WO2020168850A1 - Ube3a泛素化PP2A激活因子PTPA在治疗天使综合症和孤独症中的应用 - Google Patents
Ube3a泛素化PP2A激活因子PTPA在治疗天使综合症和孤独症中的应用 Download PDFInfo
- Publication number
- WO2020168850A1 WO2020168850A1 PCT/CN2020/070997 CN2020070997W WO2020168850A1 WO 2020168850 A1 WO2020168850 A1 WO 2020168850A1 CN 2020070997 W CN2020070997 W CN 2020070997W WO 2020168850 A1 WO2020168850 A1 WO 2020168850A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- protein
- gene
- ube3a
- pp2a
- phosphatase
- Prior art date
Links
- 0 CN(CC1)CCN1C(c1c(cc2)[o]c2c1C(*)=O)=O Chemical compound CN(CC1)CCN1C(c1c(cc2)[o]c2c1C(*)=O)=O 0.000 description 1
- SUDAHWBOROXANE-SECBINFHSA-N OC[C@H](CONC(c(c(Nc(c(F)c1)ccc1I)c1F)ccc1F)=O)O Chemical compound OC[C@H](CONC(c(c(Nc(c(F)c1)ccc1I)c1F)ccc1F)=O)O SUDAHWBOROXANE-SECBINFHSA-N 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- 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
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
-
- 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
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/118—Prognosis of disease development
Definitions
- the present invention belongs to the field of molecular biology and pharmacology. More specifically, the present invention relates to Ube3a regulating the activity of PP2A and its application in the treatment of angel syndrome and autism.
- UBE3A gene is located in the 15q11-13 region of human chromosome 15, and the encoded product Ube3a is the first E3 ubiquitin ligase found in the HECT (Homologous with E6-assiciated protein C-Terminus) family. Because there is an E6 binding domain in its protein structure, it is also called E6AP. So far, Ube3a has two functions: one is to act as an E3 ligase, which mediates the ubiquitination modification of specific substrates it recognizes, and then regulates protein degradation, transportation, and localization; the other is in the nucleus, Ube3a can It binds to hormone receptors and acts as an activator together to regulate gene transcription.
- Ube3a specifically expresses maternal genes, and the paternal gene produces a reverse RNA, which makes it not expressed or expressed very little.
- the lack of Ube3a protein ubiquitin ligase function is the most important factor in the pathogenesis of Angel syndrome (AS). Angel syndrome was first discovered and reported by pediatrician Harry Angelman in 1965. It is characterized by developmental delay, language impairment, mental retardation, and movement impairment.
- Autism spectrum disorder also known as autism spectrum disorder, is a type of neurodevelopmental disease with heterogeneous pathogenesis.
- the main characteristics of the disease are: social barriers, communication barriers, behavior repetition and stereotyped behavior, narrow range of interests, and slow language development. Children with autism usually develop before the age of 3, and the prevalence of men is four times that of women. Although the pathogenesis is not yet clear, 25% of children are accompanied by new mutations in genes or abnormal chromosome copy number.
- UBE3A is the only gene that expresses only maternal alleles. Not only that, but in the mouse model, Ube3a mice with three copies showed typical autistic characteristics. The above studies prove that UBE3A is a susceptibility gene for autism. Therefore, too much or too little protein level of Ube3a leads to abnormal development and function of the nervous system, and studying the pathogenic mechanism of Ube3a is of great significance for the treatment of neurodevelopmental diseases caused by Ube3a.
- the art also needs to find a direct substrate of Ube3a to seek more effective interventions to treat neurodevelopmental diseases caused by Ube3a.
- the purpose of the present invention is to provide Ube3a regulating the activity of PP2A and its application in the treatment of angel syndrome and/or autism-related diseases.
- an inhibitor of protein phosphatase 2A (PP2A) or phosphotyrosine phosphatase activating factor (PTPA) is provided for preparing a pharmaceutical composition for relieving or treating angel syndrome.
- the pharmaceutical composition is also used to prevent and/or treat Angel Syndrome.
- the pharmaceutical composition is also used to correct the increase of PP2A activity in mammals with Angel Syndrome.
- the mammal includes a human or non-human mammal.
- the mammal includes rodents (such as mice, rats, rabbits), and primates (such as monkeys).
- rodents such as mice, rats, rabbits
- primates such as monkeys
- the inhibitor is selected from: small molecule compounds that specifically inhibit protein phosphatase 2A or phosphotyrosine phosphatase activation factor; specifically interfere with activation of protein phosphatase 2A or phosphotyrosine phosphatase Interfering molecules for gene expression of factors; gene editing reagents that specifically knock out protein phosphatase 2A or phosphotyrosine phosphatase activator; or antibodies that specifically bind to protein phosphatase 2A or phosphotyrosine phosphatase activator Or ligand.
- the inhibitor is a small molecule compound that specifically inhibits the activity of protein phosphatase 2A, which is selected from: inhibitor LB-100.
- the inhibitor is an agent that specifically upregulates Ube3a protein (including promoting its expression or increasing its activity), which degrades phosphotyrosine phosphatase activator by upregulating Ube3a protein, thereby inhibiting the protein Phosphatase 2A.
- the reagent that specifically up-regulates Ube3a protein is an expression construct (expression plasmid) that overexpresses Ube3a.
- the inhibitor further includes Ube3a protein or a carrier expressing Ube3a protein.
- the vector for expressing Ube3a protein includes a viral vector.
- the viral vector is selected from the following group: adeno-associated virus vector, lentiviral vector, or a combination thereof.
- the protein includes a full-length protein or protein fragment.
- the Ube3a protein is derived from mammals, more preferably from rodents (such as mice, rats), primates and humans.
- the Ube3a protein also includes derivatives of Ube3a protein.
- the derivative of the Ube3a protein includes a modified Ube3a protein, a protein molecule whose amino acid sequence is homologous to the natural Ube3a protein and has the activity of the natural Ube3a protein, a dimer or multimer of the Ube3a protein, A fusion protein containing the amino acid sequence of Ube3a protein.
- the modified Ube3a protein is a PEGylated Ube3a protein.
- the "protein molecule whose amino acid sequence is homologous to the natural Ube3a protein and has natural Ube3a protein activity” means that its amino acid sequence has ⁇ 85% homology with the Ube3a protein, preferably ⁇ 90% homology, more preferably ⁇ 95% homology, and best ⁇ 98% homology; protein molecules with natural Ube3a protein activity.
- the Ube3a protein is selected from the following group:
- (C) Compared with the amino acid sequence shown in SEQ ID NO.: 18, the sequence has homology of ⁇ 90%, preferably ⁇ 95%, more preferably ⁇ 98%, and most preferably ⁇ 99% derived from Ube3a protein ⁇ , or its active fragment.
- the interference molecule that specifically interferes with the gene expression of phosphotyrosine phosphatase activator is shRNA.
- the shRNA is targeted to: CAGTTCCAGATATGGGCAAATGGAA (SEQ ID NO: 1), CCTGGTATGCC AAACTTGATCAGGA (SEQ ID NO: 2) or TCAAGGTTGTTTGATAGGTATCTT GA (SEQ ID NO: 3); preferably, the The shRNA has the sense strand of SEQ ID NO: 4 and the antisense strand of SEQ ID NO: 5, or the sense strand of SEQ ID NO: 6 and the antisense strand of SEQ ID NO: 7; or it has SEQ ID NO: 8 The sense strand and the antisense strand of SEQ ID NO: 9.
- the physiological function and mechanism of protein phosphatase 2A or phosphotyrosine phosphatase activating factor in cells are provided for preparing pharmaceutical compositions for the prevention and/or treatment of autism or related diseases.
- the use of protein phosphatase 2A or phosphotyrosine phosphatase activating factor is provided for screening drugs for relieving or treating angel syndrome.
- a method for screening potential substances for relieving or treating Angel’s syndrome comprising: (1) Treating expressed protein phosphatase 2A and/or phosphotyrosine phosphatase with candidate substances Activating factor system; and (2) detecting the expression or activity of protein phosphatase 2A and/or phosphotyrosine phosphatase activating factor in the system; wherein, if the candidate substance can reduce protein phosphatase 2A and/or The expression or activity of phosphotyrosine phosphatase activating factor indicates that the candidate substance is a potential substance for relieving or treating Angel's syndrome.
- step (1) includes: in the test group, adding the candidate substance to the system expressing protein phosphatase 2A and/or phosphotyrosine phosphatase activator;
- step (2) includes: detection test The expression or activity of protein phosphatase 2A and/or phosphotyrosine phosphatase activator in the system of the group, and compared with the control group, wherein the control group is the expression protein phosphatase 2A and/or phosphotyrosine phosphatase 2A and / Or phosphotyrosine phosphatase activating factor system; if the expression or activity of protein phosphatase 2A and/or phosphotyrosine phosphatase activating factor in the test group is statistically lower than that of the control group, it indicates the candidate It is a potential substance for relieving or curing angel syndrome.
- the said statistically lower preferably significantly lower, such as lower than 20%, preferably lower than 50%; more preferably lower than 80%.
