for example) or dilution of the denaturant, allowing immunologically and / or biologically active protein re-formation of interest. After solubilization, proteins can be separated from other bacterial proteins by standard separation techniques.
[00105] As an alternative, it is possible to purify the periplasma proteins from bacteria. Where protein is exported to the bacterial periplasm, the bacterial periplasmic fraction can be isolated by cold osmotic shock, in addition to other methods known to those skilled in the art (See, Ausubel et al, supra). To isolate recombinant proteins from the periplasm, bacterial cells are centrifuged to form a pellet. The pellet is resuspended in a buffer containing 20% sucrose. To lyse the cells, the bacteria are centrifuged and the pellet is resuspended in 5mm of cold MgSO4 and kept in an ice bath for approximately 10 minutes. The cell suspension is centrifuged and the supernatant decanted and saved. Recombinant proteins present in the supernatant can be separated from host proteins by standard separation techniques well known to those skilled in the art.
[00106] The polypeptides of the invention can be purified to substantial purity by standard techniques, including selective precipitation with substances such as ammonium sulfate; column chromatography, immunopurification and other methods (see, for example, Scopes, Protein Purification: Principles and Practice (1982); US Pat. No. 4,673,641; Ausubel et al., Current Protocols in Molecular Biology (1995 supplement); and Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., (1989)).
[00107] A number of procedures can be employed when polypeptides are being purified. For example, polypeptides can be purified using immunoaffinity columns or ion exchange.
[00108] Often as an initial step, and if the protein mixture is complex, an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the recombinant protein of interest. The preferred salt is ammonium sulfate. Ammonium sulfate precipitates proteins, effectively reducing the amount of water in the protein mixture. The proteins then precipitate based on their solubility. The more the protein is hydrophobic, the more likely it is to precipitate in low concentrations of ammonium sulfate. A typical protocol is to add saturated ammonium sulfate to a protein solution so that the resulting ammonium sulfate concentration is between 20-30%. This will precipitate the most hydrophobic proteins. The precipitate is discarded (unless the protein of interest is hydrophobic) and ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest. The precipitate is then solubilized in buffer and the excess salt removed, if necessary, by means of dialysis or diafiltration. Other methods that depend on protein solubility, such as precipitation of cold ethyl alcohol, are well known to those skilled in the art and can be used to fractionate complex protein mixtures.
[00109] Based on the calculated molecular weight, a larger and smaller protein can be isolated using ultrafiltration through membranes of different pore sizes (eg Amicon or Millipore membranes). As a first step, the protein mixture is ultrafiltered through a membrane with a pore size that has a cut molecular weight less than the molecular weight of the protein of interest. The ultrafiltration retentate is then ultrafiltered against a membrane with a molecular cut greater than the molecular weight of the protein of interest. The recombinant protein will pass through the membrane into the filtrate. The filtrate can then be chromatographed as described below.
[00110] The proteins of interest can also be separated from other proteins based on their size, liquid surface charge, hydrophobicity and affinity for ligands. In addition, antibodies produced against proteins can be conjugated to column matrices and immunopurified proteins. All of the above methods are well known in the art.
[00111] Immunoaffinity chromatography using antibodies generated for a variety of affinity tags such as hemagglutinin (HA), FLAG, Xpress, Myc, hexahistidine, glutathione S transferase (GST) and the like can be used to purify the polypeptides. Your brand will also act as a chelating agent for certain metals (for example, Ni) and therefore the metals can also be used to purify His-containing polypeptides. After purification, the mark is optionally removed by specific proteolytic cleavage.
[00112] It will be evident to those skilled in the art that chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (for example, Pharmacia Biotech). Pharmaceutical Compositions
[00113] The peptides of the present invention can be administered with a suitable pharmaceutical excipient as needed. One skilled in the art will understand that the composition will vary depending on the mode of administration and unit dosage.
The compositions typically include a conventional pharmaceutical carrier or excipient and may additionally include other medicinal agents, carriers, adjuvants, diluents, tissue permeation enhancers, solubilizers and the like. Preferably, the composition will contain about 0.01% to about 90%, about 0.1% to about 75%, about 0.1% to 50%, or about 0.1% to 10% % by weight of a conjugate of the present invention or a combination thereof, with the remainder consisting of a suitable pharmaceutical carrier and / or excipients. Suitable excipients can be adapted to the specific composition and route of administration by methods known in the art. See, for example, REMINGTON’S PHARMACEUTICAL SCIENCES, 18TH ED., Mack Publishing Co., Easton, Pa. (1990).
[00115] examples of suitable excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, acacia gum, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose and polyacrylic acids such as Carbopols, e.g., Carbopol 941, Carbopol 980, Carbopol 981 etc. The compositions may furthermore include lubricating agents such as talc, magnesium stearate and mineral oil; wetting agents; emulsifiers; suspending agents; preserving agents such as methyl-, ethyl- and propylhydroxy-benzoates (i.e., parabens); pH adjusting agents such as organic and inorganic acids and bases; sweetening agents; coloring agents; and flavoring agents. The compositions may also include biodegradable polymer granules, dextran and cyclodextrin inclusion complexes.
[00116] For oral administration, the compositions can be in the form of tablets, lozenges, capsules, emulsions, suspensions, solutions, syrups, sprays, powders and sustained release formulations. Excipients suitable for oral administration include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, gelatin, sucrose, magnesium carbonate and the like.
[00117] In some embodiments, pharmaceutical compositions take the form of a pill, tablet or capsule, and thus, the composition may contain, together with the conjugate or combination of conjugates, any of the following: a diluent such as lactose, sucrose, dicalcium phosphate and the like; a disintegrant such as starch or its derivatives; a lubricant such as magnesium stearate and the like; and / or a binder of such starch, acacia gum, polyvinylpyrrolidone, gelatin, cellulose and its derivatives. The conjugates can also be formulated in a suppository arranged, for example, in a polyethylene glycol (PEG) carrier.
[00118] Liquid compositions can be prepared by dissolving or dispersing a conjugate or a combination of conjugates and, optionally, one or more pharmaceutically acceptable adjuvants in a carrier such as, for example, aqueous saline (for example, 0% sodium chloride, 9% w / v), aqueous dextrose, glycerol, ethanol and the like, to form a solution or suspension, for example, oral, topical, or intravenous administration. The conjugates of the present invention can also be formulated into a retention enema.
[00119] For topical administration, the compositions of the present invention can be in the form of emulsions, lotions, gels, creams, jellies, solutions, suspensions, ointments and transdermal patches. For delivery by inhalation, the composition can be delivered as a dry powder or in liquid form, through a nebulizer. For parenteral administration, the compositions may be in the form of sterile injectable solutions and packaged sterile powders. Preferably, injectable solutions are formulated at a pH of about 4.5 to about 7.5.
[00120] The compositions of the present invention can also be supplied in a lyophilized form. Such compositions can include a buffer, for example, bicarbonate for reconstitution prior to administration, or the buffer can be included in the lyophilized composition for reconstitution with, for example, water. The lyophilized composition can also include an appropriate vasoconstrictor, for example, epinephrine. The lyophilized composition can be supplied in a syringe, optionally packaged in combination with the reconstitution buffer, such that the reconstituted composition can be administered immediately to the patient.
[00121] One skilled in the art understands that the dose administered will vary depending on several factors, including, but not limited to, the specific peptide composition to be administered, the mode of administration, the type of application (eg prophylactic, therapeutic etc. ), the patient's age and the patient's physical condition. Preferably, the lowest dose and concentration necessary to produce the desired result should be used. Dosage can be appropriately adjusted for children, the elderly, debilitated patients and patients with heart and / or liver disease. Further guidance can be obtained from studies known in the art using experimental animal models to assess dosage. MOTS3 or MOTS3 analogue can be formulated without undue experimentation for administration to a mammal, including humans, as appropriate for the specific application. In addition, the proper dosages of the compositions can be determined without undue experimentation using standard dose-response protocols.
