WO2019134481A1 - Utilisation d'une protéine dans la préparation d'un médicament pour la prévention et le traitement de la maladie d'alzheimer - Google Patents

Utilisation d'une protéine dans la préparation d'un médicament pour la prévention et le traitement de la maladie d'alzheimer Download PDF

Info

Publication number
WO2019134481A1
WO2019134481A1 PCT/CN2018/120172 CN2018120172W WO2019134481A1 WO 2019134481 A1 WO2019134481 A1 WO 2019134481A1 CN 2018120172 W CN2018120172 W CN 2018120172W WO 2019134481 A1 WO2019134481 A1 WO 2019134481A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
sdss1
modification
drug
amino acid
Prior art date
Application number
PCT/CN2018/120172
Other languages
English (en)
Chinese (zh)
Inventor
张英豪
付晶鹏
Original Assignee
上海清流生物医药科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海清流生物医药科技有限公司 filed Critical 上海清流生物医药科技有限公司
Publication of WO2019134481A1 publication Critical patent/WO2019134481A1/fr

Links

Images

Classifications

    • 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
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the present invention relates to the use of the sDSS1 protein drug for the preparation of a medicament for preventing and treating dementia, comprising a prophylactic medicament for the preparation of degenerative dementia and vascular dementia, or for preparing a pre-, early, intermediate or late stage of these diseases.
  • Therapeutic drugs for the preparation of a medicament for preventing and treating dementia, comprising a prophylactic medicament for the preparation of degenerative dementia and vascular dementia, or for preparing a pre-, early, intermediate or late stage of these diseases.
  • Dementia is a type of brain disease that causes long-term and progressive deterioration of patient thinking and learning memory.
  • the most common form of dementia is Alzheimer's Disease (AD), which accounts for dementia. 50-70% of patients, others include vascular dementia ( 25%), Louise-type dementia ( 15%) and so on.
  • AD Alzheimer's Disease
  • the number of people with dementia worldwide is about 46.8 million, and the number of patients worldwide is expected to reach 75 million in 2030 [1].
  • AD is a typical neurodegenerative disease with clinical manifestations of progressive learning and memory impairment and neurological damage.
  • a typical pathological feature of AD patients is the presence of ⁇ -amyloid (A ⁇ ) plaques and tau deposition in nerve tissue.
  • the ⁇ amyloid hypothesis suggests that the progressive accumulation of toxic proteins caused by the imbalance of A ⁇ protein production and clearance in nerve tissue and the resulting synaptic dysfunction and neuronal death are key mechanisms leading to disease [2] [3].
  • the researchers hope to prevent A ⁇ protein production and aggregation, or promote A ⁇ clearance, in order to prevent or treat diseases [4].
  • the results of AD studies based on animal experiments show that antibodies [5] [6], peptide drugs [7], small molecule drugs [8] can block A ⁇ aggregation or production, reduce plaque formation, and reduce animal disease indications. Therefore, the drug inhibits and clears the pathological deposition of A ⁇ or can be an effective way to treat AD.
  • 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 involved in stable genome, homologous gene recombination, DNA damage repair and cell proliferation. Etc. [9-12]. The results of the inventors of the present invention show that the DSS1 protein can be added to the oxidized protein by a energy-consuming enzymatic reaction to help the cells clear the oxidized protein [13]. These results show the important role of DSS1 protein in biological activities.
  • the transient accumulation of A ⁇ protein is one of the key factors in the pathogenesis of AD. It is one of the main ways to treat AD diseases by removing A ⁇ or inhibiting A ⁇ aggregation.
  • the sDSS1 protein provided by the invention can bind to A ⁇ protein to accelerate the clearance of A ⁇ protein, reduce the accumulation of A ⁇ protein, improve the symptoms of the disease, and has the potential for preparing clinical AD and other dementia prevention drugs and therapeutic drugs.
  • a protein for the preparation of a medicament for the prevention and treatment of dementia which is the use of the sDSS1 protein for the preparation of a medicament for the prevention and treatment of dementia.
  • the dementia refers to degenerative dementia, including Alzheimer's disease, Alzheimer's disease, Lewy body dementia, and frontotemporal dementia.
  • the dementia is vascular dementia.
  • the dementia is pre-dementia dementia, mild dementia dementia, moderate dementia dementia, and severe dementia dementia.
  • 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 in scorpionfish, 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.
  • 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 buccal gibbon sDSS1 is SEQ ID NO: 6
  • the golden monkey sDSS1 The amino acid sequence of SEQ ID NO: 7, the amino acid sequence of rhesus sDSS1 is SEQ ID NO: 8
  • the amino acid sequence of gilt monkey sDSS1 is SEQ ID NO: 9
  • the amino acid sequence of ssDSS1 is SEQ ID NO: 10, Angola.
  • amino acid sequence of simian sDSS1 is SEQ ID NO: 11
  • amino acid sequence of leucocephalus sDSS1 is SEQ ID NO: 12
  • amino acid sequence of scorpion sDSS1 is SEQ ID NO: 13.
  • the sDSS1 protein is the first protein having a similarity to the basic protein of 70% or more.
  • the sDSS1 protein is based on 58 amino acids of the nitrogen terminus of the basic protein, and fuses other polypeptide fragments at the nitrogen terminal or the carbon terminal, and the structural features or amino acid sequence characteristics of the polypeptide fragment for fusion are The second protein of the same or similar sequence of the carbon end of the basic protein.
  • the sDSS1 protein is based on the basic amino acid of the basic protein of 58 amino acids, and the other amino acid fragments are fused at the nitrogen or carbon end, and the fused protein can realize the transmembrane transport function. .
  • the sDSS1 protein is a fusion formed by using the basic protein, the first protein, the second protein or the third protein to form a protein, a carrier protein, an antibody or other amino acid fragments of any length. protein.
  • the sDSS1 protein is a polypeptide/protein modification produced by modification of the basic protein, the first protein, the second protein, the third protein or the fusion protein.
  • the polypeptide/protein modification is directed to an amino group on an amino acid side chain, a carbonyl group on an 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 polypeptide/protein modification comprises glycosylation modification, fatty acid modification, acylation modification, Fc fragment fusion, albumin fusion, polyethylene glycol modification, dextran modification, heparin modification, polyethylene Pyrrolidone modification, polyamino acid modification, polysialic acid modification, chitosan and its derivative modification, lectin modification, sodium alginate modification, carbomer modification, polyvinylpyrrolidone modification, hydroxypropyl methylcellulose modification, One or more of hydroxypropylcellulose modification, acetylation modification, formylation modification, phosphorylation modification, methylation modification, sulfonation modification, and other pharmaceutically acceptable polypeptide/protein drug modification methods.