- the Ube3a protein is also expressed in the system, and the method further comprises: detecting the expression of Ube3a protein in the system; wherein, if the candidate substance can be inhibited by increasing the expression of Ube3a (preferably Significant inhibition, such as inhibition of more than 20%, preferably more than 50%; more preferably more than 80%) Protein phosphatase 2A and/or phosphotyrosine phosphatase activator, indicating that the candidate substance is a relief or treatment The underlying substance of Angel Syndrome.
- Ube3a preferably Significant inhibition, such as inhibition of more than 20%, preferably more than 50%; more preferably more than 80%
- Protein phosphatase 2A and/or phosphotyrosine phosphatase activator indicating that the candidate substance is a relief or treatment The underlying substance of Angel Syndrome.
- the system is selected from: cell systems (such as cells or cell cultures expressing protein phosphatase 2A, phosphotyrosine phosphatase activator and/or Ube3a), subcellular systems, and solution systems , Tissue system, organ system or animal system.
- cell systems such as cells or cell cultures expressing protein phosphatase 2A, phosphotyrosine phosphatase activator and/or Ube3a
- subcellular systems such as cells or cell cultures expressing protein phosphatase 2A, phosphotyrosine phosphatase activator and/or Ube3a
- solution systems such as Tissue system, organ system or animal system.
- the candidate substance includes (but is not limited to): small molecule compounds designed for protein phosphatase 2A gene or protein, interference molecules, nucleic acid inhibitors, binding molecules (such as antibodies or ligands), etc. Or a regulatory molecule designed for the signaling pathway involved in protein phosphatase 2A gene or protein or its upstream or downstream proteins (such as Ube3a, phosphotyrosine phosphatase activator).
- the method further includes: performing further cell experiments and/or animal experiments on the obtained potential substances to further select and determine substances useful for alleviating or treating Angel Syndrome from the candidate substances.
- the use of a reagent that specifically recognizes protein phosphatase 2A gene or protein is provided for preparing reagents or kits for diagnosis or prognosis assessment of Angel syndrome.
- the reagent that specifically recognizes protein phosphatase 2A gene or protein is selected from: primers that specifically amplify protein phosphatase 2A gene; probes that specifically recognize protein phosphatase 2A gene; or specific Sex-binding protein phosphatase 2A antibody or ligand.
- the use of a reagent that specifically recognizes phosphotyrosine phosphatase activator gene or protein is provided for preparing reagents or kits for diagnosis or prognosis evaluation of Angel syndrome.
- the reagent that specifically recognizes phosphotyrosine phosphatase activator gene or protein is selected from: primers that specifically amplify phosphotyrosine phosphatase activator gene; specifically recognize phosphotyrosine An acid phosphatase activator gene probe; or an antibody or ligand that specifically binds to phosphotyrosine phosphatase activator.
- a substance for preparing a composition or preparation for preventing and/or treating autism or its related diseases wherein the substance is selected from the following group: (a) Ube3a gene Or its protein inhibitor, (b) PP2A gene, its protein or its promoter, (c) PTPA gene, its protein or its promoter, or its combination.
- the substance further includes (d) a promoter of PP2A holoenzyme assembly.
- the PP2A protein also includes active fragments of PP2A protein.
- the autism or related diseases are selected from the group consisting of autism, 15q11-13 replication syndrome, or a combination thereof.
- composition or preparation is also used for one or more purposes selected from the following group:
- the inhibitor of the Ube3a gene or its protein is selected from the group consisting of antibodies, small molecule compounds, microRNA, siRNA, shRNA, or a combination thereof.
- the PP2A promoter or PTPA promoter refers to a substance that can increase the activity and/or content of the PP2A or PTPA gene or its protein in vivo or in vitro; the substance can be synthetic or natural Compounds, proteins, nucleotides, etc.
- the PP2A or PTPA promoter includes a substance that promotes the expression of PP2A or PTPA.
- the PP2A promoter includes a PP2A protein promoter and/or a PP2A gene promoter.
- the PTPA promoter includes a PTPA protein promoter and/or a PTPA gene promoter.
- the promotion of PP2A or PTPA expression or activity refers to increasing the expression or activity of PP2A or PTPA gene or protein by ⁇ 10%, preferably, ⁇ 20%, more preferably, ⁇ 70%.
- the PP2A promoter is selected from the following group: small molecule compounds, vectors for expressing PP2A, or combinations thereof.
- the PP2A accelerator is selected from the group consisting of FTY720, C2ceramide, or a combination thereof.
- the vector expressing PP2A includes a viral vector.
- the PTPA promoter is selected from the group consisting of small molecule compounds, PTPA-expressing vectors, or combinations thereof.
- the vector expressing PTPA includes a viral vector.
- the viral vector is selected from the following group: adeno-associated virus vector, lentiviral vector, or a combination thereof.
- the protein includes a full-length protein or protein fragment.
- the PP2A gene or its protein is derived from mammals, more preferably from rodents (such as mice, rats), primates and humans.
- the PTPA gene or its protein is derived from mammals, more preferably from rodents (such as mice, rats), primates and humans.
- the PP2A protein also includes derivatives of PP2A protein.
- the PP2A protein derivative includes a modified PP2A protein, a protein molecule whose amino acid sequence is homologous to the natural PP2A protein and has natural PP2A protein activity, a dimer or multimer of PP2A protein, A fusion protein containing the amino acid sequence of the PP2A protein.
- the modified PP2A protein is a PEGylated PP2A protein.
- the "protein molecule whose amino acid sequence is homologous to the natural PP2A protein and has natural PP2A protein activity” means that its amino acid sequence has ⁇ 85% homology with the PP2A protein, preferably ⁇ 90% homology, more preferably ⁇ 95% homology, and best ⁇ 98% homology; and protein molecules with natural PP2A protein activity.
- the PP2A protein is selected from the following group:
- (C) Compared with the amino acid sequence shown in SEQ ID NO.:19, the sequence has a homology of ⁇ 90%, preferably ⁇ 95%, more preferably ⁇ 98%, and optimally ⁇ 99% derived from PP2A protein ⁇ , or its active fragment.
- the PP2A gene encodes PP2A protein.
- the PP2A gene is selected from the following group:
- the nucleotide sequence has a homology of ⁇ 95% (preferably ⁇ 98%) with the sequence shown in SEQ ID NO.: 20, and a polynucleotide encoding the polypeptide shown in SEQ ID NO.: 19;
- the PTPA protein also includes a derivative of PTPA protein.
- the derivative of the PTPA protein includes a modified PTPA protein, a protein molecule whose amino acid sequence is homologous to the natural PTPA protein and has the activity of the natural PTPA protein, a dimer or multimer of the PTPA protein, A fusion protein containing the amino acid sequence of the PTPA protein.
- the modified PTPA protein is a PEGylated PTPA protein.
- the "protein molecule whose amino acid sequence is homologous to the natural PTPA protein and has natural PTPA protein activity” means that its amino acid sequence has ⁇ 85% homology with the PTPA protein, preferably ⁇ 90% homology, more preferably ⁇ 95% homology, and best ⁇ 98% homology; protein molecules with natural PTPA protein activity.
- the PTPA protein is selected from the following group:
- (C) Compared with the amino acid sequence shown in SEQ ID NO.: 21, the sequence has a homology of ⁇ 90%, preferably ⁇ 95%, more preferably ⁇ 98%, and optimally ⁇ 99% derived from PTPA protein ⁇ , or its active fragment.
- the PTPA gene encodes a PTPA protein.
- the PTPA gene is selected from the following group:
- the nucleotide sequence has a homology of ⁇ 95% (preferably ⁇ 98%) with the sequence shown in SEQ ID NO.: 22, and a polynucleotide encoding the polypeptide shown in SEQ ID NO.: 21;
- the composition includes a pharmaceutical composition.
- the pharmaceutical composition contains (i) an inhibitor of Ube3a gene or its protein; PP2A gene, or its protein or its promoter; PTPA gene, its protein or its promoter; or a combination thereof ; And (ii) a pharmaceutically acceptable carrier.
- the pharmaceutical composition is liquid, solid, or semi-solid.
- the dosage form of the pharmaceutical composition includes tablets, granules, capsules, oral liquids, or injections.
- the component (i) accounts for 1-99wt% of the total weight of the pharmaceutical composition, preferably 10-90wt%, more preferably 30-70wt% %.
- the composition also includes additional components for preventing and/or treating autism or related diseases.
- the additional component for preventing and/or treating autism or its related diseases is selected from the group consisting of antagonist morphine drug-Nalterxone, sodium valproate, carbamazepine, Lithium carbonate, propranolol, clonazepam, chlorimipramine, imipramine, serotonin reuptake inhibitor (SSRS), buspirone, chlorpromazine, sulpiride, haloperidol, risperidone (Vestone), clozapine, olanzapine, methylphenidate (ritalin), pemoline, or a combination thereof.
- the serotonin reuptake inhibitor is selected from the group consisting of fluoxetine, sertraline, fluvoxamine (blue release), or a combination thereof.