[00122] In some embodiments, peptides comprising the compositions of the present invention are administered to a subject at a given dose of the peptide or are formulated for administering unit dosage of the peptide to a subject. In some embodiments, the dose administered to a subject is from 0.001 to about 1000 mg per day. In some embodiments, the dose administered to a subject is 0.1 to about 500 mg per day. In some embodiments, the dose administered to a subject is 0.5 to about 100 mg per day. In some embodiments, the compositions of the present invention are formulated for administering unit dosage, in which the unit dosage is from 0.001 to about 1000 mg per day. In some embodiments, the compositions of the present invention are formulated for administering unit dosage, in which the unit dosage is from 0.1 to about 500 mg per day. In some embodiments, the compositions of the present invention are formulated for administering unit dosage, in which the unit dosage is 0.5 to about 100 mg per day. Administration Methods
[00123] Administration of the peptides of the present invention with a suitable pharmaceutical excipient as needed can be carried out by any of the accepted modes of administration. Thus, administration can be, for example, oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arterioles, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, or by inhalation. Administration can be directed directly to the pancreatic tissue, for example, by injection.
[00124] The compositions of the invention can be administered repeatedly, for example, at least 2, 3, 4, 5, 6, 7, 8 or more times, or the composition can be administered by continuous infusion. Suitable sites of administration include, but are not limited to, dermal, mucosal, bronchial, gastrointestinal, anal, vaginal, eye and ear. The formulations can take the form of solid, semi-solid, lyophilized powder or liquid pharmaceutical forms, such as, for example, tablets, pills, lozenges, capsules, powders, solutions, suspensions, emulsions, suppositories, retention enemas, creams, ointments, lotions, gels, aerosols, or the like, preferably in unit dosage forms suitable for simple administration of accurate dosages.
[00125] The term "unit dosage form" refers to physically discrete units appropriate as unit doses for human subjects and other mammals, each unit containing a predetermined amount of active material calculated to produce the desired start, tolerability and / or therapeutic effects, in combination with a suitable pharmaceutical excipient (for example, an ampoule). In addition, more concentrated compositions can be prepared, of which the most diluted unit dosage compositions can then be produced. The most concentrated compositions, therefore, will contain substantially more than, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times the amount of a conjugate or combination of conjugates.
[00126] The actual methods of preparing such dosage forms are known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA. (1990). The composition to be administered contains an amount of the peptides of the invention in a pharmaceutically effective amount to improve beta islet cell survival. In addition, pharmaceutically acceptable salts of the peptides of the present invention (for example, acid addition salts) can be prepared and included in the compositions using standard procedures known to those skilled in the art of synthetic organic chemistry and described, for example, by March, Advanced Organic Chemistry: Reactions, Mechanisms and Structure, 4th Ed., New York, Wiley-Interscience (1992).
[00127] In another approach, nucleic acids encoding the polypeptides of the invention are used for transfection of cells in vitro and in vivo. These nucleic acids can be inserted into any one of a number of known vectors for the transfection of target cells and organisms, as described below. Nucleic acids are transfected into cells, either ex vivo or in vivo, through the interaction of the vector and the target cell. Nucleic acids, under the control of a promoter, then express a polypeptide of the present invention, thereby mitigating the effects of a disease associated with reduced insulin production.
[00128] Such gene therapy procedures have been used to correct inherited and acquired genetic defects, cancer and other diseases in various contexts. The ability to express artificial genes in humans facilitates the prevention and / or cure of many important human diseases, including many diseases that cannot be treated by other therapies (for a review of gene therapy procedures, see Anderson, Science, 256 : 808-813 (1992); Nabel et al., TIBTECH, 11: 211-217 (1993); Mitani et al., TIBTECH, 11: 162-166 (1993); Mulligan, Science, 926-932 (1993) ; Dillon, TIBTECH, 11: 167-175 (1993); Miller, Nature, 357: 455-460 (1992); Van Brunt, Biotechnology, 6 (10): 1149-1154 (1998); Vigne, Restorative Neurology and Neuroscience , 8: 35-36 (1995); Kremer et al., British Medical Bulletin, 51 (1): 31-44 (1995); Haddada et al., In Current Topics in Microbiology and Immunology (Doerfler & Bohm eds., 1995); and Yu et al., Gene Therapy, 1: 13-26 (1994)).).
[00129] For the delivery of nucleic acids, viral vectors can be used. Suitable vectors include, for example, herpes simplex virus vectors as described in Lilley et al., Curr. Gene Ther. 1 (4): 339-58 (2001), alphavirus DNA and particle replicons as described in, for example, Polo et al., Dev. Biol. (Basel), 104: 181-5 (2000), plasmid vectors based on Epstein-Barr virus (EBV) as described in, for example, Mazda, Curr. Gene Ther, 2 (3): 379-92 (2002), EBV replicon vector systems as described in, for example, Otomo et al., J. Gene Med., 3 (4): 345-52 (2001 ), rhesus monkey adenovirus-associated viruses, as described in, for example, Gao et al., PNAS USA, 99 (18): 11854 (2002), adenovirus and adeno-associated viral vectors as described in, for example, Nicklin et al ., Curr. Gene Ther., 2 (3): 273-93 (2002). Other appropriate adenoassociated virus (AAV) vector systems can be easily constructed using techniques well known in the art (see, for example, US Pat. Nos. 5,173,414 and 5,139,941; PCT publications No. 01070 / WO 92 and 93 WO / 03769; Lebkowski et al, Mol. Cell. Biol., 8: 3988-3996 (1988); Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, Current Opinion in Biotechnology 3: 533-539 (1992); Muzyczka, Current Topics in Microbiol. And Immunol., 158: 97-129 (1992); Kotin, Human Gene Therapy, 5: 793-801 (1994); Shelling et al., Gene Therapy, 1: 165-169 (1994); and Zhou et al., J. Exp. Med., 179: 1867-1875 (1994)). Additional suitable vectors include gene-attenuated E1B replicating adenoviruses described in, for example, Kim et al, Cancer Gene Ther., 9 (9): 725-36 (2002) and non-replicating adenovirus vectors described in, for example, Pascual et al., J. Immunol., 160 (9): 4465-72 (1998). Exemplary vectors can be constructed, as disclosed by Okayama et al, Mol. Cell. Biol., 3: 280 (1983).
[00130] In some embodiments, this invention comprises an expression vector, with a nucleic acid sequence encoding SEQ ID NO: 1. In some embodiments, this invention comprises an expression vector comprising a nucleic acid sequence that encodes a peptide at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or 100% identical to SEQ ID NO: 1. In some embodiments, this invention comprises an expression vector, comprising a cDNA of at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or 100% identical to SEQ ID NO: 2.
[00131] Molecular conjugate vectors, such as the chimeric adenovirus vectors, described in Michael et al, J. Biol. Chem., 268: 6866-6869 (1993) and Wagner et al., Proc. Natl. Acad. Sci. EUA, 89: 60996103 (1992), can also be used for the delivery of the gene according to the methods of the invention.
[00132] In an illustrative embodiment, retroviruses provide a convenient and effective platform for gene delivery systems. A selected nucleotide sequence encoding a polypeptide of the invention is inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can be isolated and delivered to a subject. Suitable vectors include lentiviral vectors as described in, for example, Scherr et al., Curr. Gene Ther., 2 (1): 45-55 (2002). Additional illustrative retroviral systems have been described (for example, US Pat. No. 5,219,740; Miller et al., BioTechniques, 7: 980-990 (1989); Miller, Human Gene Therapy, 1: 5-14 (1990); Scarpa et al., Virology, 180: 849-852 (1991); Bums et al., Proc. Natl. Acad. Sci. USA, 90: 8033-8037 (1993); and Boris-Lawrie et al., Curr. Opin. Genet. Develop., 3: 102-109 (1993).