  • the sDSS1 protein is an amino acid other than the 20 basic amino acids based on the amino acid sequence of the basic protein, the first protein, the second protein, the third protein or the fusion protein. - 31 non-natural amino acid replacement proteins substituted with any amino acid site.
  • the amino acid substitution of the non-natural amino acid replacement protein comprises replacement with hydroxyproline, hydroxylysine, selenocysteine, D-form amino acid or synthetic non-natural amino acid and derivatives thereof.
  • the sDSS1 protein is pharmaceutically applicable to the basic protein, the first protein, the second protein, the third protein, the fusion protein, the polypeptide/protein modification or the non-natural amino acid replacement protein. Part or all of the complex formed by the drug carrier.
  • the pharmaceutical carrier of the complex comprises one or one of an enteric coating preparation, a capsule, a microsphere/capsule, a liposome, a microemulsion, a double emulsion, a nanoparticle, a magnetic particle, a gelatin, and a gel. the above.
  • the base of the protein, the first protein, second protein and third protein, fusion protein, polypeptide / protein or complex modifications play operation starting concentration of toxic proteins toxic shield is not less than 0.2 ⁇ g /mL .
  • the basic protein, the first protein, the second protein, the third protein, the fusion protein, the polypeptide/protein modification or the complex exhibits a molar concentration ratio of shielding toxic protein toxicity of not less than 0.010;
  • the molar concentration ratio refers to the ratio of the molar concentration of the protein, polypeptide/protein modification or complex in the reaction system to the molar concentration of the toxic protein.
  • 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 or cerebrospinal fluid by affecting protease/peptidase activity in blood or cerebrospinal fluid.
  • the drug is the first drug formed by 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 the basic protein, the first protein, the second protein, the third protein, the fusion protein, the polypeptide/protein modification, the complex, and the first drug.
  • the sDSS1 protein is a basic protein, a first protein, a second protein, a third protein, a fusion protein, a polypeptide/protein modification, a non-natural amino acid replacement protein, a complex, A third drug formed by the combination of one, two or more of any one of the first drugs with a pharmaceutically acceptable excipient.
  • the sDSS1 protein is a fourth obtained by introducing a nucleotide sequence encoding the basic protein, the first protein, the second protein, the third protein or the fusion protein into the body and expressing the same by an expression system. Protein.
  • 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 or other medically available gene editing technology or viral vector.
  • the sDSS1 protein is a fifth protein formed by the basic protein, the first protein, the second protein, the third protein or the fusion protein obtained by the transplanted cell in the 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 a sixth protein formed by the basic protein, the first protein, the second protein, the third protein or the fusion protein obtained by transplanting tissues or organs in an individual.
  • the tissue is a whole organ or part of a tissue block of the brain, liver, kidney, spleen, islet, or blood, fat, muscle, bone marrow, skin.
  • the sDSS1 protein is a seventh protein introduced into the body by serum, cerebrospinal fluid, and interstitial fluid infusion.
  • the prophylactic agent comprises a basic protein, any of the first to seventh proteins, a fusion protein, a polypeptide/protein modification, a non-natural amino acid replacement protein, a complex, a first drug, and a second Drugs, third drugs, protein drugs in expression systems, cells, tissues, organs, body fluids, tissue fluids, peptide drugs, nucleic acid drugs, chemical small molecule drugs, cell products, commercial transplant tissues, injections, lyophilized powder, One or more of health care products and food additives.
  • the therapeutic agent comprises a basic protein, any of the first to seventh proteins, a fusion protein, a polypeptide/protein modification, a non-natural amino acid replacement protein, a complex, a first drug, and a second Drugs, third drugs, protein drugs in expression systems, cells, tissues, organs, body fluids, tissue fluids, peptide drugs, nucleic acid drugs, chemical small molecule drugs, cell products, commercial transplant tissues, injections, lyophilized powder, One or more of health care products and food additives.
  • the sDSS1 protein provided by the invention binds to A ⁇ protein, inhibits the aggregation of A ⁇ protein and reduces the binding ability of A ⁇ protein to its receptor protein, and effectively alleviates the cytotoxicity caused by A ⁇ protein.
  • the sDSS1 protein provided by the present invention can accelerate the clearance of A ⁇ protein by microglia, and shields A ⁇ oligomer toxicity and reduces microglia activation induced by A ⁇ oligomer.
  • the sDSS1 protein provided by the present invention significantly improves the disease symptoms of the AD model nematode on the CL4176 nematode and reduces the accumulation of A ⁇ protein in the nematode tissue.
  • the sDSS1 protein provided by the present invention helps mice clear A ⁇ and reduce A ⁇ levels in the blood. Long-term injection of sDSS1 protein into AD model APP/PS1 mice can effectively reduce A ⁇ plaque deposition in brain tissue.
  • the sDSS1 protein provided by the present invention is a protein possessed by humans and other primates, has a relatively small molecular weight, low immunogenicity, and has a natural protein degradation mechanism in vivo, and therefore, clinical application does not cause significant immunity.
  • the reaction or other toxic side effects are safe and reliable.
  • the present invention provides an sDSS1 protein drug for the treatment of dementia.
  • the sDSS1 protein can bind to A ⁇ protein, inhibit A ⁇ aggregation, and reduce A ⁇ protein and receptor by molecular, cell and animal level experiments.
  • the binding efficiency promotes microglia clearance of A ⁇ and reduces microglia activation.
  • sDSS1 protein promotes A ⁇ clearance in the blood, reduces the accumulation of A ⁇ protein in tissues, and alleviates disease symptoms.
  • sDSS1 protein has low immunogenicity and remarkable efficacy, and has the potential to be used for clinical preparation of dementia prevention drugs or for preparing pre-treatment, early, intermediate, and advanced dementia drugs.
  • FIG. 1A-1B Molecular experiments show that A[beta] protein binds to sDSSl protein.
  • a ⁇ 1-42 protein and A ⁇ 1-40 protein were incubated with sDSS1 protein, respectively, and a complex of A ⁇ 1-42 protein and sDSS1 protein was detected by A ⁇ protein antibody (molecular weight 10KD-37KD, Lane 3 and lane 4).
  • a ⁇ protein antibody molecular weight 10KD-37KD, Lane 3 and lane 4
  • the A ⁇ 1-40 protein is incubated alone to form a partial dimer, and incubation with the sDSS1 protein reduces dimer formation.