- composition or preparation can be used alone or in combination in the prevention and/or treatment of autism or related diseases.
- the combined use includes: combined use with other drugs for preventing and/or treating autism or related diseases.
- the other drugs for preventing and/or treating autism or related diseases are selected from the group consisting of antagonistic morphine drug-Nalterxone, sodium valproate, carbamazepine, lithium carbonate , Propranolol, clonazepam, chlorimipramine, imipramine, serotonin reuptake inhibitor (SSRS), buspirone, chlorpromazine, sulpiride, haloperidol, risperidone (vitamin (Stone), clozapine, olanzapine, methylphenidate (ritalin), pemoline, or a combination thereof.
- the serotonin reuptake inhibitor is selected from the group consisting of fluoxetine, sertraline, fluvoxamine (blue release), or a combination thereof.
- a pharmaceutical composition comprising:
- a first active ingredient for the prevention and/or treatment of autism or its related diseases the first active ingredient being selected from the following group: (i) Ube3a gene or its protein inhibitor, (ii) PP2A gene , Or its protein or its promoter, (iii) PTPA gene, or its protein or its promoter, or a combination thereof; and
- the pharmaceutical composition further includes (a2) a second active ingredient for preventing and/or treating liver fibrosis-related diseases, and the second active ingredient includes: other preventive and/or therapeutic Drugs for autism or related diseases.
- the component (a1) accounts for 1-99wt% of the total weight of the pharmaceutical composition, preferably 10-90wt%, more preferably 30-70wt% %.
- the component (a2) accounts for 1-99wt% of the total weight of the pharmaceutical composition, preferably 10-90wt%, more preferably 30-70wt% %.
- the weight ratio of the first active ingredient to the second active ingredient is 1:100 to 100:1, preferably 1:10 to 10:1.
- the pharmaceutical composition further includes an additional component that inhibits the phosphorylation level of ERK1/2.
- the additional component that inhibits the phosphorylation level of ERK1/2 is selected from the group consisting of MK-8353, U0126, SCG772984, or a combination thereof.
- the pharmaceutical composition further includes an additional component that reduces the activity of the MAPK signaling pathway.
- the additional component that reduces the activity of the MAPK signaling pathway is selected from the following group: PD98059, Selumetinib, PD0325901, or a combination thereof.
- the other drugs for preventing and/or treating autism or related diseases are selected from the group consisting of antagonistic morphine drug-Nalterxone, sodium valproate, carbamazepine, lithium carbonate , Propranolol, clonazepam, chlorimipramine, imipramine, serotonin reuptake inhibitor (SSRS), buspirone, chlorpromazine, sulpiride, haloperidol, risperidone (vitamin (Stone), clozapine, olanzapine, methylphenidate (ritalin), pemoline, or a combination thereof.
- the serotonin reuptake inhibitor is selected from the group consisting of fluoxetine, sertraline, fluvoxamine (blue release), or a combination thereof.
- the pharmaceutical composition can be a single compound or a mixture of multiple compounds.
- the pharmaceutical composition is used to prepare drugs or preparations for treating or preventing autism or related diseases.
- the pharmaceutical dosage form is oral administration or non-oral administration dosage form.
- the oral administration dosage form is a tablet, powder, granule or capsule, or an emulsion or syrup.
- the non-oral administration dosage form is injection or injection.
- the total content of the active ingredient (a1) and the active ingredient (a2) is 1-99% by weight of the total weight of the composition, more preferably 5-90% by weight.
- a medicine kit including:
- the first container, and the active ingredient in the first container (a1) Ube3a gene or its protein inhibitor; PP2A gene, its protein or its promoter; PTPA gene, its protein or its promoter ; Or a combination thereof, or a drug containing the active ingredient (a1).
- the kit further includes:
- the second container, and the active ingredient in the second container (a2) other medicines for preventing and/or treating autism or related diseases, or medicines containing the active ingredient (a2).
- first container and the second container are the same or different containers.
- the medicine in the first container is a unilateral preparation containing the active ingredient (a1).
- the medicine in the second container is a unilateral preparation containing the active ingredient (a2).
- the dosage form of the drug is an oral dosage form or an injection dosage form.
- the kit also contains instructions, which describe the combined administration of the active ingredient (a1) and the active ingredient (a2) to (i) prevent and/or treat autism or its related diseases; And/or (ii) inhibit the phosphorylation level of ERK1/2; and/or (iii) reduce the activity of the MAPK signaling pathway.
- the dosage forms of the preparation containing the active ingredient (a1) or the preparation containing the active ingredient (a2) respectively include capsules, tablets, suppositories, or intravenous injections.
- the concentration of the inhibitor of the Ube3a gene or its protein is 0.00001-10 mg/kg body weight, preferably 0.5-5 mg/kg body weight, more Jiadi 0.5-2mg/kg body weight.
- the concentration of the PP2A gene, or its protein or its promoter is 0.00001-10 mg/kg body weight, preferably 0.5-5 mg/kg body weight , More preferably 0.5-2mg/kg body weight.
- the concentration of the PTPA gene, or its protein or its promoter is 0.00001-10 mg/kg body weight, preferably 0.5-5 mg/kg body weight , More preferably 0.5-2mg/kg body weight.
- a) inhibiting the phosphorylation level of ERK1/2; and/or (b) reducing the activity of the MAPK signaling pathway including the steps:
- the brain tissue cells are derived from the cortex of a mammal.
- the mammal includes a human or non-human mammal.
- the non-human mammals include rodents, primates, preferably, mice, rats, rabbits, and monkeys.
- the brain tissue cells are brain tissue cells (such as neurons) cultured in vitro.
- the method is non-diagnostic and non-therapeutic.
- the method is therapeutic.
- the concentration of the inhibitor of the Ube3a gene or its protein is 0.00001-10 mg/kg body weight, preferably 0.5-5 mg/kg body weight, more preferably 0.5-2 mg/kg body weight.
- the action concentration of the PP2A gene, its protein or its promoter is 0.00001-10 mg/kg body weight, preferably 0.5-5 mg/kg body weight, more preferably 0.5-2 mg/kg body weight.
- the concentration of the PTPA gene, or its protein or its promoter is 0.00001-10 mg/kg body weight, preferably 0.5-5 mg/kg Body weight, more preferably 0.5-2 mg/kg body weight.
- the tenth aspect of the present invention provides a method for screening potential therapeutic agents for autism or related diseases, including:
- the "significantly lower” means E1/E2 ⁇ 1/2, preferably, ⁇ 1/3, more preferably, ⁇ 1/4.
- the cells include brain tissue cells.
- the method is non-diagnostic and non-therapeutic.
- the method includes step (c): administering the potential therapeutic agent determined in step (a) to the mammal, so as to determine its effect on autism or related diseases in the mammal.
- the mammal includes a human or non-human mammal.
- the non-human mammals include rodents, primates, preferably, mice, rats, rabbits, and monkeys.
- the eleventh aspect of the present invention provides a method for screening potential therapeutic agents for autism or related diseases, including:
- the "significantly lower” means that E1/E2 ⁇ 2, preferably, ⁇ 3, more preferably, ⁇ 4.
- the cells include brain tissue cells.
- the cells are brain tissue cells cultured in vitro.
- the method is non-diagnostic and non-therapeutic.
- the method includes step (c): administering the potential therapeutic agent determined in step (a) to the mammal, so as to determine its effect on autism or related diseases in the mammal.
- the mammal includes a human or non-human mammal.
- the non-human mammals include rodents, primates, preferably, mice, rats, rabbits, and monkeys.
- the twelfth aspect of the present invention provides a method for preventing and/or treating autism or related diseases, including the steps:
- the administration includes oral administration.
- the subject includes human or non-human mammals.
- the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.
- the administration dose of the inhibitor of the Ube3a gene or its protein is 0.00001-10 mg/kg body weight, preferably 0.5-5 mg/kg body weight, more preferably 0.5-2 mg/kg body weight.
- the frequency of administration of the inhibitor of the Ube3a gene or its protein is 15-60 times/month, preferably 20-40 days/time.
- the administration time of the inhibitor of the Ube3a gene or its protein is 10-100 days, preferably 20-80 days, most preferably 30-50 days.
- the dosage of the promoter of the PP2A gene or its protein is 0.00001-10 mg/kg body weight, preferably 0.5-5 mg/kg body weight, more preferably 0.5-2 mg/kg body weight.
- the application frequency of the PP2A gene or its protein promoter is 15-60 times/month, preferably 20-40 days/time.
- the administration time of the PP2A gene or its protein promoter is 10-100 days, preferably 20-80 days, most preferably 30-50 days.
- the dosage of the promoter of the PTPA gene or its protein is 0.00001-10 mg/kg body weight, preferably 0.5-5 mg/kg body weight, more preferably 0.5-2 mg/kg body weight.
- the administration time of the promoter of the PTPA gene or its protein is 10-100 days, preferably 20-80 days, most preferably 30-50 days.