[00133] Other known viral-based delivery systems are described in, for example, Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA, 86: 317-321 (1989); Flexner et al., Ann. N.Y. Acad. Sci., 569: 86-103 (1989); Flexner et al., Vaccine, 8: 17-21 (1990); U.S. Pat. No. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner, Biotechniques, 6: 616-627 (1988); Rosenfeld et al., Science, 252: 431-434 (1991); Kolls et al., Proc. Natl. Acad. Sci. USA, 91: 215-219 (1994); Kass-Eisler et al., Proc. Natl. Acad. Sci. USA, 90: 11498-11502 (1993); Guzman et al., Circulation, 88: 2838-2848 (1993); Guzman et al., Cir. Res., 73: 1202-1207 (1993); and Lotze et al., Cancer Gene Ther., 9 (8): 692-9 (2002). Therapeutic and prophylactic applications
[00134] In certain respects, the compositions of the invention are used for the treatment or prevention of a disease or disorder in a subject in need of it. Examples of diseases or disorders suitable for treatment with the MOTS3 compositions or MOTS3 analogues described herein include, but are not limited to, treatment and prevention of disorders that include but are not limited to type 1 and type 2 diabetes, gestational diabetes, prediabetes, insulin resistance, metabolic syndrome, impaired glucose tolerance, cancers (e.g., breast cancer, brain cancer, colon cancer, melanoma, leukemia (e.g., AML), lymphoma, pancreatic cancer, cancer prostate cancer, ovarian cancer, lung cancer and gastric cancer), obesity and fatty liver. The compositions of the invention can be used prophylactically, for example, for individuals with a genetic predisposition to diabetes.
[00135] Those skilled in the art will appreciate that the MOTS3 peptides and analogs of the invention can be administered in conjunction with other therapeutic agents for the treatment or prevention of diseases, described herein. Co-administration can be simultaneous, for example, in a single pharmaceutical composition or separate compositions. The compositions of the invention can also be administered separately from other therapeutic agents, for example, in an independent administration schedule. EXAMPLES Example 1.
[00136] Analysis of RACE 3 'revealed that MOTS3 is polyadenylated, similar to HN (figure 1A). After the initial screening, as described below, MOTS 3 was determined to have potent biological activity, and its peptide sequence is well conserved in several species (figure 1B). We generated polyclonal rabbit antibodies anti-MOTS3 that can detect endogenous and overexpressed MOTS3 by cloning ORF into a cell culture expression vector as well as GFP-labeled MOTS3 (Figure 1C). Using these validated antibodies, we can detect MOTS3 in the rat heart, liver and testicles, with similar molecular weights and in the brain at a slightly higher molecular weight (Figure 1D). The level of expression appears to be higher in the heart, an organ with one of the highest mitochondrial densities. In addition, HeLarho-O cells, which were purged of mitochondrial DNA using ethidium bromide, do not express MOTS3, as well as other well-coded mitochondria-proteins described as cytochrome oxidase I and II (COI and COII), confirming their mitochondrial origin ( figure 1E). Example 2
[00137] Exogenous treatment of synthetic MOTS3 causes a metabolic change dependent on mitochondria, as measured by the oxygen consumption rate (OCR) measured by Seahorse technology as well as a reduction in MTT (conditions below 10% and 1% FBS) ( Figure 2). Exogenous MOTS 3 treatments with synthetic peptides (Figure 3), as well as endogenous expression by cloning (Figure 4) inhibited mitochondrial activity. Notably, treatment of exogenous mots3 reduced levels of cellular ATP and mitochondrial activity (MTT), which occurred simultaneously with increased autophagy (Figure 5).
[00138] MOTS3 treatment induced an increase in glucose absorption, as measured by the residual glucose levels in culture medium and the absorption of fluorescence-labeled glucose analogue (figure 6A and figure 6B). As expected, lactate secretion was increased in proportion to high glucose intake (figure 6A). Notably, for 96 hours of MOTS3 treatment, when most of the glucose in the medium has been consumed, AMPK activation is significantly greater in MOTS3-treated cells (Figure 7), agreeing with the marked decrease in cellular ATP levels and increased autophagy , as shown in Figure 5. Impaired cell metabolism induced by exogenous MOTS3 led to reduced cell proliferation and survival in glucose medium, but not galactose medium (Figure 8); galactose forces cells to depend on mitochondrial metabolism for survival.
[00139] Overexpression of intracellular MOTS3 by transfection of expression clones also slows cell proliferation (Figure 9). Interestingly, 8 days of treatment of exogenous MOTS 3 induced cellular senescence, measured by β-galactosidase-associated senescence staining (Figure 10). Example 3.
[00140] In mice, 4 days of injections of MOTS3 (i.p .; 0.5 or 5.0 mg / kg / day) led to significant weight loss without significant change in food intake (Figure 11). According to our in vitro studies, the hepatic mitochondrial breathing capacity decreased further after the treatment of MOTS3 in both doses tested (Figure 11). Namely, MOTS3 treatment significantly reduced blood glucose levels (figure 12A) and also had higher insulin levels (figure 12B), suggesting that glucose absorption had increases similar to those seen in cell culture (Figure 6). Example 4. MOTS-c: a new peptide encoded within the mitochondrial genome that prevents obesity and regulates metabolic homeostasis
[00141] The mitochondrial genome is traditionally known to code for only 13 proteins, which are converted using a mitochondria-specific genetic code. This was challenged recently with the discovery of humanin, a peptide encoded within the mitochondrial rRNA16S. Humanin suggests the possible existence of unexplored mitochondrial genes that give rise to bioactive peptides (C. Lee, K. Yen, P. Cohen, Humanin: a harbinger of mitochondrial-derived peptides Trends in endocrinology and metabolism: TEM, (February 7 of 2013)). Here we report a new peptide encoded within mitochondrial 12S rRNA, called MOTS-c (mitochondrial open-reading-frame of the twelve SrRNA c), which has been detected in various tissues and in the circulation. MOTS-c acts as a key regulator of metabolic homeostasis, modulating nucleotide, glucose, mitochondrial and fatty acid metabolism. Notably, MOTS-c caused a> 20-fold increase in endogenous AICAR levels, via the de novo purine biosynthesis pathway, and also activated AMPK signaling in HEK293 cells and skeletal muscle in mice. MOTS-c treatment in mice prevented weight gain induced by high-fat diet and insulin resistance. In addition, coinciding with age-dependent insulin resistance in muscle, MOTS-c levels were found to decrease with age in plasma and muscle, and treatment of MOTS-c sufficiently restored insulin sensitivity in older mice. The discovery of MOTS-c, along with humanin, suggests previously unknown regulatory functions for mitochondria in coordinating critical cellular processes, including metabolism. Our results suggest that mitochondrial-derived peptide (MDP) including MOTS-c not only unveils new mitochondrial biology, but may also provide new diagnostic biomarkers and therapeutic targets that can be explored to alleviate metabolic dysfunctions associated with chronic aging and related diseases the age.
[00142] An in silico search for potential open reading frames (ORFs) encoding bioactive peptides within the mitochondrial DNA 12S rRNA (mtDNA) region was performed. MOTS-c has been identified as a 51bp open reading frame (ORF) which translates into a 16 amino acid peptide (Figure 13a), which is highly conserved (Figure 17a). This peptide is also subject to several putative post-translational modifications (figure 17b). MOTS-c translation could theoretically be mitochondrial or cytoplasmic, but because the mitochondrial genetic code produces start / stop codons in tandem (Figure 13a), MOTS-c should be subject to cytoplasmic translation, indicating that its polyadenylated transcription (figure 13b) is exported from the mitochondria by a still unknown mechanism (Y. Ninomiya, S. Ichinose, Subcellular distribution of mitochondrial ribosomal RNA in the mouse oocyte and zygote. PloS one 2, e1241 (2007) and R. Amikura, M. Kashikawa, A. Nakamura, S. Kobayashi, Presence of mitochondria-type ribosomes outside mitochondria in germ plasm of Drosophila embryos. Proceedings of the National Academy of Sciences of the United States of America 98, 9133 (Jul 31, 2001)).