  • sDSS1 protein and A ⁇ 1-42 protein (lane 3 and lane 4) or sDSS1 protein and A ⁇ 1-40 protein (lane 6 and lane 7) can also be detected by sDSS1 protein antibody ( The molecular weight is between 37 and 150 KD), and the complex formation increases as the concentration of sDSS1 protein in the reaction system increases.
  • ThT assay detects A[beta] 1-42 protein aggregation.
  • the A ⁇ 1-42 protein aggregates at 12 hours and shows an ascending curve.
  • Addition of 5% or 10% sDSS1 protein according to the molecular weight ratio inhibited the ascending curve and prolonged the detection time without observing the rising curve, suggesting that no A ⁇ 1-42 protein aggregation occurred.
  • FIG. 1 D ThT assay detects A ⁇ 1-40 protein aggregation.
  • the A ⁇ 1-40 protein began to show a rising curve at 24 hours, indicating aggregate formation.
  • Addition of 5% or 10% sDSS1 protein according to the molecular weight ratio inhibited the ascending curve, and the prolongation of the detection time did not observe a rising curve, suggesting that no A ⁇ 1-40 protein aggregation occurred.
  • FIG. 1E ThT assay detects sDSS1 protein mutant 4 inhibits A ⁇ 1-40 protein aggregation.
  • the A ⁇ 1-40 protein aggregates at 20-24 hours and shows an ascending curve.
  • 0.667%, 0, 3.33%, or 16.67% sDSS1(M4) protein was added according to the molecular weight ratio, the rise curve was inhibited, and the rise time was not observed, indicating that no A ⁇ 1-40 protein aggregation occurred.
  • FIG. 1F ThT assay detects sDSS1 protein mutant 11 inhibiting A ⁇ 1-40 protein aggregation.
  • the A ⁇ 1-40 protein aggregates at 20-24 hours and shows an ascending curve.
  • 0.667%, 0, 3.33%, or 16.67% sDSS1(M11) protein was added according to the molecular weight ratio, the rising curve was inhibited, and the rise time was not observed, indicating that no A ⁇ 1-40 protein aggregation occurred.
  • the A ⁇ 1-42 protein oligomer and the A ⁇ 1-40 protein oligomer were incubated with the sDSS1 protein, respectively, and the complex formed by the A ⁇ 1-42 protein and the sDSS1 protein was detected by the A ⁇ protein antibody (molecular weight 10KD). Between -37KD, lane 3 and lane 4). No significant difference was detected between the A ⁇ 1-42 protein and the sDSS1 protein.
  • a complex of sDSS1 protein and A ⁇ 1-42 oligomer (lane 3) or sDSS1 protein and A ⁇ 1-40 oligomer (lane 5) can be detected by using sDSS1 protein antibody (molecular weight 37-150KD) between).
  • ThT assay detects the reaction of sDSS1 protein with A ⁇ 1-40 oligomer.
  • the addition of 50% and 100% sDSS1 protein in a molecular weight ratio of 30 ⁇ M A ⁇ 1-40 oligomer showed an increase in the fluorescence value of the A ⁇ 1-40 oligomer, and the fluorescence value remained substantially unchanged during the 12-hour detection period.
  • ThT assay detects the reaction of the sDSS1 protein with the A ⁇ 1-42 oligomer.
  • the addition of 50% and 100% sDSS1 protein in a molecular weight ratio of 30 ⁇ M A ⁇ 1-42 oligomer showed an increase in the fluorescence value of the A ⁇ 1-42 oligomer.
  • the fluorescence value of the A ⁇ 1-42 oligomer also increased, and the fluorescence value increased after the addition of the sDSS1 protein.
  • FIG. 3A The sDSS1 protein is unable to interact with the RAGE receptor protein.
  • the results of molecular interaction test showed that on the binding curve, it can be seen that after the injection of sDSS1 protein, no obvious ascending binding curve was observed, and no obvious dissociation curve was observed.
  • sDSS1 protein inhibits the interaction of A ⁇ protein with RAGE receptor protein.
  • a ⁇ 1-42 protein can bind to RAGE receptor protein, and the addition of sDSS1 protein can inhibit the binding efficiency of A ⁇ protein to RAGE, the binding amount of A ⁇ protein decreases, and the effect shows a typical dose-dependent effect.
  • FIGS. 4A-4C Cell experiments demonstrate that sDSS1 protein shields A ⁇ 1-42 oligomer toxicity and protects N2a cell viability.
  • a ⁇ 1-42 oligomers cause N2a cytotoxicity, and the addition of A ⁇ 1-42 oligomers causes abnormal aggregation and rounding of cells, and sDSS1 protein can alleviate these phenomena.
  • FIG. 2B detection of cell viability revealed that sDSS1 protein can reduce cell viability by A ⁇ 1-42 oligomer. After the addition of the same amount of sDSS1 protein, cell viability is almost unaffected by the toxicity of A ⁇ 1-42 oligomer.
  • FIGS 5A-5B Cell experiments demonstrate that sDSS1 protein shields A ⁇ 1-42 oligomer toxicity and protects primary neuronal cell viability.
  • 10 ⁇ M A ⁇ 1-42 oligomers can cause a decrease in neuronal cell viability, and cell viability can be recovered after the addition of 5 ⁇ M and 10 ⁇ M sDSS1 protein.
  • sDSS1 protein can reduce the cytotoxicity level caused by A ⁇ 1-42 protein, and 10 ⁇ M sDSS1 protein can protect cells from the toxicity of A ⁇ 1-42 oligomer.
  • N 10 per group. Data were analyzed by ANOVE, *, p-value ⁇ 0.05; **, p-value ⁇ 0.01.
  • FIGS. 6A-6B The sDSS1 protein promotes microglia clearance of A ⁇ protein.
  • FIG. 6A A ⁇ 1-42 protein and sDSS1 protein were added to the medium.
  • the concentration of A ⁇ protein in microglia was significantly higher than that of the control with only A ⁇ protein added after 24 hours, with the increase of the concentration of sDSS1 protein added.
  • the cytosolic A ⁇ protein concentration is increased.
  • FIGS 6C-6D The sDSS1 protein reduces microglial activation by A ⁇ 1-42 protein oligomers.
  • FIG. 6C A ⁇ 1-42 protein oligomers caused microglia activation, which showed an increase in ROS levels in BV2 cells.
  • ROS levels in BV2 cells gradually decreased, which was close to the control group.
  • FIG. 6D A ⁇ 1-42 protein oligomer stimulates microglia to release inflammatory factor TNF- ⁇ , and sDSS1 protein reduces TNF- ⁇ release.
  • N 3 per group. Data were analyzed by ANOVE, *, p-value ⁇ 0.05; **, p-value ⁇ 0.01.
  • FIG. 7A The sDSS1 protein ameliorate the disease symptoms of CL4176 nematodes.
  • the sputum rate of the AD model CL4176 nematode gradually increased, and the addition of sDSS1 protein to the culture medium significantly slowed down the rate of sputum.
  • the mitigation efficiency showed drug dose dependence, and the mitigation effect increased with the increase of sDSS1 protein concentration.
  • the ratio of the control group to the nematode was only 15.09%, the 500 ⁇ g/mL sDSS1 protein administration group was 39.47%, and the 1000 ⁇ g/mL sDSS1 protein administration group was 65.71%.
  • the nematode in the control group was almost all sputum (the ratio of the nematode was less than 1%), while in the 500 ⁇ g/mL sDSS1 protein-administered group, the proportion of the untreated nematode was 18.42%, and the 1000 ⁇ g/mL sDSS1 protein-administered group was 48.57. %.
  • the number of nematodes per group is 90.
  • the experiment was repeated 3 times and the data was analyzed by Log-rank (Mantel-Cox) test, ***, p-value ⁇ 0.001.
  • FIG. 7B Immunohistochemical staining for the detection of A ⁇ 1-42 deposition in C14176 nematode tissue.
  • AD model 4176 elegans tissues were fixed and immunofluorescent stained, A ⁇ 1-42 specific antibodies were used to display A ⁇ protein (green fluorescence), Hoechst dye was used to stain nuclei (blue fluorescence), and white arrows indicate A ⁇ plaque deposition.