- Figure 1A Western blot to detect the changes in PTPA protein levels in the brains of Ube3a-deficient mice.
- Figure 1B Statistical bar graph of changes in PTPA protein levels in the brains of WT and AS mice.
- Figure 2B Western blot detection of UBE3A in vitro ubiquitination of PTPA.
- the PP2A inhibitor LB-100 can correct the abnormal increase in PP2A activity in the brain of AS mice.
- Figure 5 Administration time chart of PP2A inhibitor LB-100 intraperitoneal injection to treat AS mice.
- Figure 7A Morphological images of dendritic spines in Purkinje cells in the cerebellum of WT, AS, WT/LB-100 and AS/LB-100 mice detected by Golgi staining method.
- Fig. 7B a statistical diagram of the proportion of slender dendritic spines of four kinds of mice in A detected by morphological analysis.
- Figure 8A Golgi staining method to detect WT, AS, AS; Ptpa +/- and Ptpa +/- mouse cerebellar Purkinje cells morphological images of dendritic spines.
- Figure 8B a statistical diagram of the proportion of three kinds of dendritic spines in four mice in A detected by morphological analysis.
- Figure 9 Western Blot determination of the effect of three PTPA shRNAs on down-regulating PTPA protein expression.
- Figure 10 The results of exercise behavior of mice after PTPA interference shRNA virus injection.
- Figure 11 The infection range after injection of PTPA-interfering shRNA virus into the lateral ventricle.
- FIG. 12A Western Blot detection of the Ube3a overexpression vector transferred to the Ube3a protein level in HEK293T film scan.
- Figure 13A Western blot detection of the changes in the phosphorylation level of ERK1/2 in AS mice on film scans and statistical bar graphs.
- Fig. 13B Western Blot detection film scanning image of the increase in phosphorylation level of ERK1/2 in HEK293T caused by transfer into Ube3a overexpression vector.
- Figure 14 Western blot to detect the effect of PP2A inhibitors or agonists on the phosphorylation level of ERK1/2.
- PTPA phosphotyrosine phosphatase activating factor
- protein phosphatase 2A protein phosphatase 2A, PP2A
- PTPA and PP2A can be used as new targets to develop drugs to relieve or treat Angel's syndrome.
- PTPA and PP2A can also be used as markers for the diagnosis and prognosis of Angel syndrome.
- the inventors also unexpectedly discovered that by down-regulating the expression or activity of Ube3a, and/or up-regulating the expression or activity of PP2A, and/or up-regulating the expression or activity of PTPA, autism-related diseases can be prevented and/or treated. In addition It can also (i) inhibit the phosphorylation level of ERK1/2; and/or (ii) reduce the activity of the MAPK signaling pathway. On this basis, the inventor completed the present invention.
- UBE3A gene is located in the 15q11-13 region of human chromosome 15, and the encoded product UBE3A is the first E3 ubiquitin ligase of the HECT (Homologous with E6-assiciated protein C-Terminus) family to be discovered. Because there is an E6 binding domain in its protein structure, it is also called E6AP.
- HECT Homologous with E6-assiciated protein C-Terminus
- E6AP E6 binding domain in its protein structure
- UBE3A is an imprinted gene whose main expression is derived from the alleles on the maternal chromosome; while the alleles on the paternal chromosome are not expressed due to the influence of reverse transcribed non-coding RNA.
- the region 11-13 at the long arm end of human chromosome 15 is homologous to the 7C segment sequence on mouse chromosome 7.
- the present invention relates to a Ube3a protein and variants thereof.
- the amino acid sequence of the Ube3a protein is shown in SEQ ID NO.: 18.
- the Ube3a protein or its promoter of the present invention can be used as inhibitors of PP2A and/or PTPA to prevent, alleviate and/or treat Angel’s syndrome; in addition, the Ube3a gene or its protein inhibitor of the present invention can also be used ( a) preventing and/or treating autism or its related diseases; and/or (b) inhibiting the phosphorylation level of ERK1/2; (c) reducing the activity of the MAPK signaling pathway.
- the present invention also includes 50% or more of the sequence shown in SEQ ID NO.: 18 of the present invention (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more, such as 99%) homologous polypeptides or proteins with the same or similar functions.
- SEQ ID NO.: 18 is the murine Ube3a protein (the degree of homology with the human Ube3a protein is 95%); SEQ ID NO.: 23 is the human Ube3a protein.
- the "same or similar function” mainly refers to: prevention, alleviation and/or treatment of Angel syndrome.
- the protein of the present invention can be a recombinant protein, a natural protein, or a synthetic protein.
- the protein of the present invention can be a natural purified product, or a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (for example, bacteria, yeast, higher plants, insect and mammalian cells) using recombinant technology.
- a prokaryotic or eukaryotic host for example, bacteria, yeast, higher plants, insect and mammalian cells
- the protein of the present invention may be glycosylated or non-glycosylated.
- the protein of the present invention may or may not include the initial methionine residue.
- the present invention also includes Ube3a protein fragments and analogs having Ube3a protein activity.
- fragment and analogs having Ube3a protein activity refer to a protein that substantially maintains the same biological function or activity as the natural Ube3a protein of the present invention.
- the mutein fragment, derivative or analogue of the present invention may be (i) a mutein in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acids
- the residue may or may not be encoded by the genetic code, or (ii) a mutein with a substitution group in one or more amino acid residues, or (iii) a mature mutein and another compound (such as an extended mutein) Half-life compounds, such as polyethylene glycol) fused to form a mutant protein, or (iv) additional amino acid sequence fused to the mutant protein sequence to form a mutant protein (such as leader sequence or secretory sequence or used to purify the mutant protein)
- the sequence or proprotein sequence, or the fusion protein formed with the antigen IgG fragment According to the teachings herein, these fragments, derivatives and analogs belong to the scope well known to those skilled in the art.
- conservatively substituted amino acids are preferably generated by amino acid substitution
- the present invention also includes that the natural Ube3a protein of the present invention has 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more, such as 99 %) Homologous polypeptides or proteins with the same or similar functions.
- the protein variant can be obtained by substituting, deleting or adding at least one amino acid by several (usually 1-60, preferably 1-30, more preferably 1-20, most preferably 1-10) Derivative sequence, and adding one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal.
- the substitution of amino acids with similar or similar properties usually does not change the function of the protein, and adding one or several amino acids to the C-terminal and/or ⁇ terminal usually does not change the function of the protein.
- the present invention includes that the difference between the natural Ube3a protein analog and the natural Ube3a protein can be the difference in the amino acid sequence, the difference in the modification form that does not affect the sequence, or both.
- Analogs of these proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, site-directed mutagenesis or other known biological techniques.
- Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as ⁇ , ⁇ -amino acids). It should be understood that the protein of the present invention is not limited to the representative proteins exemplified above.
- Modified (usually not changing the primary structure) forms include: chemically derived forms of proteins in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those that undergo glycosylation modifications during protein synthesis and processing. This modification can be accomplished by exposing the protein to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). In addition, the mutant protein of the present invention can also be modified.
- Modified (usually not changing the primary structure) forms include: chemically derived forms of mutein in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modifications during the synthesis and processing of the mutant protein or during further processing steps. This modification can be accomplished by exposing the mutein to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). It also includes mutant proteins that have been modified to improve their resistance to proteolysis or optimize their solubility.
- the present invention also provides a polynucleotide sequence encoding Ube3a protein.
- the polynucleotide of the present invention may be in the form of DNA or RNA.
- DNA forms include: DNA, genomic DNA, or synthetic DNA.
- DNA can be single-stranded or double-stranded.
- a polynucleotide encoding a mature polypeptide includes: a coding sequence that only encodes the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence (and optional additional coding sequence) of the mature polypeptide and non-coding sequences.
- polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or a polynucleotide that also includes additional coding and/or non-coding sequences.
- the present invention also relates to variants of the above polynucleotides, which encode fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the present invention.
- the variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants.
- These nucleotide variants include substitution variants, deletion variants and insertion variants.
- an allelic variant is an alternative form of a polynucleotide. It may be a substitution, deletion or insertion of one or more nucleotides, but it will not substantially change the function of the encoded polypeptide. .
- the coding nucleic acid sequence of the present invention can be constructed by the method of synthesizing the nucleotide sequence in segments and then performing overlap extension PCR.
- the present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
- the present invention particularly relates to polynucleotides that can hybridize with the polynucleotide of the present invention under stringent conditions (or stringent conditions).
- stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, and more Fortunately, hybridization occurs when more than 95%.
- the protein and polynucleotide of the present invention are preferably provided in an isolated form, and more preferably, purified to homogeneity.
- the full-length sequence of the polynucleotide of the present invention can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
- primers can be designed according to the relevant nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA prepared by a conventional method known to those skilled in the art can be used.
- the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
- the recombination method can be used to obtain the relevant sequence in large quantities. This usually involves cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
- artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain a very long fragment.
- the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis.
- the DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.
- mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
- the method of amplifying DNA/RNA using PCR technology is preferably used to obtain the polynucleotide of the present invention. Especially when it is difficult to obtain full-length cDNA from the library, the RACE method (RACE-cDNA end rapid amplification method) can be preferably used.