[00143] The possibility of MOTS-c may also be of nuclear origin, due to a phenomenon known as nuclear mitochondrial DNA transfer (M. Ricchetti, F. Tekaia, B. Dujon, Continued colonization of the human genome bymitochondrial DNA. PLoS biology 2, E273 (September 2004)) has been recognized. Using the NCBI basic local nucleotide search tool (BLAST), it was found that none of its peptide products or NUMTs had complete homology to the mitochondrial MOTS-c sequence (Figure 13 c). To confirm its mitochondrial origin, MOTS-c expression in HeLa cells that were selectively depleted of mitochondrial DNA (p0) was measured. In HeLa-p0 cells, both 12S rRNA and MOTS-c transcription were undetectable by qRT-PCR (figure 13d), and immunoblots using specific MOTS-c antibodies (figure 17 c) showed undetectable levels of cytochrome oxidase I and II ( COI / II) mitochondrial-encoded and MOTS-c, but unchanged expression of nuclear-encoded GAPDH (figure 13e). To study its subcellular distribution pattern, HEK293 cells were immunostained for MOTS-c and mitochondrial colocalization was observed (figure 13f; Figure 17d, 17e). MOTS-c was detected in various tissues in mice and rats (Figure 13g), as well as circulating in human and rodent plasma (Figure 13h; figure 17f). Notably, fasting altered the endogenous expression of MOTS-c in certain metabolically active tissues (figure 13i) and in plasma (figure 13j).
[00144] To unravel the biological role of MOTS-c, microarray analysis in HEK293 cells after 4 and 72 hours of MOTS-c treatment (10 μM) was performed. Principal component analysis (PCA) showed that MOTS-c promoted a clear change in global gene expression profile for 72 hours (Figure 14a). To further highlight the differences between functional pathways modified by MOTS-c, parametric analysis of joint gene enrichment (PAGE) was used to show that gene expression significantly altered for 4 hours after MOTS-c treatment, which became remarkably distinct for 72 hours (figure 14b). There was a modest overlap between gene signatures at 4 and 72 hours, suggesting a time-dependent progression in response to MOTS-c treatment (Figure 14c). MOTS-c was found to have a profound effect on gene expression in pathways involved in cellular metabolism and inflammation (Figure 14d; Figure 30).
[00145] Next, the effect of MOTS-c on global metabolism was studied using impartial metabolomic profiling in HEK293 cells or stably transfected with MOTS-c or empty expression of clones (MOTS-c-ST and MOTS-c-EV cells , respectively) or exogenously treated with synthetic MOTS-c (10 Hum) for 24 to 72 hours. Of the 356 named metabolites, 194 were found to be significantly altered in the MOTS-c-ST cells and 49 and 177 were significantly altered for 24 and 72 hours, respectively (figure 31). Interestingly, the de novo purine biosynthetic pathway was found to be significantly altered (figure 14e, figure 20a), including a> 20-fold increase in endogenous AICAR concentration (5-aminoimidazole-4-carboxamide ribonucleotide) 72-hour MOTS treatment -c exogenous (figure 18a). Consistent with the metabolomic findings, microarray analysis also showed MOTS-c purine biosynthesis changed again as early as 4 hours post-treatment (figure 18b). Purines are synthesized from ribose-5-phosphate (R5P), produced from pentose phosphate (PPP) and also by ADP-ribose derived from NAD + (figure 14e). It accelerated glycolysis / PPP and increased levels of NAD + (figures 18a, 19b), but there was a reduction in the levels of ADP-ribose and R5P in cells treated with MOTS-c. These data are further supported by reduced glutamine and increased levels of pyrophosphate (PPi) (Figure 14 g), which indicate that MOTC-s stimulated increased flow of these pathways in HEK293 cells. This accumulation in AICAR consequently blocked and reduced downstream intermediates such as inosine, adenyl succinate, AMP and also the final purine products, including adenine, guanine and xanthine (figures 14g, 18 and 20a). MOTS-c can promote an accumulation of AICAR, changing the metabolism of folic acid. A significant reduction in 5-methyl-tetrahydrofolate (THF-5Me) was observed (figure 20b), which is necessary to catalyze the formulation of AICAR for its downstream F-AICAR intermediate. Previous reports of fact show that inhibiting the metabolism of folic acid by methotrexate increases intracellular levels of AICAR (E. S. Chan, B. N. Cronstein, Methotrexate - how does it really work Nature reviews. Rheumatology 6, 175 (Mar, 2010)). Impaired folic acid metabolism, especially 5Me-THF, has also been implicated as a mechanism underlying the activation of AMPK by the antidiabetic drug metformin (B. Corominas-Faja et al., Metabolomic fingerprintreveals that metformin impairs one-carbon metabolism in a similar manner to the antifolate class of chemotherapy drugs. Aging 4, 480 (Jul, 2012)).
[00146] AICAR is a potent activator of the master energy regulator AMPK, shown to stimulate fatty acid oxidation through inactivation induced by acetyl-CoA (ACC) carboxylase phosphorylation, relieving allosteric inhibition of carnitine palmitoyltransferase 1 (CPT-1) , the central transport mechanism of fatty acids in the mitochondria for β-oxidation. (G. Hasko, B. Cronstein, Regulation of inflammation by adenosine. Frontiers in immunology 4, 85 (2013)). Activation of AMPK induced by AICAR increases glucose absorption in muscle, in part, increasing GLUT4 transcription comparable to that achieved by exercise (GR Steinberg, BE Kemp, AMPK in Health and Disease. Physiological reviews 89, 1025 (Jul, 2009). 72 hours of MOTS-c treatment (10 μM) led to phosphorylation of AMPKα (Thr172) and ACC (Ser79) and also increased CPT-1 and GLUT4 protein levels (Figure 14h) in a time- and dose form -dependent (figure 14i). In addition, MOTS-c was found to promote Akt phosphorylation in Ser-473 (figure 14i). AMPK activation was notably observed under low levels of AMP and high levels of ADP and ATP ( figures 19a, 20a) Increased ATP can be attributed to enhanced glycolysis, which is a fast but inefficient method of producing ATP (PS Ward, CB Thompson, Metabolic reprogramming: a cancer hallmark even warburg did not anticipate. Cancer cell 21 , 297 (Mar 20, 2012)). These findings suggest that MOTS-c promotes AMP-i activation independent of AMPK, similar to that achieved by salicylate (K. Hashiguchi, Q. M. Zhang-Akiyama, Establishment of human cell lines lacking mitochondrial DNA. Methods in molecular biology 554, 383 (2009)), leptin (T. Ohta et al., Untargeted metabolomic profiling as an evaluative tool of fenofibrate-induced toxicology in Fischer 344 male rats. Toxicologic pathology 37, 521 (Jun, 2009)) , and metformin (N. Fujii, N. Jessen, LJ Goodyear, AMP-activated protein kinase and the regulation of glucose transport. American journal of physiology. Endocrinology and metabolism 291, E867 (Nov, 2006)).
[00147] Next, the effect of MOTS-c on cell metabolism, focusing on glucose and fatty acid metabolism and cell respiration, was investigated. It was found that it stimulated MOTS-c glycolysis reflected by increased glucose absorption (figure 15a; Figure 21a, b) and lactate production (figure 15b; figure 21a). In addition, intracellular glucose levels were lower, along with other glycolytic intermediates (Figure 15c; Figure 22), supporting further increased glycolytic flow. MOTS-c also changed the use of other sugar substrates (Figure 22). To directly test the rate of glycolysis in real time, extracellular acidification rate (ECAR) was measured. MOTS-c-ST cells had a higher basal glycolysis rate and showed an improvement in the glycolytic response when stimulated with glucose (Figure 15 d). In addition, the total glycolytic capacity estimated by the oligomycin treatment was higher in these cells (Figure 15d). Intracellular lactate levels were identical between MOTS-c-ST and MOTS-c-EV cells (Figure 15c), as excess production by MOTS-c-ST cells was released in the medium (figure 15b; figure 21a). Consistent with observations in MOTS-c-ST cells, mass spectrometry analysis provided additional evidence of increased glycolysis in HEK293 cells within 24 hours of MOTS-c treatment (figure 23a). Pentose-phosphate (PPP), an alternative branch of glycolysis, provides R5P for de novo purine biosynthesis. Reduction in the levels of PPP intermediates, including R5P, was observed in MOTS-c-ST cells and, also, MOTS-c-EV cells after 72 hours of treatment of exogenous MOTS-c (10 μM) (figure 23b, c ).
