WO2019179338A1 - Application de protéine dans la préparation d'un médicament pour la prévention ou le traitement d'une complication du diabète - Google Patents

Application de protéine dans la préparation d'un médicament pour la prévention ou le traitement d'une complication du diabète Download PDF

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WO2019179338A1
WO2019179338A1 PCT/CN2019/077857 CN2019077857W WO2019179338A1 WO 2019179338 A1 WO2019179338 A1 WO 2019179338A1 CN 2019077857 W CN2019077857 W CN 2019077857W WO 2019179338 A1 WO2019179338 A1 WO 2019179338A1
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protein
sdss1
modification
amino acid
use according
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Chinese (zh)
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张英豪
闫桂蕊
付晶鹏
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上海清流生物医药科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the invention belongs to the technical field of medicine and relates to the application of the sDSS1 protein in preparing a medicament for preventing and treating diabetic complications.
  • Diabetes is a metabolic disease characterized by chronic hyperglycemia.
  • IDF International Diabetes Federation
  • 1 out of every 11 people in the world have diabetes, and the total number of patients in 2017 reached 425 million, including mainland Chinese adults. Diabetes accounts for about 114.4 million, ranking first in the world.
  • the probability of complicated cardiovascular disease in diabetic patients is 2 to 3 times that of non-diabetic patients.
  • the incidence of end-stage renal disease in diabetic patients is 10 times higher than that of non-diabetic patients. Every 30 seconds in the world, some people lose part of their lower limbs or lower limbs because of diabetes.
  • drug treatment is difficult to reverse. Therefore, preventing or delaying the complications of diabetes as soon as possible, protecting the terminal organs involved in diabetes, improving the quality of life of patients and reducing the social and economic burden have become the ultimate goal of treating diabetes.
  • Urinary microalbumin is the earliest marker of diabetic nephropathy. Urinary microalbumin is also a powerful predictor of cardiovascular complications [1], and its excretion is positively associated with late diabetic complications [2]. Urinary microalbumin is therefore clinically recommended as a marker for early screening for diabetic complications (IDF DIABETES ATLAS Eighth edition 2017). The pathogenesis of diabetic complications is complex. The current research results suggest that metabolic disorders, oxidative stress, hemodynamic changes, chronic low-grade inflammatory reactions, and genetic factors are involved in the progression of the disease [3].
  • Terminal glycosylation products are stable covalent additions of macromolecules such as proteins, lipids or nucleic acids that spontaneously react with excessive glucose or other reducing monosaccharides under non-enzymatic conditions. Things. It can be considered as an oxidative stress-assisted oxidative and/or glycated complex. On the one hand, circulating AGEs can act on RAGE receptors to cause oxidative stress, inflammation and apoptosis; on the other hand, long-term high glucose and oxidative stress can cause glycosylation and cross-linking of extracellular matrix proteins, making blood vessels Hardening; third, intracellular polysaccharide modification can interfere with a variety of proteins, enzymes or receptors to exert their biological functions [3,4].
  • the Shfm1 (split hand/split foot malformation type 1) gene is one of the key genes in human crab claw disease. It is highly conserved in evolution and its encoded protein DSS1 is a universal endogenous multifunctional disorder protein. Participate in the process of stable genome, homologous gene recombination, DNA damage repair, RNA splicing, protein degradation and cell proliferation [11]. The results show that the DSS1 protein can be added to the oxidized protein by energy-consuming enzymatic reaction to help the cells clear the oxidized protein [12]. These results show the important role of DSS1 protein in biological activities.
  • the sDSS1 protein provided by our (inventors) can bind to AGEs and reduce the cytotoxicity caused by AGEs.
  • Long-term administration of sDSS1 protein can reduce urinary albumin excretion in diabetic model mice and significantly improve renal filtration function. Therefore, the sDSS1 protein can be clinically prepared for the prevention and treatment of diabetic complications.