- the primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein. And can be synthesized by conventional methods.
- the amplified DNA/RNA fragments can be separated and purified by conventional methods such as gel electrophoresis.
- PP2A is a highly conserved serine/threonine phosphatase, accounting for nearly 1% of the total cell protein content.
- PP2A is a heterotrimer.
- the 35kD catalytic subunit C (PP2Ac) and the 65kD structural subunit A (PR65) constitute the core dimer of PP2A.
- the core dimer combines with a regulatory subunit B through the A subunit to form the PP2A holoenzyme. Complex.
- the present invention relates to a PP2Ac protein, a catalytic subunit of PP2A, and variants thereof.
- the amino acid sequence of the PP2A protein is shown in SEQ ID NO.: 19.
- the PP2A protein or its promoter of the present invention can be used to (a) prevent and/or treat autism or its related diseases; and/or (b) inhibit the phosphorylation level of ERK1/2; (c) reduce the activity of MAPK signaling pathway .
- the present invention also includes 50% or more of the sequence shown in SEQ ID NO.: 19 of the present invention (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more, such as 99%) homologous polypeptides or proteins with the same or similar functions.
- SEQ ID NO.: 19 is mouse PP2A protein (the homology degree with human PP2A protein is 99%);
- SEQ ID NO.: 24 is human PP2A protein.
- the "same or similar function” mainly refers to: (a) prevention and/or treatment of autism or its related diseases; and/or (b) inhibition of the phosphorylation level of ERK1/2; (c) reduction of the MAPK signal pathway active.
- PTPA encoded by the PPP2R4 gene, is an ATP/Mg 2+ dependent peptide proamide trans/cis isomerase, which is commonly found in mammalian tissues and cells.
- PTPA enhances the activity of PP2A phosphatase by changing the conformation of proline 190 of PP2Ac subunit and increasing the methylation level of Leu309.
- the present invention relates to a PTPA protein and variants thereof.
- the amino acid sequence of the PTPA protein is shown in SEQ ID NO.:21.
- the PTPA protein or its promoter of the present invention can be used to (a) prevent and/or treat autism or its related diseases; and/or (b) inhibit the phosphorylation level of ERK1/2; (c) reduce the activity of MAPK signaling pathway .
- the present invention also includes 50% or more of the sequence shown in SEQ ID NO.: 21 of the present invention (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more, such as 99%) homologous polypeptides or proteins with the same or similar functions.
- SEQ ID NO.: 21 is a murine PTPA protein (the homology degree with human PTPA protein is 87%); SEQ ID NO.: 25 is a human PTPA protein.
- the "same or similar function” mainly refers to: (a) prevention and/or treatment of autism or its related diseases; and/or (b) inhibition of the phosphorylation level of ERK1/2; (c) reduction of the MAPK signal pathway active.
- the promoter includes a substance capable of increasing the activity and/or content of the PP2A or PTPA gene or its protein in vivo or in vitro.
- the expression of PP2A or PTPA can be increased by the following methods: the tissue itself secretes a large amount of PP2A or PTPA protein, or artificially overexpresses PP2A or PTPA protein, or artificially delivers PP2A or PTPA protein (for example, using a viral vector, such as adeno-associated virus). Carrier) or PP2A or PTPA accelerator.
- the PP2A or PTPA promoter is not particularly limited, as long as it can promote the expression of PP2A or PTPA or enhance the activity of PP2A or PTPA protein, all within the protection scope of the present invention.
- the PP2A or PTPA accelerator includes a small molecule compound.
- the four types of genetic mutations that cause Angel Syndrome are: the deletion of the maternal chromosome of the 15q11-13 segment (75%); the diploid of the paternal chromosome 15 (2%); the deletion of the imprinting center of chromosome 15 ( imprinting defect, ID) (2%); mutation of UBE3A gene (20%). These chromosomal abnormalities lead to decreased UBE3A protein levels or loss of activity, which in turn leads to the occurrence of Angel syndrome.
- autism or related diseases include but are not limited to: autism, 15q11-13 replication syndrome.
- Autism spectrum disorder is a neurodevelopmental disease with heterogeneous pathogenesis.
- the main characteristics of the disease are: social barriers, communication barriers, behavior repetition and stereotyped behavior, narrow range of interests, and slow language development. Children with autism usually develop before the age of 3, and the prevalence of men is four times that of women. Although the pathogenesis is not yet clear, 25% of children are accompanied by new mutations in genes or abnormal chromosome copy number.
- chromosome 15q11-13 The abnormal copy number of chromosome 15q11-13 accounts for 1%-3% of all autism patients. Some people with maternal 15q11-13 chromosomal duplication (double) will show autism phenotypes, while those with isobaric dicentric chromosome 15 (triple) mutations will all show autism. This type of chromosomal abnormality is also called 15q11-13 replication syndrome.
- UBE3A is the only gene that expresses only maternal alleles. Not only that, but in the mouse model, Ube3a mice with three copies showed typical autistic characteristics.
- UBE3A is a susceptibility gene for autism.
- UBE3A can cause Angel syndrome, and the abnormal increase of UBE3A can cause autism. Therefore, more or less bacteria of UBE3A caused neurodevelopmental diseases.
- the present invention provides a use of an inhibitor of PP2A or PTPA to prepare a pharmaceutical composition for preventing, relieving and/or treating Angel's syndrome.
- inhibitors of PP2A or PTPA include down-regulators, antagonists, blockers, blockers, degradants and the like.
- the inhibitor of PP2A or PTPA gene or protein refers to any inhibitor that can reduce the activity of PP2A or PTPA protein, reduce the stability of PP2A or PTPA gene or protein, down-regulate the expression of PP2A or PTPA protein, and reduce the effective effect of PP2A or PTPA protein.
- Time, or substances that inhibit the transcription and translation of PP2A or PTPA genes, can be used in the present invention, as substances useful for down-regulating PP2A or PTPA, and thus can be used to relieve or treat Angel syndrome.
- the inhibitors are: interfering RNA molecules or antisense nucleotides that specifically interfere with the expression of the PP2A or PTPA gene; or antibodies or ligands that specifically bind to the protein encoded by the PP2A or PTPA gene, and so on.
- the inhibitor of the present invention can also simultaneously inhibit the expression or activity of PP2A and PTPA genes or their proteins.
- the inhibitor is a small molecule compound targeting PP2A or PTPA.
- PP2A inhibitor LB-100 the small molecule compound is PP2A inhibitor LB-100.
- the inhibitor can be PP2A or PTPA-specific interfering RNA molecules (shRNA).
- shRNA interfering RNA molecules
- the use of interfering RNA molecules can significantly down-regulate PP2A or PTPA and play a role in relieving or treating diseases.
- the present invention has no particular limitation on the preparation method of interfering RNA molecules, including but not limited to: chemical synthesis method, in vitro transcription method and the like.
- the interfering RNA can be delivered into the cell by using an appropriate transfection reagent, or can also be delivered into the cell by using various techniques known in the art.
- the CRISPR/Cas9 system can be used for targeted gene editing to specifically knock out the PP2A or PTPA genes.
- Common methods for knocking out PP2A or PTPA genes include: co-transferring sgRNA or nucleic acid capable of forming the sgRNA, Cas9 mRNA, or nucleic acid capable of forming the Cas9 mRNA into a target region or target cell. After the target site is determined, known methods can be used to introduce sgRNA and Cas9 into the cell.
- the nucleic acid capable of forming the sgRNA is a nucleic acid construct or an expression vector
- the nucleic acid capable of forming the Cas9 mRNA is a nucleic acid construct or an expression vector, and these expression vectors are introduced into the cell so as to Active sgRNA and Cas9 mRNA are formed inside.
- the inhibitor is an agent that specifically upregulates the Ube3a protein (including promoting its expression or increasing its activity), which degrades PTPA by upregulating the Ube3a protein, thereby inhibiting the activity of PP2A.
- An upregulator of Ube3a refers to any substance that can increase the activity of Ube3a, increase the stability of Ube3a, up-regulate the expression of Ube3a, and increase the effective time of Ube3a. These substances can be used in the present invention as a substance useful for up-regulating Ube3a.
- PTPA thereby inhibiting PP2A.
- They can be compounds, small chemical molecules, or biological molecules.
- the biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level.
- the Ube3a upregulator is an expression system or expression construct that expresses Ube3a.
- the expression system or expression construct is not particularly limited, and any expression system that can express similar activities of Ube3a or its analogs or mimics can be applied to the present invention.
- the expression system or expression construct includes, but is not limited to: an expression vector, a host cell or virus containing the expression vector; the virus includes adenovirus, adeno-associated virus, lentivirus and the like.
- Those skilled in the art know how to express glycerol kinase or its analogues or mimetics, and there are already some commercial expression systems that can be applied in the present invention.