[00148] Then, mitochondrial respiration was measured because glycolytic end products are transported to the mitochondria to extract additional energy through oxidative phosphorylation. In line with increased glycolysis, treatment of MOTS-c reduced the rate of baseline oxygen consumption (OCR) as well as the maximum respiratory capacity in HEK293 cells (figure 15e). This is consistent with the "Crabtree effect", a phenomenon by which rapidly dividing cells including: cancer cells, lymphocytes, stem cells and sperm, exhibit suppressed respiration in response to high glucose concentrations (KH Ibsen, The Crabtree effect: a Cancer research 21, 829 (Aug 1961); T. Wang, C. Marquardt, J. Foker, Aerobic glycolysis during lymphocyte proliferation. Nature 261, 702 (Jun 24, 1976); and V. Gogvadze, S. Orrenius, B. Zhivotovsky, Mitochondria in cancer cells: what is so special about them Trends in cell biology 18, 165 (Apr, 2008)). Decreased electron transport chain activity was also indicated by reduced proton leakage (M. Jastroch, AS Divakaruni, S. Mookerjee, JR Treberg, MD Brand, Mitochondrial proton and electron leaks. Essays in biochemistry 47, 53 (2010)) (Figure 15f). Reduced oxidative capacity was associated with depletion of TCA cycle intermediates in MOTS-c-ST cells and HEK293 cells treated with exogenous MOTS-c (Figure 15g; figure 24). Transient transfection of MOTS-c expression clones into HEK293 cells also decreased OCR (figure 25). Despite reduced mitochondrial metabolism, MOTS-c did not disturb cellular redox homeostasis, as determined by the reduced to oxidized glutathione (GSSG / GSH) ratio, total glutathione levels were nevertheless decreased in MOTS-c-ST cells and HEK293 cells 72 hours after MOTS-c treatment (Figure 26). To test whether MOTS-c suppresses mitochondrial respiration by increasing glycolytic flow or targeting mitochondrial metabolism per se, HEK293 cells were cultured in medium with glucose or galactose as the main source of carbon. In mammalian cells, galactose is largely metabolized by the mitochondria, displacing the dependence on oxidative phosphorylation for energy production (LD Marroquin, J. Hynes, JA Dykens, JD Jamieson, Y. Will, Circumventingthe Crabtree effect: replacing media glucose with galactose increases susceptibility of HepG2 cells to mitochondrial toxicants.Toxicological sciences: an official journal of the Society of Toxicology 97, 539 (Jun, 2007)). MOTS-c reduced cell proliferation under abundant glucose conditions, but did not affect cells cultured in galactose medium. These data suggest that respiratory suppression induced by MOTS c is probably secondary to increased glucose absorption (Figure 15h).
[00149] Lipid metabolism was also significantly affected by MOTS-c (figure 27a). MOTS-c-ST cells exposed to higher levels of carnitine transports, as well as increased concentrations of carnitine and deoxycarnitine compared to MOTS-c-EV cells (figure 15i). These results were also observed, but to a lesser extent, in cells treated with exogenous MOTS-c (figure 27b). Consistent with increased carnitine transports, we found a reduction in the levels of essential fatty acids in the MOTS-c-ST cells (figure 15j), as well as in HEK293 cells treated with MOTS-c (figure 27C). After entering the mitochondria, fatty acids undergo e-oxidation to extract the reduction potential and acetyl-CoA. Miristoil-CoA, an early e-oxidation intermediate, increased significantly in MOTS-c-ST cells (Figure 15k), as well as HEK293 cells treated exogenously with MOTS-c (10 µM) (Figure 27d). In addition, other long-chain fatty acids were also significantly reduced, suggesting an increased use of fatty acids (Figure 28). Notably, e-oxidation itself is not an aerobic reaction, and increased fatty acid oxidation was observed under reduced respiration during treatment with AICAR (LD Marroquin, J. Hynes, JA Dykens, JD Jamieson, Y.Will, Circumventing the Crabtree effect: replacing media glucose with galactose increases susceptibility of HepG2 cells to mitochondrial toxicants.Toxicological sciences: an official journal of the Society of Toxicology 97, 539 (Jun, 2007)) and metformin (A. Martin-Montalvo et al., Metformin improves healthspan and lifespan in mice.Nature communications 4, 2192 (Jul 31, 2013)).
[00150] Acute treatment of MOTS-c in male mice exocrated CD-1 for 4 days (5 mg / kg / day; IDB) caused modest reductions in body weight, food intake and blood glucose levels (figure 29a-c ), but significantly reduced baseline levels of IL-6 and TNFa plasma (Figure 16a; Figure 29 d), which are implicated in obesity and insulin resistance (MF Gregor, GS Hotamisligil, Inflammatory mechanisms in obesity. Annual review of immunology 29 , 415 (2011)). Considering our findings in cell cultures, the effects of MOTS-c on metabolism in exocrated CD-1 mice fed a high-fat diet (HFD; 60% per calorie) were tested (LA Scrocchi, DJ Drucker, Effects of aging and a high fat diet on body weight and glucose tolerance in glucagon-like peptide-1 receptor - / - mice Endocrinology 139, 3127 (Jul, 1998) and WL Breslin, K. Strohacker, KC Carpenter, L. Esposito, BK McFarlin, Weight gain in response to high-fat feeding in CD-1 male mice. Laboratory animals 44, 231 (Jul, 2010)). 8 weeks of treatment with MOTS-c had no effect on body weight in mice fed a normal diet, but it remarkably prevented HFD-induced obesity (figure 16b). This difference in weight was not attributed to food intake, as caloric intake was identical between groups (figure 29e-f). High-fat food promotes hyperinsulinemia in an attempt to overcome peripheral insulin resistance to maintain glucose homeostasis (M. Hou et al., Protective effect of metformin in CD1 mice placed on a high carbohydrate- high fat diet. Biochemical and biophysical research communications 397, 537 (Jul 2, 2010)). Importantly, MOTS-c treatment prevented hyperinsulinemia (Figure 16c, d) and improved hepatic lipid accumulation in mice fed with HFD (figure 16e). Notably, in line with our in vitro studies, MOTS-c promoted AMPK activation and GLUT4 expression in the muscle of these HFD-fed mice (figure 16f).