  • the diabetic complications include diabetic nephropathy, diabetic ocular complications, diabetic foot, diabetic cardio-cerebral vascular complications or diabetic neuropathy.
  • the diabetic ocular complications include diabetic retinopathy, diabetic macular edema (DME), diabetic cataract or glaucoma.
  • DME diabetic macular edema
  • the diabetic cardio-cerebral vascular complications include coronary artery disease (CAD) caused by diabetes, angina pectoris, myocardial infarction, arteriosclerosis, stroke, brain atrophy, peripheral arterial disease (PAD) or congestive heart failure.
  • CAD coronary artery disease
  • angina pectoris myocardial infarction
  • arteriosclerosis stroke
  • brain atrophy brain atrophy
  • PDA peripheral arterial disease
  • congestive heart failure congestive heart failure.
  • the diabetic complications include type I diabetes complications and type II diabetes complications.
  • the sDSS1 protein comprises human, chimpanzee, bonobo, gorilla, orangutan, white-cheeked gibbons, golden monkey, rhesus monkey, golden monkey, East African pheasant, Angora simian, white-tailed white-browed monkey, ghost A basic protein formed by any sDSS1 protein sequence of scorpion or porpoise monkey, wherein the amino acid sequence of human sDSS1 is SEQ ID NO: 1, the amino acid sequence of chimpanzee sDSS1 is SEQ ID NO: 2, and the amino acid sequence of porcine chimpanzee sDSS1 is SEQ ID NO: 3, the amino acid sequence of gorilla sDSS1 is SEQ ID NO: 4, the amino acid sequence of orangutan sDSS1 is SEQ ID NO: 5, the amino acid sequence of white cheek gibbon sDSS1 is SEQ ID NO: 6, amino acid of Rhinopithecus sDSS1 The sequence is as
  • the sDSS1 protein is any first protein having a similarity to the basic protein of 70% or more.
  • the sDSS1 protein is any structural or amino acid sequence characteristic of a polypeptide fragment fused to another polypeptide fragment based on 58 amino acids of the basic protein nitrogen base at the nitrogen terminal or the carbon terminal. Thirty-one second proteins of the same or similar sequence.
  • the sDSS1 protein is any third protein which is based on 58 amino acids of the basic protein nitrogen end, and fuses other amino acid fragments at the nitrogen terminal or the carbon terminal, and the fusion protein can realize transmembrane transport function.
  • the sDSS1 protein is formed by linking any one of a basic protein, a first protein, a second protein and a third protein to the protein itself, a carrier protein, an antibody or other amino acid fragments of any length. Fusion protein.
  • the sDSS1 protein is a protein modification produced by modification based on any one of a basic protein, a first protein, a second protein, and a third protein.
  • the modification of the protein modification is directed to an amino group on the amino acid side chain, a carbonyl group on the amino acid side chain, a nitrogen terminal amino group, a carbon terminal carbonyl group, a cysteine, a tyrosine, a serine, a tryptophan.
  • the method for modifying the protein modification comprises glycosylation modification, fatty acid modification, acylation modification, Fc fragment fusion, albumin fusion, polyethylene glycol modification, dextran modification, heparin modification, polyvinylpyrrolidone modification , polyamino acid modification, polysialic acid modification, chitosan and its derivative modification, lectin modification, sodium alginate modification, carbomer modification, polyvinylpyrrolidone modification, hydroxypropyl methylcellulose modification, hydroxypropyl
  • a cellulose modification, acetylation modification, formylation modification, phosphorylation modification, methylation modification or sulfonation modification and other pharmaceutically acceptable polypeptide/protein drug modification methods.
  • the sDSS1 protein is one based on amino acids other than the 20 essential amino acids based on the amino acid sequence of any of the basic protein, the first protein, the second protein, and the third protein.
  • a non-natural amino acid replacement protein substituted with 31 arbitrary amino acid positions.