- the present invention provides a method for screening potential substances for alleviating or treating Angel’s syndrome, the method comprising: treating a system expressing PP2A with a candidate substance; and detecting the expression or activity of PP2A in the system;
- the candidate substance can inhibit the expression or activity of PP2A, indicating that the candidate substance is a potential substance for relieving or treating Angel's syndrome.
- the system for expressing PP2A is preferably a cell (or cell culture) system, and the cell may be a cell that expresses PP2A endogenously; or it may be a cell that expresses PP2A recombinantly.
- Ube3a protein is also expressed in the system, and the method further comprises: detecting the expression of Ube3a protein in the system; wherein, if the candidate substance can inhibit the protein by increasing the expression of Ube3a Phosphatase 2A indicates that the candidate substance is a potential substance for relieving or treating Angel's syndrome.
- PTPA is also expressed in the system, and the method further comprises: detecting the expression of PTPA in the system; wherein, if the candidate substance can inhibit PP2A by inhibiting the expression of PTPA, then It shows that the candidate substance is a potential substance for relieving or treating Angel's syndrome.
- a control group in order to make it easier to observe changes in the expression or activity of Ube3a, PP2A or PTPA during screening, a control group can also be set, and the control group may be one without adding the candidate substance.
- the method further includes: performing further cell experiments and/or animal experiments on the obtained potential substances to further select and determine substances that are really useful for relieving or treating Angel Syndrome.
- the present invention also provides potential substances obtained by the screening method.
- These preliminarily screened substances can constitute a screening library, so that people can finally screen out substances that can inhibit the expression and activity of PP2A or PTPA, thereby alleviating or treating Angel syndrome.
- the present invention also provides a pharmaceutical composition for relieving or treating angel syndrome, which contains an effective amount (such as 0.000001-50wt%; preferably 0.00001-20wt%; more preferably, 0.0001-10wt%) of all
- an effective amount such as 0.000001-50wt%; preferably 0.00001-20wt%; more preferably, 0.0001-10wt% of all
- the inhibitor of PP2A or PTPA gene or protein and a pharmaceutically acceptable carrier. Any of the aforementioned inhibitors of PP2A or PTPA genes or proteins can be used in the preparation of the composition.
- compositions for relieving or treating Angel’s syndrome contains an effective amount of the inhibitor LB-100 of the present invention, and a pharmaceutically acceptable Carrier.
- the "effective amount” refers to an amount that can produce function or activity on humans and/or animals and can be accepted by humans and/or animals.
- the "pharmaceutically acceptable carrier” refers to a carrier used for the administration of a therapeutic agent, and includes various excipients and diluents. The term refers to such pharmaceutical carriers: they are not essential active ingredients themselves, and they do not have excessive toxicity after administration. Suitable carriers are well known to those of ordinary skill in the art.
- the pharmaceutically acceptable carrier in the composition may contain liquid, such as water, saline, and buffer. In addition, these carriers may also contain auxiliary substances, such as fillers, lubricants, glidants, wetting or emulsifying agents, and pH buffering substances.
- the vector may also contain a cell transfection reagent.
- the inhibitor of the PP2A or PTPA gene or protein After learning the use of the inhibitor of the PP2A or PTPA gene or protein, a variety of methods well known in the art can be used to administer the inhibitor or its encoding gene, or its pharmaceutical composition to a mammal . Including but not limited to: subcutaneous injection, intramuscular injection, transdermal administration, topical administration, implantation, sustained-release administration, etc.; preferably, the administration method is parenteral administration.
- the inhibitor of PP2A or PTPA can be directly administered to the subject by methods such as injection; or, the expression unit (such as an expression vector or virus, etc.) carrying the inhibitor of PP2A or PTPA can be delivered through a certain route.
- siRNA is delivered to the target and expresses the active PP2A or PTPA inhibitor. The specific situation depends on the type of the inhibitor, and these are well known to those skilled in the art.
- the effective amount of the inhibitor of PP2A or PTPA gene or protein of the present invention can vary with the mode of administration and the severity of the disease to be treated.
- the selection of the preferred effective amount can be determined by a person of ordinary skill in the art according to various factors (for example, through clinical trials).
- the factors include, but are not limited to: the pharmacokinetic parameters of the PP2A or PTPA gene or protein inhibitor, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, The patient’s immune status, route of administration, etc.
- PP2A or PTPA can be used as markers for the diagnosis or prognosis of Angel’s syndrome: (i) Performing the classification and/or differential diagnosis of Angel’s syndrome; (ii) Assessing related populations Disease risk, drug efficacy, prognosis, and selection of appropriate treatment methods. For example, people with abnormal expression of PP2A or PTPA genes can be isolated, so that more targeted treatment can be carried out.
- the prognosis of Angel’s syndrome of the subject who provided the sample can be predicted by judging the expression or activity of PP2A or PTPA in the sample to be assessed, and appropriate drugs can be selected for treatment.
- a threshold value for PP2A or PTPA can be specified. When the expression of PP2A or PTPA is higher than the specified threshold value, a treatment that inhibits PP2A or PTPA is considered.
- the threshold is easy to determine for those skilled in the art. For example, it can be obtained by comparing the expression of PP2A or PTPA in the microenvironment of normal human tissue with the expression of PP2A or PTPA in the microenvironment of the patient.
- the threshold for abnormal expression of PP2A or PTPA can be predicted by judging the expression or activity of PP2A or PTPA in the sample to be assessed, and appropriate drugs can be selected for treatment.
- a threshold value for PP2A or PTPA can be specified. When the expression of PP2A or
- Various techniques known in the art can be used to detect the presence or absence and expression of the PP2A or PTPA gene, and these techniques are all included in the present invention.
- existing techniques such as Southern blotting, Western blotting, DNA sequence analysis, PCR, etc. can be used, and these methods can be used in combination.
- the present invention also provides reagents for detecting the presence or absence and expression of PP2A or PTPA genes in an analyte.
- primers that specifically amplify PP2A or PTPA can be used; or a probe that specifically recognizes PP2A or PTPA to determine the presence or absence of PP2A or PTPA gene; when performing protein level detection At this time, antibodies or ligands that specifically bind to the protein encoded by PP2A or PTPA can be used to determine the expression of PP2A or PTPA protein.
- a specific probe for PP2A or PTPA gene is a technique well known to those skilled in the art.
- a probe can be prepared that can specifically bind to a specific site on the PP2A or PTPA gene, but not with PP2A or PTPA. Genes other than genes specifically bind, and the probe carries a detectable signal.
- the method of using an antibody that specifically binds to PP2A or PTPA protein to detect the expression of PP2A or PTPA protein in an analyte is also a technique well known to those skilled in the art.
- the present invention also provides a kit for detecting the presence or absence and expression of the PP2A or PTPA gene in an analyte.
- the kit includes: a primer for specifically amplifying the PP2A or PTPA gene; specifically identifying the PP2A or PTPA gene Probes; or antibodies or ligands that specifically bind to the protein encoded by the PP2A or PTPA gene.
- the kit can also include various reagents required for DNA extraction, PCR, hybridization, color development, etc., including but not limited to: extraction solution, amplification solution, hybridization solution, enzyme, control solution , Color developing liquid, lotion, etc.
- kit may also include instructions for use and/or nucleic acid sequence analysis software.
- the present invention provides an inhibitor containing active ingredients (a) Ube3a gene or its protein; PP2A gene, its protein or its promoter; PTPA gene, its protein or its promoter; or a combination thereof; and (b) pharmacy
- the compound pharmaceutical composition of the above acceptable carrier include (but are not limited to): saline, buffer, dextrose, water, glycerol, ethanol, powder, and combinations thereof.
- the pharmaceutical preparation should match the mode of administration.
- the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods.
- compositions such as injections, solutions, tablets and capsules should be manufactured under sterile conditions.
- the pharmaceutical combination of the present invention can also be made into a powder for inhalation.
- the dosage forms of the pharmaceutical composition of the present invention are injections, oral preparations (tablets, capsules, oral liquids), transdermal agents, and sustained-release agents.
- the amount of active ingredient administered is a therapeutically effective amount.
- the pharmaceutical preparation of the present invention can also be made into a sustained-release preparation.
- the pharmaceutical composition of the present invention is preferably an injection preparation.
- the pharmaceutical composition of the present invention can also be used together with other therapeutic agents.
- the pharmaceutical composition of the present invention may also include additional components selected from the following group: other (a) prevention and/or treatment of autism or related diseases; and/or (b) inhibition The phosphorylation level of ERK1/2; and/or (c) substances that reduce the activity of the MAPK signaling pathway, such as MK-8353, U0126 and SCG772984, PD98059, Selumetinib and PD0325901.
- the effective amount of the active ingredient of the present invention can vary with the mode of administration and the severity of the disease to be treated.
- the selection of the preferred effective amount can be determined by a person of ordinary skill in the art according to various factors (for example, through clinical trials).
- the factors include, but are not limited to: the pharmacokinetic parameters of the active ingredients such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, and administration Way etc.
- the active ingredient of the present invention is administered at a dose of about 0.00001 mg-10 mg/kg animal body weight (preferably, 0.5 mg-5 mg/kg animal body weight), satisfactory effects can be obtained.