  • Typical A ⁇ 1-42 plaques and diffuse A ⁇ precipitates were seen in the control nematode tissues. After treatment with 500 ⁇ g/mL sDSS1 protein, A ⁇ plaques and diffuse deposition were significantly reduced. The 1000 ⁇ g/mL sDSS1 protein administration group showed almost no plaque and less diffuse deposition.
  • FIG. 7C - Figure 7D 4176 Reduction of A[beta] 1-42 aggregates and total A[beta] protein content in nematode tissue mills.
  • FIG. 7C Western blotting was used to detect A ⁇ 1-42 aggregates in nematode tissue mills. It can be seen that after treatment with sDSS1 protein, most A ⁇ aggregate bands decreased or disappeared completely (black arrow indicates position).
  • Fig. 7D the total A ⁇ protein in the tissue slurry was detected by ELISA, and it was found that the total A ⁇ protein of the nematode tissue of the sDSS1 protein administration group was significantly decreased, and the content of the 1000 ⁇ g/mL sDSS1 protein administration group was only 59.07% of the control group.
  • the number of nematodes used in each group was 400 in the experiment.
  • FIGS 8A-8B sDSS1 protein promotes A ⁇ protein clearance in mouse blood.
  • Figure 8A shows a model of acute A ⁇ rise by intravenous injection of A ⁇ protein into mice. The results show that sDSS1 protein administration accelerates A ⁇ protein clearance. As the amount of sDSS1 protein administered increased, the A ⁇ content decreased significantly.
  • FIG. 8B Injecting sDSS1 protein into AD model APP/PS1 mice, a significant decrease in A ⁇ levels was also detected in the blood.
  • FIGS 9A-9C sDSS1 protein reduces A ⁇ plaque deposition in brain tissue of APP/PS1 mice.
  • Fig. 9A after staining with FSB dye, a large amount of A ⁇ plaque precipitate (blue fluorescence) was observed in the brain tissue of APP/PS1 mice at 11 months of age. After administration of sDSS1 protein, plaque deposition in the hippocampus of brain tissue decreased. The number of plaques is reduced and the brightness is reduced.
  • Fig. 9B after the photo was processed by Phototshop CS software, the plaque area was counted. The results showed that the plaque area of brain tissue after sDSS1 protein administration was significantly lower than that of the control group.
  • FIG 9C the number of small plaques (marked as 1 in Figure 9A), medium-sized plaques (marked as 2 in Figure 9A), and large plaques (marked as 3 in Figure 9A) were counted according to plaque size. In contrast, the number of medium-sized plaques and large plaques in tissues was significantly decreased after administration of sDSS1 protein, and there was no significant difference in small plaques.
  • APP/PS1 mice were 3 in each group. The data was analyzed by ANOVE, n.s., p-value>0.5; **, p-value ⁇ 0.01.
  • the sDSS1 protein used in the following examples is the human sDSS1 protein produced by the company itself, and the protein sequence is shown in SEQ ID NO: 1.
  • the company controls the quality of protein.
  • the purity of the tested protein is greater than 95%.
  • the endotoxin (less than 3EU/mg protein) and other impurities are in compliance with the standard and can be used in animal experiments without causing significant animal toxicity.
  • Example 1 The sDSS1 protein inhibits A ⁇ protein aggregation.
  • sDSS1 protein, sDSS1 mutants 4 (sDSS1 (M4)) (amino acid sequence is shown in SEQ ID 15), sDSS1 mutants 11 (amino acid sequence see SEQ ID 16) (sDSS1 (M11 )) were diluted to 1mg / mL in PBS (100 ⁇ M).
  • the experiment was carried out in a PBS environment. First, a PBS was added in a 1.5 mL centrifuge tube, and then sDSS1 protein or mutant protein, A ⁇ protein and ThT dye were sequentially added, and the sDSS1 protein molar concentration was added according to a preset concentration gradient.
  • the molar concentrations of the A ⁇ protein and the ThT dye were 30 ⁇ M and 5 ⁇ M, respectively; for the A ⁇ 1-40 protein, the molar concentrations of the A ⁇ protein and the ThT dye were 30 ⁇ M and 10 ⁇ M, respectively.
  • the solution was shaken and mixed on the suspension, and then 200 ⁇ L was added to a black fluorescence detection plate (Costar, 3792) for two duplicate wells per group.
  • the black fluorescence detection plate was placed on a multi-function microplate reader (Molecular Devices, SpectraMax i3x) to detect the fluorescence value, and the excitation light was set to 450 nm, the emission light was 485 nm, the detection interval was 30 minutes, and the detection was continued for 48 hours.
  • a multi-function microplate reader Molecular Devices, SpectraMax i3x
  • Protein Interactions A ⁇ 1-42 and A ⁇ 1-40 protein monomers were diluted to 20 ⁇ M with PBS and mixed with 10 ⁇ M or 20 ⁇ M sDSS1 protein, and 20 ⁇ M A ⁇ protein was used as a positive control.
  • the protein solution was mixed and incubated overnight at 4 °C. 5x loading buffer was added to the incubated protein solution, mixed and heated at 100 ° C for 10 minutes, and the prepared samples were used for Western Blotting analysis.
  • a ⁇ 1-42 or A ⁇ 1-40 protein was co-incubated with sDSS1 protein, and the mixture after incubation was detected. It was found that A ⁇ 1-42 protein forms a complex with sDSS1 protein (molecular weight 10KD-37KD). Co-incubation of the A ⁇ 1-40 protein with the sDSS1 protein significantly reduced the formation of dimers (Fig. 1A). The A ⁇ protein and sDSS1 protein complex signals were detected by the same technique using the sDSS1 protein antibody, and the complex signal was enhanced as the sDSS1 protein concentration was increased (Fig. 1B). A ⁇ 1-42 or A ⁇ 1-40 monomeric proteins aggregate to form an amyloid deposit, which is enhanced by binding to ThT dye.
  • Example 2 The sDSS1 protein binds to an A ⁇ protein oligomer.
  • a ⁇ 1-42 protein solubilization and oligomer preparation 1 mg A ⁇ 1-42 or A ⁇ 1-40 protein was added to 500 ⁇ L of 20 mM NaOH solution, shaken and mixed for 10 minutes, and then sonicated for 10 minutes to help dissolve the protein. Dilute to 100 ⁇ M with PBS solution, incubate at 4 ° C for 24 hours, centrifuge at 12000 g for 10 minutes to remove possible precipitates, and obtain A ⁇ 1-42 or A ⁇ 1-40 oligomers. A ⁇ oligomers are labeled according to the concentration of A ⁇ monomer protein prior to incubation.
  • ThT assay The sDSS1 protein was diluted to 1 mg/mL (100 ⁇ M) with PBS. The experiment was carried out under PBS conditions. First, PBS was added in a 1.5 mL centrifuge tube, and then sDSS1 protein, A ⁇ protein oligomer and ThT dye were sequentially added, and the molar concentration of sDSS1 protein was added according to a preset concentration gradient. For the A ⁇ 1-42 protein, the molar concentrations of the A ⁇ protein and the ThT dye were 30 ⁇ M and 5 m ⁇ , respectively; for the A ⁇ 1-40 protein, the molar concentrations of the A ⁇ protein and the ThT dye were 30 ⁇ M and 10 m ⁇ , respectively.
  • the solution was shaken and mixed on the suspension, and then 200 ⁇ L was added to a black fluorescence detection plate (Costar, 3792) for two duplicate wells per group.
  • the black fluorescent detection plate was placed on a multi-function microplate reader to detect the fluorescence value, and the excitation light was set to 450 nm, the emission light was 485 nm, the detection interval was 30 minutes, and the detection was continued for 12 hours.