  • the amino acid substitution of the non-natural amino acid replacement protein comprises hydroxyproline, hydroxylysine, selenocysteine, D-form amino acid or synthetic non-natural amino acid and derivatives thereof.
  • the sDSS1 protein is formed by combining a basic protein, a first protein, a second protein, a third protein, a fusion protein, a protein modification or a non-natural amino acid substitute with a pharmaceutically acceptable drug carrier. Part or all of the complex.
  • the pharmaceutical carrier comprises one or more of an enteric coating formulation, a capsule, a microsphere/capsule, a liposome, a microemulsion, a double emulsion, a nanoparticle, a magnetic particle, a gelatin or a gel.
  • the sDSS1 protein targets the individual's own sDSS1 protein, and affects the level of the individual's own sDSS1 protein by the exogenous drug.
  • the drug is a drug target of a sDSS1 protein, a gene of sDSS1 protein, a regulatory element of a gene of sDSS1 or a gene of sDSS1.
  • the drug modulates the amount of sDSS1 protein in the blood by affecting protease/peptidase activity in the blood.
  • the drug is a chemical small molecule drug, an antibody, a polypeptide/protein drug, a nucleic acid drug or a nano drug.
  • the sDSS1 protein is any one of a basic protein, a first protein, a second protein, a third protein, a fusion protein, a protein modification, an unnatural amino acid substitute, a complex, and a drug. a combination of two or more of the ingredients.
  • the sDSS1 protein is any one of a basic protein, a first protein, a second protein, a third protein, a fusion protein, a protein modification, an unnatural amino acid substitute, a complex, and a drug.
  • a pharmaceutically acceptable excipient One, two or more of the ingredients in combination with a pharmaceutically acceptable excipient.
  • the sDSS1 protein is a protein obtained by introducing a nucleotide sequence encoding any one of a basic protein, a first protein, a second protein, a third protein and a fusion protein into an expression system by expression system. .
  • the expression system is eukaryotic expression plasmid vector, adenovirus, adeno-associated virus, lentivirus, retrovirus, baculovirus, herpes virus, pseudorabies virus, ZFN gene editing technology, TALEN gene editing technology, CRISPR/Cas gene editing technology and other medically available gene editing techniques or viral vectors.
  • the sDSS1 protein is any one of a basic protein, a first protein, a second protein, a third protein, and a fusion protein obtained by the transplanted cell in an individual.
  • the cell is a stem cell, a precursor cell or an adult cell of any one of humans.
  • the stem cells are embryonic stem cells, induced pluripotent stem cells, cells obtained by transdifferentiation, or stem cells derived from primary culture, pluripotent or pluripotent stem cells differentiated from mother cells.
  • the sDSS1 protein is an sDSS1 protein introduced into an individual by serum or interstitial fluid infusion.
  • the sDSS1 protein is any one of a basic protein, a first protein, a second protein, and a third protein obtained in an individual by transplanting tissues or organs.
  • the tissue is a whole organ or part of a tissue block of the brain, liver, kidney, spleen, pancreatic islet, or blood, fat, muscle, bone marrow, skin.
  • the prophylactic agent comprises a basic protein, a first protein, a second protein, a third protein, a fusion protein, a protein modification, a non-natural amino acid replacement protein, a complex, a drug combination, an expression system, Protein, tissue, organ, body fluid, tissue fluid protein drug, peptide drug, nucleic acid drug, chemical small molecule drug, cell product, commercial transplantation tissue, injection, lyophilized powder, health care product or food additive.
  • the therapeutic drug comprises a basic protein, a first protein, a second protein, a third protein, a fusion protein, a protein modification, a non-natural amino acid substitution protein, a complex, a drug combination, an expression system, Protein, tissue, organ, body fluid, tissue fluid protein drug, peptide drug, nucleic acid drug, chemical small molecule drug, cell product, commercial transplantation tissue, injection, lyophilized powder, health care product or food additive.