- several divided doses can be given every day, or the dose can be reduced proportionally.
- the pharmaceutically acceptable carriers of the present invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or Its combination.
- the choice of carrier should match the mode of administration, which are well known to those of ordinary skill in the art.
- the present invention also provides a method that can be used to (a) prevent and/or treat autism or its related diseases; and/or (b) inhibit the phosphorylation level of ERK1/2; and/or (c) reduce the MAPK signal pathway Active pill box, which contains:
- the first container, and the active ingredient in the first container (a1) Ube3a gene or its protein inhibitor; PP2A gene, its protein or its promoter; PTPA gene, its protein or its promoter ; Or a combination thereof, or a drug containing the active ingredient (a1); and
- compositions and kit of the present invention are suitable for (a) preventing and/or treating autism or its related diseases; and/or (b) inhibiting the phosphorylation level of ERK1/2; and/or (c) reducing MAPK signal Pathway activity.
- the preparation of the present invention can be taken three times a day to once every ten days, or once every ten days in a sustained-release manner.
- the preferred way is to take it once a day, because it facilitates the patient's adherence, thereby significantly improving the patient's compliance with medication.
- the total daily dose in most cases should be lower (or equal to or slightly greater than) the daily daily dose of each single drug in most cases.
- the effective dose of the active ingredient used can vary depending on the mode of administration and the expected dose. The severity of the disease to be treated will vary.
- the present invention also provides the use of the active ingredients of the present invention or corresponding drugs to (a) prevent and/or treat autism or its related diseases; and/or (b) inhibit the phosphorylation level of ERK1/2; and/or ( c) A method for reducing the activity of the MAPK signaling pathway, which comprises administering to a mammal an effective amount of an active ingredient (a1) inhibitor of Ube3a gene or its protein; PP2A gene, or its protein or its promoter; PTPA gene, or its Protein or its promoter; or a combination thereof, or administration of a pharmaceutical composition containing the active ingredient (a1).
- the active ingredient of the present invention can be mixed with one or more pharmaceutically acceptable carriers or excipients, such as solvents, diluents, etc., and can be administered orally in the form of tablets: , Pills, capsules, dispersible powders, granules or suspensions (containing, for example, about 0.05-5% suspending agent), syrups (containing, for example, about 10-50% sugar), and elixirs (containing about 20-50% ethanol), or Parenteral administration is in the form of a sterile injectable solution or suspension (containing about 0.05-5% suspension in an isotonic medium).
- these pharmaceutical preparations may contain about 0.01-99%, more preferably about 0.1%-90% by weight of the active ingredient mixed with the carrier.
- the two active ingredients or pharmaceutical compositions of the present invention can be administered by conventional routes, including (but not limited to): intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, oral, intratumoral or topical administration .
- the preferred route of administration includes oral administration, intramuscular administration or intravenous administration.
- the preferred pharmaceutical composition is a liquid composition, especially an injection.
- the active ingredients or drugs of the present invention can also be combined with other (a) prevention and/or treatment of autism or its related diseases; and/or (b) inhibit the phosphorylation level of ERK1/2; and/or (c) reduce Active components of the MAPK signaling pathway or drugs (such as MK-8353, U0126, SCG772984, PD98059, Selumetinib, PD0325901, etc.) are used in combination.
- Active components of the MAPK signaling pathway or drugs such as MK-8353, U0126, SCG772984, PD98059, Selumetinib, PD0325901, etc.
- mice 30 days after birth P30: wild-type (WT) and littermate Ube3a maternally deleted AS mice (obtained from Duke University, introduce a termination under the second exon of Ube3a Codons, resulting in the non-expression of Ube3a), anesthetize, inject 0.7% sodium pentobarbital into the intraperitoneal cavity according to the standard of 70mg/kg; after it is anesthetized, quickly open its head with scissors to take out its entire brain, and quickly release it.
- WT wild-type
- Ube3a maternally deleted AS mice obtained from Duke University, introduce a termination under the second exon of Ube3a Codons, resulting in the non-expression of Ube3a
- anesthetize inject 0.7% sodium pentobarbital into the intraperitoneal cavity according to the standard of 70mg/kg; after it is anesthetized, quickly open its head with scissors to take out its entire brain, and quickly release it.
- the Tiangen BCA protein quantification kit was used to determine the protein concentration in the tissue. First take out 5ul of the protein lysate and dilute it by 10 times. According to the instructions, mix A and B in the kit at a ratio of 1:50, prepare 50ul each of BSA protein standard, and separate the sample and the standard. Mix 25ul and 200ul of the AB mixture into a 96-well plate (produced by Corning, USA). After reacting at 37° for 30 minutes, take it out and place it in a microplate reader to measure the absorbance at 562nm. After calculating the standard curve, bring the obtained absorbance value of the sample into the formula, calculate its corresponding concentration, and then expand it by 10 times to get the concentration of the tested sample.
- the samples were uniformly adjusted to a concentration of 2ug/ul, and the protein sample and an equal volume of 2 ⁇ protein loading buffer (dithiothreitol (DTT): 0.1572g bromophenol blue; 0.01g Tris-HCl( 1M pH 6.8): 0.5ml 10% SDS: 2ml glycerol: 1ml H 2 O: constant volume to 10ml) After mixing, cook at 80°C for 7 minutes.
- DTT dithiothreitol
- SDS 2ml glycerol
- 1ml H 2 O constant volume to 10ml
- Tris (1M pH 6.8) buffer Weigh 24.228g of Tris Base and dissolve it in about 160ml of ultrapure water, stir to dissolve, adjust the pH to 6.8 with hydrochloric acid, and dilute to 200ml with ultrapure water.
- Tris (1.5M pH 8.8) preparation method Weigh 36.342g of Tris Base and dissolve it in about 160ml ultrapure water, stir to dissolve it, adjust the pH to 8.8 with hydrochloric acid, and dilute to 200ml with ultrapure water;
- Tris (0.5M pH 6.8) buffer solution Weigh 12.114g Tris Base and dissolve in about 160ml ultrapure water, stir to dissolve, adjust the pH to 6.8 with hydrochloric acid, and dilute to 200ml with ultrapure water;
- SDS preparation method Weigh 5g sodium dodecyl sulfate (SDS) and dissolve it in ultrapure water to 50ml;
- APS preparation method Weigh 1g of ammonium persulfate (APS) and dissolve it in ultrapure water to 10ml;
- TEMED refers to N,N,N’,N’-Tetramethylethylenediamine, the Chinese name is N,N,N’,N’-tetramethylethylenediamine.
- Electrophoresis prepare 10 ⁇ electrophoresis solution (Tris: 30.3g, glycine: 144.0g, SDS: 10.0g deionized water, constant volume to 1000ml), mix 100ml 10 ⁇ electrophoresis solution with 900ml H 2 O 2 to get 1000ml 1 ⁇ Electrophoresis solution.
- Transfer membrane Prepare 10 ⁇ transfer membrane solution (Tris: 30.3g glycine: 144.0g H 2 O: constant volume to: 1000ml). Next, prepare 1 ⁇ transfer solution (10 ⁇ transfer solution: 80ml methanol: 160ml H 2 O: constant volume to 800ml). Prepare the membrane transfer device, two sets of 8.5 ⁇ 8cm filter paper, two sheets in each set. Soak the PVDF membrane in methanol for about 1 minute until it is saturated with no white spots, then put it in the transfer liquid for equilibrium, and then transfer it to a small box containing the transfer liquid for use. Then use a plastic sheet to gently cut away the concentrated glue, and then cut both ends of the separating glue and the surface in contact with the glass.
- Blocking Prepare 10 ⁇ TBS buffer (1L contains 24.23g Tris, 80.06g NaCl, and adjust pH to 7.4 with HCl), then dilute 10 times with deionized water to obtain 1 ⁇ TBS, and add 0.5%(v/v) Tween-20 is formulated as TBST.
- Prepare 5% skimmed milk dissolve 5g of skimmed milk (China Inner Mongolia Yili Industrial Group Co., Ltd.) in 100ml TBST solution, which is the blocking solution. After the film transfer is completed, take out the PVDF film and put it into a plastic box, pour 30ml of 5% skimmed milk, and place it on a decolorizing shaker for one hour.
- Secondary antibody incubation first wash 3 times with 1 ⁇ TBST for 10 minutes each time. Pipette horseradish peroxidase labeled rabbit immunoglobulin antibody (manufactured by Santa Cruz company, dilution ratio 1:3000) or mouse immunoglobulin antibody (manufactured by Santa Cruz company, dilution ratio 1:3000), and add to 3ml Shake well in blocking solution. Put the washed film into a ziplock bag, pour in the second antibody working solution, seal, and shake on a shaker for 120 minutes.
- IPTG isopropylthio- ⁇ -D-galactoside
- PBS preparation KH2PO4 0.24g, Na2HPO4 1.44g, NaCl 8.0g, KCl 0.2g, add water to 1000mL.
- elution buffer 0.615g glutathione, 10mL Tris-HCl (1M, pH 8.0), 90mL double distilled water.