  • a ⁇ 1-42 or A ⁇ 1-40 protein oligomer was diluted to 20 ⁇ M with PBS and mixed with 10 ⁇ M or 20 ⁇ M sDSS1 protein, and 20 ⁇ M A ⁇ oligomer was used as a positive control.
  • the protein solution was mixed and incubated overnight at 4 °C. 5x loading buffer was added to the incubated protein solution, mixed and heated at 100 ° C for 10 minutes, and the prepared samples were used for Western Blotting analysis.
  • the oligomer of A ⁇ 1-40 or A ⁇ 1-42 is the major form of A ⁇ protein producing cytotoxicity.
  • the A ⁇ protein was first incubated to form an oligomer, and then incubated with sDSS1 protein for western blotting and ThT detection.
  • the results showed that the reaction of sDSS1 protein with A ⁇ 1-42 oligomer form was detected by A ⁇ protein antibody, and the complex content increased with the increase of sDSS1 protein concentration in the reaction system (Fig. 2A).
  • a complex formed by the sDSS1 protein and the A ⁇ protein can also be detected using the sDSS1 protein antibody (Fig. 2B).
  • the sDSS1 protein can react with the amyloid precipitation of A ⁇ 1-40 or A ⁇ 1-42, and the fluorescence intensity increases after the sDSS1 protein reacts with the A ⁇ 1-40 amyloid precipitate (Fig. 2C); After the reaction of the sDSS1 protein with the A ⁇ 1-42 amyloid precipitate, the fluorescence intensity was significantly reduced (Fig. 2D).
  • the above results indicate that the sDSS1 protein can also form a complex with the A ⁇ protein oligomer form, and these interactions may inhibit the cytotoxicity of the A ⁇ protein.
  • Example 3 sDSS1 protein reduces the binding efficiency of A ⁇ protein to receptor protein.
  • the biomacromolecular interaction instrument (GE, Biacore 3000) was used to detect the binding efficiency of RAGE protein to sDSS1 protein.
  • the chip was first rinsed with PBS for 20 minutes until the baseline was stable, and the protein was coupled to the sensor chip NTA chip (GE) with a His tag on 1 ⁇ g/mL RAGE protein with a coupling amount of approximately 50 RU.
  • the sDSS1 protein was diluted to 10 ⁇ g/mL, the loading amount was 20 ⁇ L, and the loading time was 200 seconds. After completion, rinse with PBS solution and measure the elution curve.
  • RAGE protein was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. (11629-H02H).
  • the blank plate was washed once with PBS, and 100 ⁇ L of 0.5 ⁇ g/mL RAGE protein diluted with PBS was added to each well overnight at 4 ° C, and washed once with PBS.
  • the PBS diluted 2% BSA in PBST (0.05% Tween 20) was added and blocked at 37 ° C for 1 hour, and then washed once with PBS.
  • a ⁇ 1-42 protein or A ⁇ 1-42 protein and sDSS1 protein mixture set A ⁇ 1-42 protein concentration to 0.625 ⁇ g/mL and 0.3125 ⁇ g/mL, respectively, and sDSS1 protein concentration of 0.2 ⁇ g/mL and 1 ⁇ g/mL, respectively, incubate at room temperature. 2 hours.
  • PBST 0.05% Tween 20
  • 100 ⁇ L of Rabbit-anti-A ⁇ (1:10000 diluted in PBST solution containing 0.5% BSA) was added, and the mixture was incubated at room temperature for 1 hour, and then washed three times with PBST.
  • HRP horseradish peroxidase
  • sDSS1 protein could not bind to the RAGE protein and showed no significant binding peak on BIAcore (Fig. 3A).
  • ELISA experiments showed that A ⁇ 1-42 protein as a ligand can bind to RAGE protein, showing a higher binding amount.
  • the addition of 0.2 ⁇ g/mL and 1 ⁇ g/mL sDSS1 protein reduced the binding amount of A ⁇ 1-42 to RAGE, and this effect exhibited a significant concentration-dependent effect ( FIG. 3B ).
  • Example 4 sDSS1 protein shields cytotoxicity caused by A ⁇ oligomers.
  • N2a Cell Culture Mouse-derived neuroblastoma cells
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal calf serum
  • mice that were pregnant for 16 days were deeply anesthetized with ether and then sacrificed by cervical dislocation.
  • the E16 stage fetus was dissected.
  • the fetal rats were anesthetized with ice
  • the cervical vertebrae were sacrificed, the rat brain was dissected, and immersed in a pre-cooled DMEM solution (containing 2% double-antibody).
  • the pia mater was removed by ophthalmology and repeatedly washed to remove blood.
  • the frontal cortex was dissected and the tissue was cut with an ophthalmic scissors. All tissues were collected into a centrifuge tube and digested with 5 mL of 0.05% trypsin for 15 minutes at room temperature.
  • the digestion was terminated by adding an equal volume of DMEM complete medium, and the tissue was repeatedly beaten and filtered with a 40 ⁇ m cell strainer to obtain a cell suspension. After the cells were counted, they were added to a 96-well plate at a rate of 3.0 x 10 4 cells per well, 200 ⁇ L of DMEM complete medium, and the plate was incubated at 37 °C. After 24 hours, switch to 200 ⁇ L of neuronal medium containing 98% Neurobasal (Gibco, 21103049), 2% B27 supplement (Gibco, 17504044), 1% GlutaMAX-1supplement (Gibco, 35050061). Fresh neuronal medium was changed every two days thereafter. Cultured neurons matured after 8 days and can be used in subsequent experiments.
  • a ⁇ 1-42 protein solubilization and oligomer preparation 1 mg of A ⁇ 1-42 was diluted to 100 ⁇ M with serum-free DMEM-/- medium, and the dilution was incubated at 4 ° C for 24 hours to obtain A ⁇ 1-42 oligomer protein.
  • the A ⁇ oligomer protein is labeled according to the concentration of A ⁇ monomer protein before incubation.
  • Cytotoxicity assay N2a cells were seeded into 96-well plates at 2x10 4 cells per well. After the cells were attached for 12 hours, they were replaced with serum-free DMEM medium, and the cells were starved for 24 hours.
  • the A ⁇ 1-42 oligomer was diluted to a 20 ⁇ M solution in serum-free DMEM.
  • the negative control group was treated with serum-free DMEM, and the positive control group was added with 20 ⁇ M A ⁇ protein solution.
  • the experimental group contained 10 ⁇ M and 20 ⁇ M sDSS1 protein in addition to 20 ⁇ M A ⁇ protein.
  • a ⁇ was diluted with serum-free DMEM according to the N2a cell experiment and the same sDSS1 protein gradient was set. Neurons were processed for 48 hours. The supernatant of the cells in the 96-well plate was collected, centrifuged at 100 g for 10 minutes, and the supernatant was used for cytotoxicity detection; the cells were used for cell viability assay.
  • the lactate dehydrogenase cytotoxicity test kit was purchased from Biyuntian Biotechnology Co., Ltd. (C0016). When detecting the enzyme activity, first configure a sufficient amount of the working fluid according to the kit instructions. The supernatant collected in a 120 ⁇ L cytotoxicity experiment was added to each well of a 96-well plate, then 60 ⁇ L of the test working solution was added, mixed slightly, and incubated at 37 ° C for 30 minutes. The absorbance at 490 nm was measured on a microplate reader, and the absorbance was directly proportional to the cytotoxicity level.
  • the cell proliferation/cytotoxicity assay kit (CCK-8) was purchased from Dongren Chemical Technology (Shanghai) Co., Ltd. (CK04). The working solution was prepared by diluting the CCK-8 solution with 1:20 (volume ratio, v/v) in serum-free DMEM. After completion of the cell treatment in the 96 wells, the old medium was discarded, and 100 ⁇ l of CCK-8 working solution was added to each well. The plate was incubated in an incubator for 1-2 hours, and then the 450 nm absorbance value was measured on a multi-function microplate reader to reflect the cell viability.