  • a protein in a nursing device for improving diabetic complications which is the use of any of the above-mentioned methods in the preparation of a medicament for preventing and treating diabetes complications, and then using the prepared medicament for improving medical equipment related to diabetes complications care Performance.
  • the medical device comprises blood collection equipment and consumables, blood purification equipment and consumables, blood purification equipment auxiliary equipment and consumables, body fluid processing equipment and consumables, kidney dialysis equipment and consumables, peritoneal dialysis equipment and consumables, blood perfusion One or more of a device, an infusion set, an insufflator, a sustained release device, and an artificial kidney.
  • the sDSS1 protein provided by the present invention binds to AGEs and effectively alleviates the cytotoxicity caused by AGEs.
  • the sDSS1 protein provided by the present invention significantly reduces urinary microalbumin levels in one of the markers of diabetic complications in db diabetic model mice.
  • the sDSS1 protein provided by the present invention improves the compensatory increase in glomerular filtration rate in db diabetic model mice, improves renal function and reduces blood glycosylated hemoglobin levels.
  • the sDSS1 protein provided by the present invention is an endogenous protein or a derivative thereof of human and other primates, has a relatively small molecular weight, low immunogenicity, and has a natural protein degradation mechanism in vivo, and therefore, clinical application The probability of causing a significant immune response or other toxic side effects is not high, safe and reliable.
  • the present invention provides a method for the prevention and treatment of diabetic complications by using sDSS1 protein.
  • the sDSS1 protein can be combined with AGEs to reduce cell damage caused by AGEs by molecular, cellular and animal level experiments.
  • sDSS1 protein can effectively reduce the excretion of urinary microalbumin in db diabetic mice, improve renal function, reduce glycosylated hemoglobin levels, and alleviate disease symptoms.
  • the sDSS1 protein has the potential to be used clinically to prepare drugs for the prevention and treatment of diabetic complications.
  • Figure 1A-1B Figure 1A. Molecular level experiments show that sDSS1 interacts with AGEs. The AGEs protein or AGEs protein was incubated with sDSS1 and stained with Coomassie blue. The results showed that AGEs could interact with sDSS1 protein and the SDSS1 protein band became lighter (L3vs L2, L6vs L5).
  • Figure 1B AGEs protein or AGEs protein and sDSS1 incubation products were separated by SDS-PAGE and labeled with anti-AGEs antibody, and developed in the darkroom by developer and fixer. The results showed that AGEs protein strips recognized by sDSS1 interacted with AGEs. The band is lighter and the degree of lightening is proportional to the concentration of sDSS1 protein, showing a significant concentration dependence.
  • Figures 3A-3B Molecular level experiments show that sDSS1 interacts with CML-BSA.
  • Figure 3A SDS-PAGE separation and staining with Coomassie brilliant blue showed that CML-BSA interacted with sDSS1 protein, and the band at the corresponding position of sDSS1 protein became lighter (L3 vs L2, L5 vs L4).
  • Figure 3B Western blotting shows that the amount of CML-BSA protein recognized by the antibody after sDSS1 interacts with CML-BSA is reduced, showing a shallow band. And the degree of lightening is directly proportional to the concentration of sDSS1 protein, showing a significant concentration dependence.
  • Figure 4A-4B sDSS1 protein shields cytotoxicity caused by CML-BSA.
  • Figure 4A 33 ⁇ g/mL CML-BSA was added to rat kidney cell culture, and the state of the cells was observed under a microscope and photographed. When 33 ⁇ g/mL CML-BSA was added to the culture medium, the number of cells was significantly reduced, the cells became round, and the intercellular connections disappeared. As the concentration of sDSS1 protein was increased, the cell status gradually returned to normal.
  • Figure 4B By measuring cell viability, the addition of 33 ⁇ g/mL CML-BSA in rat kidney cell culture can significantly reduce the cell viability level.