[00151] Next, the effects of MOTS-c on glucose homeostasis in the C57BL / 6 strain of mice, prone to obesity and commonly studied, were tested by performing a glucose tolerance test (GTT). MOTS-c-treated mice showed rapid glucose release, suggesting improved insulin sensitivity (Figure 16 g). Thereafter, hyperinsulinemic-euglycemic clamp studies were performed to quantify the effects of 7-day MOTS-c treatment on insulin sensitivity of the whole body regardless of changes in body weight that occur with prolonged treatment durations. MOTS-c improved whole-body insulin sensitivity, as reflected by a ~ 30% increase in the exogenous glucose infusion rate (GIR) needed to maintain euglycemia during insulin stimulation (Figure 16h). Insulin promotes the elimination of glucose in peripheral tissues and suppresses hepatic glucose production (HGP) to maintain homeostasis during periods of increased glucose availability (CR Kahn, Banting Lecture. Insulin action, diabetogenes, and the cause of type II diabetes Diabetes 43, 1066 (Aug, 1994)). Tritiated glucose was infused during clamp to determine the tissue specificity of MOTS-c's action on insulin sensitivity. Although MOTS-c treatment was found to significantly improve the insulin-stimulated glucose elimination rate (IS-GDR) (figure 16i), no MOTS-c effect on hepatic insulin sensitivity was detected, depending on the rate of production hepatic glucose (HGP) was comparable between groups (figure 16j). Considering that 70-85% of insulin-stimulated glucose elimination is in skeletal muscle, actions of MOTS-c to improve insulin sensitivity and glucose homeostasis can be mediated by this tissue. In fact, this notion was supported by the strong activation of AMPK and increased expression of GLUT4 in skeletal muscle of mice fed with HFD after the treatment of MOTS-c (figure 16f). In addition, MOTS-c also enhanced glucose absorption in the stimulated soleus muscle with a physiological dose of human insulin ex vivo. Because the levels of MOTS-c in muscle (Figure 16k) and in circulation (Figure 16l) decline concomitantly with the development of insulin resistance during aging ((CR Kahn, Banting Lecture. Insulin action, diabetogenes, and the cause of type Diabetes II Diabetes 43, 1066 (Aug, 1994), it was determined that MOTS-c could reverse age-dependent deficiencies in insulin action by measuring insulin-stimulated glucose uptake (2-deoxyglucose) in soleus muscles of C57BL / 6 mice middle-aged males (12 months) and young (3 months) Signs of insulin resistance begin to appear around 12 months of age in C57BL / 6 mice (CR Kahn, Banting Lecture. Insulin action, diabetogenes, and the cause of type II diabetes. Diabetes 43, 1066 (Aug, 1994)) In fact, the muscles of older rats were more resistant to insulin, but 7 days of MOTS-c treatment restored sensitivity comparable to young animals (Figure 16m) . DISCUSSION
[00152] Technological advances have revealed previously unknown properties of mitochondrial genetics, suggesting the existence of small ORFs in mitochondrial DNA (T. R. Mercer et al., The human mitochondrialtranscriptome. Cell 146, 645 (Aug 19, 2011)). In particular, several small bioactive ORFs have been reported in the nuclear genome in Drosophila (EG Magny et al., Conserved regulation of cardiac calcium uptake by peptides encoded in small open readingframes. Science 341, 1116 (Sep 6, 2013); J. Savard, H Marques-Souza, M. Aranda, D. Tautz, A segmentation gene in tribolium produces a polycistronic mRNA that codes for multiple conserved peptides. Cell 126, 559 (Aug 11, 2006); T. Kondo et al., Small peptides switch the transcriptional activity of Shavenbaby during Drosophila embryogenesis.Science 329, 336 (Jul 16, 2010) .; and MI Galindo, JI Pueyo, S. Fouix, SA Bishop, JP Couso, Peptides encoded by short ORFs control development and define a new eukaryotic gene family. PLoS biology 5, e106 (May, 2007)). There is evidence in the literature discussing the potential existence of factors encoded within mitochondrial DNA (mtDNA). For example, polyadenylated mitochondrial rRNA clones were cloned in the early 1980s as part of a cDNA library constructed from interferon-induced human myeloblast cells (J. Villegas, P. Araya, E. Bustos-Obregon, LO Burzio , Localization of the 16S mitochondrial rRNA in the nucleus of mammalian spermatogenic cells Molecular human reproduction 8, 977 (Nov, 2002), suggesting a strong interferon-induced factor encoded in mtDNA. Also, in humans, 16S rRNA was found located in the nucleus of human sperm cells (J. Durieux, S. Wolff, A. Dillin, The cell-non-autonomous nature of electron transport chain-mediated longevity. Cell 144, 79 (Jan 7, 2011), while in Drosophila, mitochondrial rRNAs have been found in the cytoplasm where they play a role in the establishment of germ cells (R. Amikura, M. Kashikawa, A. Nakamura, S. Kobayashi, Presence of mitochondria-type ribosomes outside mitochondria in germ plasm of Drosophila embryos. Proceedings of the National Academy of Sciences of the United States of America 98, 9133 (Jul 31, 2001)).
[00153] MOTS-c may be a product of adaptation for effective bilateral communication between mitochondria and the distant cell or organs such as skeletal muscle, especially considering that its translation requires the genetic code of mammals. Sufficient evidence is provided in this document to suggest that MOTS-c and humanin as a new class of inherent mitochondrial signals that regulate global physiology (C. Lee, K. Yen, P. Cohen, Humanin: a harbinger of mitochondrial-derived peptides Trends in endocrinology and metabolism: TEM, (February 7, 2013)). A recent report, using the C. elegans nematode, showed that mitochondrial disturbance in neurons causes a mitochondrial unfolded protein (UPR) response in the intestine and extends useful life, an effect mediated by an unidentified circulation signal that allows communication of inter-organ stress (DK Woo, GS Shadel, Mitochondrial stress signals review an old aging theory. Cell 144, 11 (Jan. 7, 2011) and C. Cheadle, MP Vawter, WJ Freed, KG Becker, Analysis of microarray data using Z score transformation.The Journal of molecular diagnostics: JMD 5, 73 (May, 2003)).
[00154] While the specific mechanistic details of MOTS-c's action have yet to be fully identified, its impact on metabolism is likely to have important implications for aging and age-related diseases including diabetes, cancer, atherosclerosis and neurodegeneration. The emerging biology of MDPs and the exclusive involvement of AICAR and AMPK in MOTS-c action (figure 16n) provide an interesting opportunity to expand our understanding of the role of mitochondria in physiological and pathological conditions, as well as to identify new diagnostic and therapeutic targets . METHODS Cell Culture
[00155] HEK293 and HeLa cells were routinely cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37oC, with 5% CO2. P0 cells, devoid of mitochondrial DNA, were generated by culturing HeLa cells in low doses of ethidium bromide (EtBr; 100ng / mL) as described above (K. Hashiguchi, QM Zhang-Akiyama, Establishment of human cell lines lacking mitochondrial DNA Methods in molecular biology 554, 383 (2009)). MOTS-c-ST cells were generated by selecting and maintaining G418 (500 μM; Sigma) in DMEM with 10% FBS. Mice
[00156] All animal work was carried out in accordance with the University of Southern California and University of California Los Angeles Institutional Animal Care and Use Committee. MOTS-c (Genscript, USA) was injected daily through intraperitoneal injections in all in vivo experiments. Old 8-week CD-1 (ICR) mice were purchased from Harlan. C57Bl / 6 mice were purchased from Jackson Laboratories. The mice were fed a high-fat diet (60% calories) and corresponding control diet purchased from Research Diets (Cat # D12492 and D12450J, respectively) for 8 weeks. Pellets were replaced twice a week, and food consumption and body weight were recorded daily (N = 10). Cloning
[00157] Directional cloning using restriction enzymes EcoRI (5 ') and XhoI (3') were performed to construct MOTS-c expression clones. Nucleotide sequences of MOTS-c and MOTS-c-HA flanked by restriction enzymes were synthesized and 5 'phosphorylated (IDT, USA), hybridized and ligated with digested pcDNA3.1 (+) (Invitrogen, USA), or pcDNA-IRES GFP (present from Nir Barzilai at AECOM). All enzymes were purchased from NEB (USA), unless otherwise specified. Immunocytochemistry
[00158] HEK293 cells plated on the coverslip were fixed with 4% paraformaldehyde for 15 min at room temperature. After fixation, the cells were permeabilized with 0.2% Triton X-100 in phosphate buffered saline (PBS) for 10 minutes at room temperature and were blocked in PBS containing 0.2% Triton X-100 and 1% bovine serum albumin (BSA) for 1 hour at room temperature. The cells were then incubated with rabbit anti-MOTS-c antibodies (1:50) and goat anti-hsp60 antibody (1: 100; Santa Cruz Biotechnology, USA) in PBS containing 0.2% Triton X-100 and 1 % BSA at 4 ° C overnight. After three washes with PBS, the cells were further incubated with IgG Alexa Fluor 488- donkey anti-rabbit conjugate (1: 200; Invitrogen, USA) and IgG Alexa Fluor 568-donkey anti-goat conjugate (1: 200; Invitrogen ) in PBS containing 0.2% Triton X-100 and 1% BSA for 1 hour at room temperature. Cores were stained for 5 minutes at room temperature in PBS containing Hoechst 33258 (2mg / ml; Invitrogen). Specificity of immunostaining was demonstrated using MOTS-c antibody after incubation with MOTS-c peptide at 69 μg / ml (Genscript, USA) for 1 hour at room temperature. Coverslips were mounted with ProLong Gold anti-wear reagent (Invitrogen) and observed under an ELYRA PS.1 (Carl Zeiss, Germany) using wide field laser. For microscopy images (SIM) of structured illumination, sections of Z-stack were collected with five grid rotations and images were reconstructed using ELYRA PS.1 software. Immunoassays
[00159] The entire MOTS-c peptide was conjugated to keyhole limpet hemocyanin (KLH) and injected into rabbits. Purified IgG serum was used for western blotting and ELISA. Circulating MOTS-c levels were measured from serum, plasma and CSF by in-house sandwich Elisa developed at USC. Customized polyclonal MOTS-c rabbit antisera were produced at YenZym Antibodies, LLC (South San Francisco, CA). IgG subclasses purified with protein A / G column chromatography (Pierce, Rockford, IL) were used as a capture antibody. The anti-MOTS-c IgG was labeled with biotin and used as an antibody of detection. To measure endogenous levels of MOTS-c, synthetic MOTS-c (GenScript) was used as a standard within the range of 25 pg / mL to 6400 pg / mL. Before the test, MOTS-c was extracted in 90% acetonitrile and 10% 1N HCl. Soon, 200μL of extraction reagent was added to 100 μL of plasma, gently mixed and incubated at room temperature for 30 minutes. The mixture was centrifuged and the supernatant was removed and dried. The dry extract was reconstituted with 200 μL of phosphate buffer with 0.5% Tween 20 and then used for testing. 96-well microtiter plates were coated with capture antibody at 0.5 μg / well and incubated 4 hours at room temperature on a shaker. Detected and pre-titrated detection standards, controls or samples were added to the appropriate wells and incubated overnight after 2 washes with wash buffer and 2 washes with Superblock buffer (PierceChemicals, Rockford, IL). Wells were washed 3 times and then added with streptavidin-HRP and further incubated for 30 minutes at room temperature. After washing, 200 μL / well of OPD solution (1 mg / mL in hydrogen peroxide substrate) was added and incubated for 10-20 minutes. The reaction was terminated by the addition of 2N H2SO4, and the absorbance was measured on a plate spectrophotometer (Molecular Designs, Sunnyvale, CA) at 490 nm. The intra- and inter-trial coefficient (CV) variations were less than 10%. Plasma insulin levels were detected using a mouse insulin assay kit (MSD; Cat # K112BZC-1) and Sector Imager 2400A (MSD, USA).