  • sDSS1 protein can significantly block the decrease of cell viability caused by A ⁇ 1-42 oligomer, and 20 ⁇ M sDSS1 protein can protect cells from A ⁇ 1-42 oligomer (Fig. 4B). ).
  • the cytotoxicity level was measured by the LDH kit, and the results showed that the cytotoxicity level of the sDSS1 protein group was significantly decreased (Fig. 4C).
  • the sDSS1 protein On mouse primary neuronal cells, the sDSS1 protein also showed a shielding effect on the toxicity of A ⁇ oligomers, the cell viability of neurons was protected by sDSS1 protein (Fig. 5A), and the level of cytotoxicity was decreased (Fig. 5B).
  • Example 5 The sDSS1 protein promotes microglia clearance of A[beta] protein and reduces microglial activation.
  • BV2 Cell Culture Mouse microglia
  • DMEM medium containing 10% FBS
  • a ⁇ clearance assay BV2 cells were seeded into 6-well plates at 3x10 5 per well and cells were adhered for 12 hours.
  • the A ⁇ protein was previously diluted with DMEM complete medium to a 1 ⁇ M solution.
  • the negative control group was not added with protein, and the positive control group was added with 1 ⁇ M A ⁇ protein solution.
  • the experimental group contained 0.5 ⁇ M and 2 ⁇ M sDSS1 protein except 1 ⁇ M A ⁇ protein. After 24 hours of treatment, 500 ⁇ L of the supernatant was collected, centrifuged at 100 g for 10 minutes, and the supernatant was taken for ELISA assay to detect A ⁇ 1-42 levels.
  • BV2 cells were seeded per well in accordance 1.5x10 5 to 6 well plates, DMEM medium was changed to serum-free 12 hours, cells were starved for 24 hours.
  • the A ⁇ 1-42 oligomer was diluted to a 20 ⁇ M solution in serum-free DMEM.
  • the negative control group was not added with protein, and the positive control group was added with 20 ⁇ M A ⁇ protein solution.
  • the experimental group contained 10 ⁇ M and 20 ⁇ M sDSS1 protein except 20 ⁇ M A ⁇ protein.
  • the supernatant was collected, centrifuged at 100 g for 10 minutes, and the supernatant was taken for ELISA assay to detect the TNF ⁇ level of the solution. All cells were collected for ROS level detection.
  • ELISA assay The culture solution obtained in the above A ⁇ scavenging experiment was used in an ELISA experiment, and the culture solution sample was diluted 50 times with a standard diluent buffer provided by the kit for subsequent detection. According to the instructions of ELISA kit (Invitrogen, Human A ⁇ 42 ELISA kit, KHB3441), the standard protein and the nematode protein to be tested were sequentially incubated and washed, and the chromogenic substrate was added for 20 minutes. The stop solution was added and detected on the microplate reader. 450 nm absorbance value. The A ⁇ protein concentration in the protein sample was calculated from the standard curve.
  • ROS level detection Active oxygen detection kit was purchased from Biyuntian Biotechnology Co., Ltd. (S0033). Positive control cells were pretreated with 1 ⁇ M Rosup for 20 minutes at 37 °C. 300 ⁇ L of the cell suspension was taken, centrifuged at 100 g for 5 minutes, and the cells were washed twice with PBS. 300 ⁇ L of serum-free DMEM-diluted fluorescent probe (DCFDA) (1:1000) was added and incubated at 37 ° C for 20 minutes. After centrifugation at 100 g for 5 minutes, the cells were washed three times with PBS and detected by flow cytometry (Millipore, guava Easycyte). Green fluorescence (488 nm) intensity reflects cellular ROS levels.
  • DCFDA serum-free DMEM-diluted fluorescent probe
  • TNF ⁇ level The supernatant collected in the BV2 cell activation experiment was diluted 10-fold with Standard Dilute buffer provided in the kit for ELISA detection. According to the instructions of ELISA kit (Invitrogen, TNF alpha Mouse ELISA Kit, KMC3011C), the standard protein and the nematode protein to be tested were sequentially incubated and washed, incubated with chromogenic substrate for 20 minutes, and the stop solution was added to the microplate reader. The 450 nm absorbance value was measured. The concentration of TNF ⁇ in the supernatant was calculated from the standard curve.
  • Microglia are the main cells in nerve tissue that remove toxic proteins such as A ⁇ protein.
  • 1 ⁇ M A ⁇ 1-42 protein and different concentrations of sDSS1 protein were added to cultured mouse microglia.
  • the results showed that A ⁇ protein was detected in the cytoplasm of microglia, and the concentration of cytosolic A ⁇ protein was significantly increased with the addition of sDSS1 protein (Fig. 6A).
  • the A ⁇ protein residue in the culture solution decreased, and the A ⁇ protein concentration became lower and lower as the concentration of sDSS1 protein in the solution increased (Fig. 6B).
  • sDSS1 protein in the culture medium can significantly reduce the number of activated microglia, and the cell ROS level is significantly reduced (Fig. 6C).
  • the release of the inflammatory factor TNF- ⁇ gradually decreased as the concentration of sDSS1 protein increased (Fig. 6D).
  • Example 6 The sDSS1 protein reduces disease symptoms in the AD model nematode.
  • AD model nematode detection The nematode experimental platform was provided by Shanghai Southern Model Biotechnology Co., Ltd., and the AD nematode model was provided by the nematode experimental platform.
  • the adult CL4176 nematode was picked into a solid medium and placed in a 16 ° C incubator. After 2 hours of culture, all the adults were picked and the eggs were placed in a 16 ° C incubator. After 48 hours, the culture dishes were transferred to a 25 ° C incubator for further culture and treated with different concentrations of sDSS1 protein, 0 ⁇ g/ml in the control group, 500 ⁇ g/ml in the low dose group, and 1000 ⁇ g/ml in the high dose group.
  • the standard for nematodes is that when the wire is lightly nematode, the nematode is stiff and incapable of moving.
  • elegans lysate containing 62 mM Tris-HCl, 2% SDS, 10% glycerol, 4% ⁇ -mercaptoethanol, cocktail protease inhibition) Agent
  • liquid nitrogen freeze-thaw 2 times 60Hz ultrasound for 5 minutes, centrifugation at 12000rpm for 10 minutes, take the supernatant is the total protein of nematodes.
  • the nematode protein was added to a loading buffer and heat treated at 100 ° C for 10 minutes to prepare a protein sample for Wetern blotting analysis.
  • the completed membrane was first blocked with 5% skim milk powder (dissolved in PBST) for 1 hour at room temperature, then sequentially incubated with rabbit-derived A ⁇ antibody dilution at 4 ° C overnight (1:3000 dilution in blocking solution), washed three times with PBST, HRP coupling The conjugated anti-rabbit antibody dilution was incubated for 1 hour at room temperature (1:5000 dilution into PBST). After the ECL color is developed, the tablet is compressed and photographed and saved.
  • the internal reference used tubulin protein (antibody was purchased from Biyuntian Biotechnology Co., Ltd., AF0001).
  • the total nematode protein extracted from the above Western blotting experiment was used in the ELISA experiment, and the protein sample was diluted three times with the standard diluent buffer provided by the kit for subsequent detection, and the protein dilution was added. The volume was adjusted according to the data of the internal reference in the Western blotting experiment to ensure that the total amount of protein added was consistent. According to the instructions of the ELISA kit, the standard protein and the nematode protein to be tested were sequentially incubated and washed, incubated with a chromogenic substrate for 20 minutes, and the stop solution was added to measure the absorbance at 450 nm on a microplate reader. The A ⁇ protein concentration in the protein sample was calculated from the standard curve.