  • the 30 ⁇ M sDSS1 protein can substantially block the decrease in cell viability caused by 33 ⁇ g/mL CML-BSA, and this effect exhibits a typical dose-dependent effect.
  • the data was analyzed by ANOVA, #, blank control group vs only CML-BSA group, *, all added CML-BSA and sDSS1 group vs only CML-BSA group; #, *, p-value ⁇ 0.05;##,* *, p-value ⁇ 0.01; ###, ***, p-value ⁇ 0.001.
  • sDSS1 improves urinary albumin levels in diabetic mice.
  • the urinary albumin excretion of db mouse urinary albumin was increased compared with wild littermate mice. After sDSS1 administration, the urinary albumin excretion symptoms were improved.
  • the 10 mpk (mg protein per kilogram body weight) dose group The effect of prolonged administration time was increased, and the amount of urinary albumin excretion was significantly reduced after 40 days of administration. Data were analyzed by ANOVA, *P ⁇ 0.05, **P ⁇ 0.01 vs.db/db.
  • sDSS1 improves symptoms of diabetes complicated with kidney disease.
  • the glomerular filtration function of db mice was compensatoryly increased compared to wild littermates.
  • sDSS1 was able to dose-dependently alleviate this compensatory increase.
  • Data were analyzed by ANOVA, *P ⁇ 0.05, **P ⁇ 0.01 vs.db/db.
  • FIG. 7 sDSS1 improves glycated hemoglobin levels in diabetic mice.
  • the blood glycosylated hemoglobin of db mice was significantly increased compared to wild littermate mice, and the dose of 10 mpk decreased the ratio of glycated hemoglobin by about 1.3% after administration of sDSS1.
  • Data were analyzed by ANOVA, *P ⁇ 0.05, ****P ⁇ 0.0001 vs.db/db.
  • the sDSS1 protein used in the following examples is self-produced and quality controlled by the company.
  • the purity of the tested protein is greater than 95%. Endotoxin and other impurity residues meet the standards and can be used in animal experiments without causing significant animal toxicity.
  • Example 1 The sDSS1 protein interacts with AGEs.
  • Bovine serum albumin (Aladdin, A104912)
  • ribose (Aladdin, D1608050)
  • anti-AGEs antibody was purchased from TransGenic, 50489-M08H.
  • AGEs protein Bovine serum albumin and ribose were incubated in PBS for 14 days to prepare AGEs protein.
  • 2 ⁇ g of AGEs, 4 ⁇ g of sDSS1, 2 ⁇ g of AGEs, 4 ⁇ g of sDSS1, 2 ⁇ g of AGEs, 8 ⁇ g of sDSS1, 2 ⁇ g of AGEs and 8 ⁇ g of sDSS1 were respectively added to a 1.5 mL EP tube, and reacted at 37 ° C overnight.
  • the incubation product was added to the loading buffer, mixed, and denatured at 100 ° C for 10 minutes to prepare a sample for loading.
  • the samples were separated by polyacrylamide gel electrophoresis (SDS-PAGE), and the PAGE gel was stained with Coomassie blue to reveal the protein bands.
  • HRP horseradish peroxidase
  • the obtained AGEs protein has a molecular weight of 75 KD-150 KD, forming a diffuse band (L1, Fig. 1A).
  • L1, Fig. 1A a diffuse band
  • the AGEs were mixed with the sDSS1 protein, it was observed that the light color of the sDSS1 protein was light, suggesting that AGEs interacted with the sDSS1 protein and decreased the number of sDSS1 proteins.
  • the two bands of the AGEs protein were significantly lighter, and as the sDSS1 ratio increased, the bands became lighter and lighter (L2). -L4, Figure 1B).
  • Example 2 sDSS1 protein blocks cytotoxicity caused by AGEs.
  • MATERIALS NRK-52E cell line (Chinese Academy of Sciences, Culture Collection Committee Cell Bank, catalog number: GNR 8), DMEM medium (HyClone, AC10210629), SpectraMax Plus 384 multi-function microplate reader (Molecular Devices), cell proliferation / Toxicity test kit (Dojindo, CK04).