[00160] For western blotting, protein samples were prepared in 1% Triton X-100 with EDTA-free protease and phosphatase inhibitors (Roche, USA), heated at 95oC for 5 minutes, performed in 4-20% gradient gels. tris-glycine (TGX; Bio-Rad, USA) and transferred to PVDF membranes (Bio-Rad) using a semi-dry turbo transfer system (Transblot Turbo; Biorad) at 9V for 15-30 minutes. Membranes were blocked with 5% BSA for 30 minutes and incubated with primary antibody overnight at 4oC, followed by secondary antibodies conjugated to HRP for 1 hour at room temperature. Chemiluminescence was detected and using enhanced ECL image (Immun-Star WesternC; Bio-Rad) and Chemidoc XRS system (Bio-Rad). Microarray
[00161] RNA was isolated using the RNeasy kit (Qiagen, Valencia, CA) and then hybridized to BD-103-0603 Illumina Beadchips. Raw data were subjected to Z-normalization, as previously described (C. Cheadle, MP Vawter, WJ Freed, KG Becker, Analysis of microarray data using Z score transformation. The Journal of molecular diagnostics: JMD 5, 73 (May, 2003) ). Principal component analysis, performed on the standardized Z-scores of all probes detectable in the samples, was performed with DIANE 6.0 software (http://www.grc.nia.nih.gov/branches/rrb/dna/diane software. pdf). Significant genes were selected by z-test <0.05, false discovery rate <0.30, as well as z-ratio> 1.5 in both directions and ANOVA p value <0.05. Parametric analysis of joint gene enrichment (PAGE) was analyzed as previously described (S. Y. Kim, D. J. Volsky, PAGE: parametric analysis of gene set enrichment. BMC bioinformatics 6, 144 (2005)). Gene regulatory network and canonical pathway analysis was performed using Ingenuity Pathways Analysis © (Ingenuity Systems; Redwood City, CA) (N = 6). Metabolomics
[00162] HEK293 cells were treated with MOTS-c (10 μM) or water (vehicle) for 24 to 72 hours. In addition, HEK293 cells were used, which were stably transfected with a validated MOTS-c expression vector, or empty (control) vector. The cells were grown in 10 cm plates in 7 ml DMEM without phenol supplemented with 10% FBS. For collection, cells were washed twice with cold phosphate buffered saline (PBS) and immediately scraped, centrifuged, and frozen instantly after aspiration of supernatant. Frozen pellets were used for metabolomic analysis (Metabolon). Instrumentation of non-targeted metabolic profile employed a combination of three independent platforms: ultra high performance liquid chromatography / tandem mass spectrometry (MS / UHPLC / MS2) optimized for basic species, UHPLC / MS / MS2 optimized for acidic species and spectrometry of mass / liquid chromatography (GC / MS). The samples were processed essentially as previously described (T. Ohta et al., Untargeted metabolomic profiling as an evaluative tool of fenofibrate-induced toxicology in Fischer 344 male rats. Toxicologic pathology 37, 521 (Jun, 2009) and AM Evans, CD DeHaven , T. Barrett, M. Mitchell, E. Milgram, Integrated, nontargeted ultrahigh performance liquid chromatography / electrospray ionization tandem mass spectrometry platform for the identification and relative quantification of the small-molecule complement of biological systems. Analytical chemistry 81, 6656 (Aug 15, 2009)). In a minimal volume of water, cells were lysed using a Covaris adaptive focused acoustics E-series tissue homogenizer / disruptor. From the homogenate, a 100 μl aliquot was taken by subsequent mass spectrometry and 25 μL used to measure total proteins (Bradford assay). Using an automatic liquid handler (Hamilton LabStar, Salt Lake City, UT), the protein was precipitated from homogenate with methanol that contained four standards for reporting extraction efficiency. The resulting supernatant was divided into equal aliquots for analysis on the three platforms. Aliquots, dried under nitrogen and vacuum-dehydrated were later reconstituted in 50μL to 0.1% formic acid in water (acidic conditions) or 50μL of 6.5 mM ammonium bicarbonate in water, pH 8 (basic conditions). two UHPLC / MS / MS2 analyzes or derivatized to a final volume of 50μL for GC / MS analysis using equal parts of bistrimethyl-silyl-trifluoroacetamide and solvent mixture of acetonitrile: dichloromethane: cyclohexane (5: 4: 1) with 5% triethylamine at 60 ° C for one hour. In addition, three types of controls were analyzed in conjunction with the experimental samples: aliquots of a sample derived from aliquots of experimental samples served as technical replicates throughout the data set, extracted water samples served as blanks for the process, and a cocktail of patterns embedded in each sample analyzed allowed monitoring of instrument performance. Controls and experimental samples were randomized on platform execution days.
[00163] For UHLC / MS / MS2 analysis, aliquots were separated using Waters Acquity UPLC (Waters, Millford, MA) and analyzed using an LTQ mass spectrometer (Thermo Fisher Scientific, Inc., Waltham, MA) which consisted of a source electrospray ionization (ESI) and linear ion-trap mass analyzer (LIT). The MS instrument scanned 991000 m / z and alternated between MS and MS2 scans using dynamic exclusion with approximately 6 scans per second. Samples derived by GC / MS were separated on a 5% phenyldimethyl silicone column with helium as the carrier gas and a temperature ramp from 60 ° C to 340 ° C and then analyzed on a Thermo-Finnigan Trace DSQ MS (Thermo Fisher Scientific, Inc.) operated on mass unit resolution energy with electron impact ionization and a scanning range of 50-750 atomic mass units.
[00164] Metabolites were identified by automated comparison of ion characteristics in experimental samples to a reference library of standard chemical inputs that included retention time, preferred adducts, molecular weight (m / z) and source code fragments as well as associated MS spectra and were cured by visual inspection for quality control using software developed in Metabolon (CD Dehaven, AM Evans, H. Dai, KA Lawton, Organization of GC / MS and LC / MS metabolomics data into chemical libraries. Journal of cheminformatics 2, 9 (2010)).