  • C. elegans immunohistochemical staining CL4176 nematodes were treated in different concentrations of drugs for 20 hours, and after completion, nematodes were collected, with 50-100 nematodes in each group. The nematodes were washed once with PBS and placed in a 1.5 mL centrifuge tube, and the supernatant was discarded, and 4% paraformaldehyde (dissolved in PBS, pH 7.2) was added at 4 ° C overnight. The fixed nematode was first rinsed once with wash buffer (containing 10 mm Tris/HCl, 1% Triton-X100, 1 mM EDTA, pH 7.4).
  • wash buffer containing 10 mm Tris/HCl, 1% Triton-X100, 1 mM EDTA, pH 7.4
  • the nematodes were sequentially incubated with a washing solution containing 1% ⁇ -mercaptoethanol for 1 hour at room temperature, boric acid buffer (containing 25 mM H 3 BO 3 , 12.5 mM NaOH, 10 mM DTT, pH 9.2) was incubated for 1 minute at room temperature for 5 minutes. 1% H 2 O 2 wash buffer was incubated for 15 minutes at room temperature. After completion, the nematodes were washed once with wash buffer and blocked by adding blocking solution (containing 1% BSA, 0.5% Triton-X100, 1 mM EDTA, PBS) for two hours at room temperature.
  • blocking solution containing 1% BSA, 0.5% Triton-X100, 1 mM EDTA, PBS
  • the treated nematodes were sequentially incubated overnight at 4 °C with a primary antibody dilution (A ⁇ antibody, diluted 1:100 in blocking solution), and the secondary antibody dilution (added Hoechst at a ratio of 1:1000) (Donkey-anti-Rabbit-Alex Fluor) 488 (purchased from Life Technology, Inc., A-11008) was incubated for 1 hour at room temperature with a dilution of 1:5000 in PBST. The PBST was washed three times, and after completion, it was mounted and observed with a Confocol laser confocal microscope (Nikon, Nikon C1Plus), and photographed.
  • CL4176 nematode was used as an animal model for AD to detect toxicity caused by accumulation of acute A ⁇ 1-42 protein and was used for drug evaluation.
  • the addition of different concentrations of sDSS1 protein significantly reduced the nematode sputum level, and the effect showed a significant concentration dependence (Fig. 7A).
  • the proportion of the non-nematode in the control group was less than 1%, while the nematodes with 18.42% (500 ⁇ g/mL sDSS1 protein) and 48.57% (1000 ⁇ g/mL sDSS1 protein) in the administration group were normal.
  • the median time of occurrence of sputum in half of the nematodes was 22 hours in the control group, 24 hours in the low concentration group, and 42 hours in the high concentration group.
  • sDSS1 protein reduced the content of A ⁇ protein multimer (Fig. 7C).
  • the A ⁇ 1-42 protein content in the tissue lysate was detected by ELISA. It can be seen that the A ⁇ protein in the sDSS1 protein-treated nematode was decreased, and the A ⁇ protein content in the 1000 ⁇ g/mL sDSS1 protein treatment group was only 59.09% of the control group (Fig. 7D). .
  • sDSS1 protein can reduce the formation of A ⁇ 1-42 aggregates, reduce the accumulation of tissue A ⁇ , and alleviate the disease symptoms of AD model nematodes in the AD nematode model.
  • Example 7 The sDSS1 protein accelerates clearance of A ⁇ protein in mice.
  • APP/PS1 mouse blood A ⁇ clearance test APP/PS1 mice (2 months old, male) were purchased from the Model Animal Center of Nanjing University. The APP/PS1 mice were injected with sDSS1 protein at a dose of 25 mg/kg body weight for 8 weeks from the age of 8 months. After completion, the mice were sacrificed, blood was taken and centrifuged to obtain plasma for ELISA to detect A ⁇ 1-42 levels in the blood.
  • a ⁇ protein acute clearance test ICR mice (6-8 weeks old, male) were purchased from Shanghai Slack Laboratory Animals Co., Ltd. The mice were divided into four groups according to their body weight, including a negative control group (injected with normal saline), a positive control group (administered with A ⁇ protein alone), a low-dose group (A ⁇ -administered and 5 mg/kg body weight injected with sDSS1 protein), and high dose.
  • the administration group (A ⁇ administration and 30 mg/kg body weight injection of sDSS1 protein).
  • the mice were first injected with sDSS1 protein according to the tail vein of the group.
  • the negative control group and the positive control group were injected with the same amount of PBS.
  • the positive control group and the drug-administered group were injected with A ⁇ protein at the tail vein of 10 mg/kg body weight.
  • the control group was injected with the same amount of normal saline. After the injection was completed, the mice were normally reared, and the mice were sacrificed 2 hours later. Blood was taken from the heart, and the cells were centrifuged at 3,500 g for 20 minutes to obtain plasma for ELISA to detect A? 1-42 levels.
  • ELISA test Plasma samples obtained in the above experiment were used for the ELISA experiment. According to the instructions of the ELISA kit, the standard protein and the plasma sample to be tested are sequentially incubated and washed, and the chromogenic substrate is added for 20 minutes, the stop solution is added, and the 450 nm absorbance value is detected on the microplate reader. The A ⁇ protein concentration in plasma was calculated from the standard curve.
  • sDSS1 protein can help clear A ⁇ protein in the blood of mice.
  • high concentrations of A ⁇ 1-42 protein were injected through the tail vein while the sDSS1 protein was injected.
  • the results of the experiment showed that different concentrations of sDSS1 protein administration significantly reduced blood A ⁇ protein levels, and the effect was dose-dependent (Fig. 8A).
  • AD model APP/PS1 mice were injected with sDSS1 protein for a long time in the tail vein, and it was found that sDSS1 protein also reduced blood A ⁇ protein levels (Fig. 8B).
  • Example 8 sDSS1 protein reduces plaque deposition in brain tissue of AD model mice.
  • AD model mice administration APP/PS1 mice (2 months old, male) were purchased from the Model Animal Center of Nanjing University. The APP/PS1 mice were injected with sDSS1 protein at a dose of 50 mg/kg body weight for 8 weeks from the age of 9 months. After completion, the mice were used for behavioral experiments. After the behavioral experiment was completed, the mice were sacrificed, and plasma was taken for blood index detection. Brain tissue was taken and fixed in 4% paraformaldehyde for immunohistochemical staining to analyze plaque deposition levels.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Genetics & Genomics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Immunology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Epidemiology (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Vascular Medicine (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Urology & Nephrology (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Hospice & Palliative Care (AREA)
  • Psychiatry (AREA)
  • Toxicology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