  • NRK-52E cells were seeded into 96-well plates at 2x10 4 cells per well. After adhering to the cells overnight, the cells were replaced with serum-free medium and starved for 24 hours. Each group was divided into blank medium, 50 ⁇ M AGEs, 50 ⁇ M AGEs and 25 ⁇ M sDSS1, 50 ⁇ M AGEs and 50 ⁇ M sDSS1, 50 ⁇ M AGEs and 75 ⁇ M sDSS1, 50 ⁇ M AGEs and 100 ⁇ M sDSS1. After 48 hours, cell viability assay was performed using the cell proliferation toxicity test kit. .
  • Example 3 sDSS1 can interact with CML-BSA.
  • CML-BSA was purchased from Cell Biolab (STA-314); anti-AGEs antibody was purchased from TransGenic (50489-M08H).
  • Method 1 ⁇ g of CML-BSA was mixed with 0.5, 5 ⁇ g of sDSS1. A separate CML-BSA was also included in comparison with sDSS1. After all samples were incubated at 37 °C overnight in an EP tube, the incubation product was added to the loading buffer, mixed, and denatured at 100 ° C for 10 minutes to prepare a sample for loading. The samples were separated by polyacrylamide gel electrophoresis (SDS-PAGE), and the PAGE gel was stained with Coomassie blue to reveal the protein bands.
  • SDS-PAGE polyacrylamide gel electrophoresis
  • the above protein samples were separated by SDS-PAGE, transferred to a PVDF membrane, and blocked with 5% skim milk. Then, anti-AGEs were performed and overnight at 4 °C. The membrane was washed sequentially, the secondary antibody was added, the membrane was washed, the color developed, developed, and fixed to show the protein band of interest.
  • CML is one of the representatives of the AGEs complex.
  • sDSS1 protein band became lighter (Fig. 3A).
  • Western-Blot results showed that the two bands corresponding to the CML-BSA protein were significantly lighter, and as the sDSS1 ratio increased, the band color became lighter (Fig. 3B). Therefore, western blot results indicate that sDSS1 protein can interact with CML-BSA and reduce the number of AGE proteins recognized by antibodies.
  • the results of the Octet molecular interrogation test showed that the affinity constant KD of sDSS1 protein and CML-BSA was 5.26 ⁇ 10 -7 , and the binding constant was 3.71 ⁇ 10 3 , and the dissociation constant was 1.95 ⁇ 10 -3 ; while sDSS1-M1
  • the binding of the protein to CML-BSA is more tight, and the affinity constant KD is 2.96 ⁇ 10 -8 , wherein the binding constant is 9.35 ⁇ 10 3 and the dissociation constant is 2.77 ⁇ 10 -4 . Therefore, the sDSS1 protein can interact with CML-BSA, and the affinity of mutant 1 is higher.
  • Example 4 sDSS1 protein shields cytotoxicity caused by CML-BSA.
  • NRK-52E cell line NRK-52E cell line, DMEM medium (HyClone, AC10210629), SpectraMax Plus 384 multi-function microplate reader (Molecular Devices), cell proliferation/toxicity test kit (Dojindo, CK04), CML-BSA (Cell Biolabs) , STA-314).
  • NRK-52E cells were seeded into 96-well plates at 2x10 4 cells per well. The cells were starved for 24 hours after adherence. Each group was separately added with blank medium, 33 ⁇ g/mL CML-BSA, 33 ⁇ g/mL CML-BSA and 3 ⁇ M sDSS1, 33 ⁇ g/mL CML-BSA and 10 ⁇ M sDSS1, 33 ⁇ g/mL CML-BSA and 30 ⁇ M sDSS1. After completion, the cells were continued for 48 hours. The cell morphology was observed under the microscope of the completed cells, and the cell viability level test was performed using the cell proliferation toxicity test kit.