[00165] For the purpose of data display and statistical analysis, any missing values were assumed to be below the detection limits and these values were imputed with the minimum of compounds (imputation of the minimum value). In addition, each value has been normalized to a total protein value on an example basis. Statistical analysis of log-transformed data was performed using "R" (http://cran.r-project.org/), which is a freely available open source software package. Student's t test was performed to compare data between experimental groups. Glucose and Lactate Assay
[00166] Extracellular glucose and lactate from the cell culture medium were measured using glucose and D-lactate test kits according to the manufacturer's instructions (Eton Bioscienes, USA). Intracellular glucose was determined by microscopy using 2-NBDG (Invitrogen, USA) according to the manufacturer's instructions. Oxygen consumption and extracellular acidification rate
[00167] Oxygen consumption rates in real time (OCR) were measured using the XF24 / 96 Extracellular Flux Analyzer (Hippocampus Bioscience). ATP renewal and maximum respiratory capacity were estimated by challenge to cells with oligomycin and FCCP. Respiratory capacity was determined by 'maximum respiratory rate / baseline respiratory rate', proton leakage by 'non-mitochondrial ATP renewal OCR-OCR (rotenone / antimycin)' and coupling efficiency by '1 - (ATP renewal OCR / OCR) baseline respiratory rate) '. Glycolytic rate was determined using the extracellular acidification rate (ECAR). Cells were stimulated with glucose to determine the active glycolytic rate, with oligomycin to determine maximum glycolytic capacity and with 2-DG to determine glycolytic capacity. Ex-vivo glucose absorption from soleus muscle strip
[00168] Ex-vivo whole muscle glucose absorption was evaluated in 12-week-old mice using 2-deoxyglucose (Perkin Elmer) and 60μl / ml human insulin (Novo Nordisk Pharmaceutical Industries), with minor changes to that previously described ( V. Ribas et al., Myeloid-specific estrogen receptor alpha deficiency impairments metabolic homeostasis and accelerates atherosclerotic lesion development. Proc Natl Acad Sci USA 108, 16457 (Sep 27, 2011) and CE McCurdy, GD Cartee, Akt2 is essential for the full effect of calorie restriction on insulin-stimulated glucose uptake in skeletal muscle.Diabetes 54, 1349 (May, 2005) Briefly, soleus and EDL muscles were carefully excised from anesthetized animals and immediately incubated for 30 min in the Krebs-Henseleit buffer complete with or without insulin at 60μU / ml at 35 ° C. The muscles were transferred to the same buffer containing 3mCi / ml of 3H-2-deoxyglucose and 0.053mCi / ml of 14C-mannitol and incubated for 20 min before instant freezing. The muscles were homogenized in lysis buffer and counted by radioactivity or subjected to western blotting. Glucose absorption was standardized for non-specific absorption of mannitol and estimated as mmol of glucose absorption per gram of soleus muscle. Studies of euglycemic-hyperinsulinemic clamp
[00169] Double catheters were surgically placed in the right jugular vein and glucose clamp studies were performed 3 days after surgery as previously described (V. Ribas et al., Myeloid-specific estrogen receptor alphadeficiency impairs metabolic homeostasis and accelerates atherosclerotic lesion development. Proc Natl Acad Sci USA 108, 16457 (Sep 27, 2011); AL Hevener et al., Muscle-specific Pparg deletion causes insulin resistance. Nature medicine 9, 1491 (Dec, 2003); and AL Hevener et al., Macrophage PPAR gamma is required for normal skeletal muscle and hepatic insulin sensitivity and full antidiabetic effects of thiazolidinediones. J Clin Invest 117, 1658 (Jun, 2007)). All animals were fasted for 6 h and the last dose of MOTS-c was administered 4 h before clamp. The animals were studied in the conscious state. Basal glucose turnover was determined after a constant 90-minute infusion of (5.0 μCi / h, 0.12 ml / h) [3-3 h] D-glucose (Perkin Elmer). After the baseline period, glucose (50% dextrose, Abbott Laboratories) and insulin (8 mU / kg / min), Novo Nordisk Pharmaceutical Industries) in addition to tracer infusions (5.0 μCi / h) were started simultaneously, and levels of glucose fixed in euglycemia using a variable glucose infusion rate (GIR). At steady state, the rate of total glucose elimination (RDA), measured by the marker dilution technique, is equal to the sum of the rate of hepatic or endogenous glucose (HGP) production and the exogenous (cold) glucose infusion rate (GIR) (AL Hevener et al., Muscle-specific Pparg deletion causes insulin resistance. Nature medicine 9, 1491 (Dec, 2003); and AL Hevener et al., Macrophage PPAR gamma is required for normal skeletal muscle and hepatic insulin sensitivity and full antidiabetic effects of thiazolidinediones. J Clin Invest 117, 1658 (Jun, 2007); and R. Steele, Influences of glucose loading and of injected insulin on hepatic glucose output. Ann NY Acad Sci 82, 420 (Sep 25, 1959) ). The component of total insulin-stimulated GDR (IS-GDR) is equal to that of total GDR minus the basal glucose turnover rate. Glucose tolerance test (GTT)
[00170] Blood glucose was measured using a glucometer (Freestyle, Abbott). 12-week-old C57BL / 6 male mice were treated with MOTS-c (0.5mg / kg / day; IP), or sterile pure water (vehicle), daily, for 7 days. Then the mice were injected with D-glucose (1g / kg; IP) and blood was sampled from the tail 0, 15, 30, 60, 90 and 120 minutes after glucose injection.
[00171] The examples defined above are provided to give those skilled in the art a full disclosure and description of how to make and use the modalities of the compositions, systems and methods of disclosure and are not intended to limit the scope of what inventors consider their disclosure . Modifications of the methods described above for carrying out the disclosure which are obvious to those skilled in the art are intended to be within the scope of the following claims. All patents and publications mentioned in the specification are indicative of the levels of those versed in the technique for which this disclosure is intended. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference, in its entirety, individually.
[00172] All titles and section names are used for clarity and reference purposes only and should not be considered limiting in any way. For example, those skilled in the art will appreciate the usefulness of combining various aspects of different titles and sections, as appropriate, in accordance with the spirit and scope of the invention described here.
[00173] All references cited in this document are incorporated by reference into this document in its entirety and for all purposes, to the same extent as if each individual publication or patent or patent application had been specifically and individually indicated to be incorporated by reference, in its entirety, for all purposes.
[00174] Many of the modifications and variations of this request can be made without deviating from its spirit and scope, as will be evident to those skilled in the art. The specific modalities and examples described herein are offered by way of example only, and disclosure will be limited only by the terms of the added claims, together with the full scope of equivalents to which the claims are entitled.

权利要求:
Claims (4)
[0001]
1. Pharmaceutical composition, characterized by the fact that it comprises an isolated polypeptide comprising the sequence of SEQ ID NO: 1 and a pharmaceutically acceptable carrier.
[0002]
2. Pharmaceutical composition according to claim 1, characterized by the fact that it is for use in the manufacture of a medicine for the treatment of cancer, in which the cancer is a cancer selected from a group consisting of breast cancer, brain cancer, colon cancer, melanoma, leukemia (eg, AML), lymphoma, pancreatic cancer, ovarian cancer, lung cancer, and gastric cancer.
[0003]
Pharmaceutical composition according to claim 1, characterized by the fact that it is for use in the manufacture of a medication for the treatment of diabetes, in which the diabetes is type I diabetes or type II diabetes.
[0004]
Pharmaceutical composition according to claim 1, characterized by the fact that it is for use in the manufacture of a medicine for the treatment of obesity and / or fatty liver.

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-02-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-10-15| B07E| Notice of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2019-10-29| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-05-12| B07A| Technical examination (opinion): publication of technical examination (opinion) [chapter 7.1 patent gazette]|

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2020-10-13| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-01-12| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 14/03/2014, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201361801474P| true| 2013-03-15|2013-03-15|

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US61/801,474|2013-03-15|

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PCT/US2014/028968|WO2014144521A1|2013-03-15|2014-03-14|Mitochondrial-derived peptide mots3 regulates metabolism and cell survival|

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