Utilisation d'une protéine dans la préparation d'un médicament pour la prévention et le traitement de la maladie d'Alzheimer, ladite protéine étant une protéine sDSS1 utilisée dans la préparation d'un médicament pour la prévention de maladies neurodégénératives et démences vasculaires ou d'un médicament pour le traitement desdites maladies au stade précoce, initial, intermédiaire, en phase avancée ou terminale. La protéine sDSS1 interagit avec une protéine bêta-amyloïde (A bêta) de manière à supprimer A bêta tout en assurant la protection contre la toxicité A bêta. Par ailleurs, le dépôt de A bêta est efficacement réduit chez les animaux et la concentration de A bêta soluble s'en trouve réduite aussi, ce qui se traduit par un soulagement des symptômes de la maladie.
PCT/CN2018/120172 2018-01-02 2018-12-11 Utilisation d'une protéine dans la préparation d'un médicament pour la prévention et le traitement de la maladie d'alzheimer WO2019134481A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810002892.7A CN109985231A (zh) 2018-01-02 2018-01-02 一种蛋白在制备预防和治疗痴呆症的药物中的应用
CN201810002892.7 2018-01-02

Publications (1)

Publication Number Publication Date
WO2019134481A1 true WO2019134481A1 (fr) 2019-07-11

Family

ID=67128294

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/120172 WO2019134481A1 (fr) 2018-01-02 2018-12-11 Utilisation d'une protéine dans la préparation d'un médicament pour la prévention et le traitement de la maladie d'alzheimer

Country Status (2)

Country Link
CN (1) CN109985231A (fr)
WO (1) WO2019134481A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AT506819B1 (de) * 2008-06-12 2011-06-15 Affiris Forschungs Und Entwicklungs Gmbh Vakzin zur behandlung von alzheimer-krankheit
CN102895659B (zh) * 2011-07-29 2014-10-29 复旦大学 一种防治老年痴呆症复合疫苗及其制备方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GONG, CHENGXIN: "DSSylation, a Novel Guide for Protein Degradation", PROTEIN & CELL, vol. 5, no. 2, 31 December 2014 (2014-12-31), pages 91 - 93, XP035734440, DOI: doi:10.1007/s13238-014-0020-9 *
ZHANG, YINGHAO ET AL.: "DSSylation, a Novel Protein Modification Targets Proteins Induced by Oxidative Stress, and Facilitates Their Degradation in Cells", PROTEIN & CELL, vol. 5, no. 2, 31 December 2014 (2014-12-31), pages 124 - 140, XP035734439, DOI: doi:10.1007/s13238-013-0018-8 *

Also Published As

Publication number Publication date
CN109985231A (zh) 2019-07-09

Similar Documents

Publication Publication Date Title
US20180318383A1 (en) Peptide having neuronal loss prevention and regeneration effects, and composition containing same
AU2016271635B2 (en) Compositions and methods for degradation of misfolded proteins
Schlenzig et al. N‐Terminal pyroglutamate formation of Aβ38 and Aβ40 enforces oligomer formation and potency to disrupt hippocampal long‐term potentiation
JP6751097B2 (ja) 聴力損傷予防用ペプチド及びそれを含む組成物
JP6843625B2 (ja) 様々な疾患の処置のためのｊｎｋ阻害剤分子の新規使用
Wang et al. Neuroinflammation in Alzheimer’s disease: microglia, molecular participants and therapeutic choices
JP2001501972A (ja) アミロイドベータタンパク質（球状アッセンブリーおよびその使用）
CN113855804A (zh) 用于治疗衰老相关病症的方法和组合物
JP7384672B2 (ja) Ｃ末端ｃｄｎｆ断片及びｃ末端ｍａｎｆ断片、それらを含む医薬組成物、並びにそれらの使用
JP2022514088A (ja) ハイブリッドプロモーターおよび療法における使用、特にｉｉ型コラーゲン異常症を治療するための使用
CN110302362B (zh) 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
US20230365626A1 (en) Alloferon Peptide and Method Using the Same
WO2019134481A1 (fr) Utilisation d'une protéine dans la préparation d'un médicament pour la prévention et le traitement de la maladie d'alzheimer
JP5219168B2 (ja) アルツハイマー病の予防および／または治療用βシート破壊ペプチド
EP2694977B1 (fr) Cible d'amyloïdose utile dans des méthodes pour le criblage de composés
JP6397122B2 (ja) 血管新生関連疾患を治療するためのペプチドの使用
WO2015077578A1 (fr) Compositions et procédés d'administration de protéines bioencapsulées à travers les barrières hémato-encéphalique et rétinienne
Lantz et al. The neuroprotective N-terminal amyloid-β core hexapeptide reverses reactive gliosis and gliotoxicity in Alzheimer’s disease pathology models
KR101131512B1 (ko) 퇴행성신경질환 예방 또는 치료용 약제학적 조성물
US20200230205A1 (en) Compositions and methods for treating myelin disorders
WO2023246847A1 (fr) Peptide bloquant b2m-glun1, composition pharmaceutique et son utilisation
WO2023143610A2 (fr) FRAGMENT C-TERMINAL D'APOLIPOPROTÉINE E ET SON APPLICATION DANS L'INHIBITION DE L'ACTIVITÉ DE LA γ-SÉCRÉTASE
JP2014144933A (ja) アミロイドβの除去
CN117229384A (zh) 新型生长激素释放激素类似肽单体、二聚体或四聚体及其制备和应用
Sansa Zaragoza et al. Calpain Inhibition Increases SMN Protein in Spinal Cord Motoneurons and Ameliorates the Spinal Muscular Atrophy Phenotype in Mice

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18897999

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18897999

Country of ref document: EP

Kind code of ref document: A1