  • Example 5 sDSS1 can alleviate complications of db diabetic mice
  • mice of C57BLKS/J background were purchased from Nanjing University-Nanjing Institute of Biomedical Research, male. Mice were grouped according to body weight and blood glucose after adaptive feeding, followed by intravenous administration of solvent control or sDSS1 protein every two days. Monitor both weight and diet. At 21 days of administration, the mice were placed in a metabolic cage and urine was collected for 24 hours to measure the urinary albumin concentration. After 37 days of administration, FITC-labeled inulin was intravenously administered, and plasma inulin concentrations were measured at 3/7/10/15/35/70 minutes, respectively, and glomerular filtration rate was calculated.
  • mice were again placed in a metabolic cage and urine was collected for 24 hours to measure the urinary albumin concentration.
  • the mice were sacrificed after 6 weeks of administration, and plasma, heart, eyeball, kidney, and brain were collected.
  • the mouse urinary albumin assay kit was purchased from Bethyl Laboratories, Inc.
  • the mouse glycated hemoglobin Elisa kit was purchased from Shanghai Langton Biotechnology Co., Ltd. (Cat. No. BPE20512).
  • db diabetic mice showed increased urinary albumin excretion compared with their littermate wild control mice, suggesting impaired renal function.
  • sDSS1 had a trend to improve urinary albumin excretion, but there were no statistical differences.
  • sDSS1 10mpk significantly improved urinary albumin excretion, and the effect of sDSS1 10mpk was stronger than that of 3mpk ( Figure 5).
  • the glomerular filtration rate function was tested and the results showed that db mice had a compensatory increase in glomerular filtration function compared to their wild control mice.
  • sDSS1 proteins 3mpk and 10mpk alleviated the increased glomerular filtration rate in db mice (Fig. 6).
  • Blood glycated hemoglobin data showed that the 10 mpk dose reduced the glycated hemoglobin ratio by about 1.3%, suggesting that sDSS1 can affect glycosylated hemoglobin levels (Figure 7).
  • sDSS1 can improve the symptoms of renal complications in db diabetic mice, and this improvement has a time-dependent and dose-effect relationship.

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  • Marine Sciences & Fisheries (AREA)
  • Vascular Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne une application de la protéine sécrétoire DSS1 dans la préparation d'un médicament pour la prévention ou le traitement d'une complication du diabète. La DSS1 sécrétoire peut se lier à des produits finaux de glycation avancée et bloquer la cytotoxicité induite par ceux-ci, ce qui permet de soulager un symptôme dans un modèle animal présentant une complication du diabète.
PCT/CN2019/077857 2018-03-20 2019-03-12 Application de protéine dans la préparation d'un médicament pour la prévention ou le traitement d'une complication du diabète WO2019179338A1 (fr)

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CN201810231927.4A CN110302362B (zh) 2018-03-20 2018-03-20 一种蛋白在制备预防和治疗糖尿病并发症的药物中的应用

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CN112553323B (zh) * 2020-12-25 2022-05-13 福建农林大学 Col1a1作为一种2型糖尿病的生物标志物及其应用
IL311448A (en) * 2021-09-15 2024-05-01 Shanghai Puyou Biomedical Co Ltd polypeptide

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CN107573412A (zh) * 2016-07-04 2018-01-12 上海清流生物医药科技有限公司 一种新型天然蛋白及其应用

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KRAGELUND, BB.: "DSS1/Sem1, a Multifunctional and Intrinsically Disordered Protein", TRENDS IN BIOCHEMICAL SCIENCES, vol. 41, no. 5, 31 May 2016 (2016-05-31), pages 446 - 459, XP029523207 *
KRISTENSEN, CN: "Depletion of DSS1 protein disables homologous recombinational repair in human cells", MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS, vol. 694, 10 December 2010 (2010-12-10), pages 60 - 64, XP027516240 *

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