- the purification steps of His tag protein are similar to the purification steps of GST protein.
- the purification steps are Ni-NTA beads (Qiagen), and the purification steps follow the instructions.
- the in vitro ubiquitination reaction was performed according to the instructions provided in the E6AP (UBE3A) Ubiquitin Ligation Kit (Boston Biochem, K-240).
- the specific reaction is as follows: Mix 3 ⁇ L 10X E1, 3 ⁇ L 10X E2, 3 ⁇ L 10X His6-UBE3A, 3 ⁇ L 10X Mg2+-ATP, 3 ⁇ L 10X His6-PTPA enzyme (2 ⁇ g), 3 ⁇ L 10X reaction buffer and 6 ⁇ L water. In the negative control group, water was substituted for Mg2+-ATP.
- 3 ⁇ L of 10X ubiquitin solution was added, the reaction started. The reaction was carried out at 37 degrees Celsius. After 1.5 hours, the loading buffer was added to terminate the reaction, and the ubiquitination level of PTPA was detected by western blotting.
- Example 2 Take out the entire brain of the mouse according to the method in Example 1, put it into a pre-cooled glass homogenizer, add 1ml imidazole lysis solution (20mM imidazole hydrochloric acid (PH7.0), 2mM EDTA, 2mM EGTA, 1mM PMSF) , 5ul Protease Inhibitor Cocktail (P8430)), homogenize evenly for about 20 times, pipette the lysate into a 1.5ml centrifuge tube, and insert it into the ice box. Put the centrifuge tube in a refrigerated centrifuge, centrifuge at 3000g at 4°C for 5min. Remove the supernatant to a new centrifuge tube, and determine the protein concentration according to the method in Example 1.
- 1ml imidazole lysis solution (20mM imidazole hydrochloric acid (PH7.0), 2mM EDTA, 2mM EGTA, 1mM PMSF) , 5ul Protease Inhibitor Cocktail (P
- Example 4 PP2A inhibitor corrects the increase of PP2A activity in the brain of angel syndrome mice
- LB-100 is a water-soluble PP2A inhibitor with a water solubility of 53mg/mL and its molecular formula is C 13 H 20 N 2 O 4 , and its molecular structure is as follows:
- the LB-100 used was purchased from Selleck, with a purity of 95%. Dissolve the drug in physiological saline to prepare the mother liquor with a concentration of 10mg/ml. After 20ul aliquots, it is stored in a refrigerator at -20°C.
- AS model mice came from Duke University, and introduced a stop codon under the second exon of Ube3a, resulting in the non-expression of Ube3a.
- the mice that do not express Ube3a are model mice of Angel syndrome.
- WT mice Take wild-type mice and AS mice and divide them into 4 groups with 4-6 mice in each group. Take out the whole brain of the mouse according to the method in Example 1, and put it into tissue fluid 1. After taking out the cerebellum, Put it in artificial cerebrospinal fluid containing oxygen. Among them, the WT component is the control group and the LB-100 administration group: the artificial cerebrospinal fluid is added to the physiological saline group or the LB-100 administration group with a final concentration of 300 nM. Similarly, AS mice were divided into control group and LB-100 administration group. After the drug was treated for 2 hours, the tissue was taken out, and the activity of PP2A was measured according to the method in Example 3.
- Tissue fluid 1 N-Methyl-D-glucamine (NMDG), 93mM; KCl, 2.5mM; NaH2PO4, 1.2mM; NaHCO3, 30mM; HEPES, 20mM; Glucose, 25mM, Sodium pyruvate, 3mM; MgSO4, 10mM; CaCl2, 0.5 mM, adjust pH 7.3-7.4.
- NMDG N-Methyl-D-glucamine
- KCl 2.5mM
- NaH2PO4 1.2mM
- NaHCO3 30mM
- HEPES 20mM
- Glucose 25mM
- Sodium pyruvate 3mM
- CaCl2 0.5 mM, adjust pH 7.3-7.4.
- Example 5 Effect of PP2A inhibitor on dyskinesia in mice with Angel syndrome
- the PP2A inhibitor LB-100 was prepared according to the method in Example 4. Dissolve the drug in physiological saline to prepare the mother liquor with a concentration of 10mg/ml. After 20ul aliquots, it is stored in a refrigerator at -20°C.
- AS model mice came from Duke University, and introduced a stop codon under the second exon of Ube3a, resulting in the non-expression of Ube3a.
- the mice that do not express Ube3a are model mice of Angel syndrome.
- WT wild-type mice and 8 AS mice each, and divide them into 4 groups: WT for saline group (WT), AS for saline group (AS), WT for LB-100 drug group (WT/LB- 100), AS gives the LB-100 drug group (AS/LB-100).
- WT saline group
- AS saline group
- AS WT for LB-100 drug group
- AS/LB-100 LB-100 drug group
- the administration procedure is shown in Fig. 5, from 14 days after birth (P14), the medicine is given every two days, until 30 days after birth (P30), conduct behavioral testing.
- String suspension experiment This experiment is mainly to detect the muscle strength and movement coordination of mice. Take a string with a diameter of 0.2cm and a length of 54.4cm, fix it on the brackets at both ends, adjust its height to 50cm above the ground, and place a foam cushion under it to prevent mice from falling and falling. . The mice were first put in the room to adapt to the environment for 30 minutes and then the experiment was started. In the experiment, after lifting the mouse, hang the forelimbs on the string and release the hand, record the time that the mouse does not fall on the wire, and the maximum time is recorded to 120s. Repeat three times for each mouse with an interval of 15 minutes.
- Rotating rod experiment This experiment is mainly to detect the movement coordination and learning ability of mice.
- the experiment takes six days: on the first day, the mouse is placed on the rotating rod instrument, 4 revolutions per minute, and after 2 minutes of exercise, it is taken out and placed in the cage; the second day starts the formal experiment: the setting of the rotating rod instrument is adjusted to the gradual acceleration program , From 5 revolutions per minute to 30 revolutions per minute, the acceleration time is 5 minutes.
- This experiment records the time it spends on the rotating rod instrument. If the mouse falls or holds the column and rotates twice, it is regarded as a failure. The time at this time is the time the mouse stops on the rotating rod instrument. Continue this step for five days. The five-day performance of each mouse was made into a curve, and the exercise ability of the mice was compared.
- Example 6 Effect of PP2A inhibitor in treating abnormal dendritic spines in mice with Angel syndrome
- mice in Example 5 After the mice in Example 5 completed the behavioral experiment, they were subjected to Golgi silver staining to observe the dendritic spines of Purkinje cells in the mouse cerebellum.
- the staining kit used in this experiment is FD Golgi stain kit (Consulting & Services, PK401).
- the mice were anesthetized by the same method as in Example 1.
- the brain tissue of the mice was placed in ddH 2 O, and the blood and debris on the surface were removed by simple rinsing.
- the mouse brain tissue into a 15ml centrifuge tube (produced by Corning) containing 5ml AB mixture, and soak the brain tissue. After storing in the dark for 24 hours, replace with 5ml of the AB mixture, and continue to store in dark at room temperature for 14 days. After 14 days, the AB mixed solution was replaced with the C solution in the kit, and stored at 4°C in the dark. After 24 hours, the C solution was replaced and kept at 4°C for 3 days. After 3 days, the cerebellum in the brain tissue was cut off, placed sagittal in a 24-well plate, poured into the pre-dissolved 3% agar, and placed in the refrigerator, and waited for it to solidify.
- a 15ml centrifuge tube produced by Corning
- Preparation of 3% agar solution Weigh 3g of agar powder into a glass Erlenmeyer flask, add 110ml of water, heat it in a microwave oven, wait for it to boil once, shake it a few times, and put it in the microwave again. Wait for it to boil again. Finally, after the solution is in a uniform state, it is quickly poured into a 50ml centrifuge tube (purchased by Cornin) and placed in a 60°C water bath to keep it in a dissolved state.
- a 50ml centrifuge tube purchased by Cornin
- the embedded brain tissue was taken out and put into a vibrating microtome (Leica) for sectioning. Specifically, after taking out the brain tissue and trimming it, fix it on the base of the microtome with super glue, pour C solution quickly, adjust the slice thickness of the microtome to 150um, and take the cut brain slice directly Affixed to the gelatin-coated glass slide, prepare a glass slide for each cerebellum. When finished, put it in a dark box and let it dry. After about 2 days, Golgi staining was performed.
- the film to be shot was taken under the bright field of a Nikon A1 inverted confocal microscope with a 60x objective lens. After adjusting the light source to the optimal level, the dendrites of Purkinje cells away from the cell body were selected for shooting. Finally, the dendritic spines in the resulting picture are divided into three categories according to their morphology: mushroom, stubby, and thin. The three types of dendritic spines in each dendritic trunk are calculated. And finally calculate the proportion of each category.
- Example 7 Knockdown of PTPA gene expression can improve neuronal morphological abnormalities in mice with Angel syndrome
- the present inventors combined female paternal Ube3a-deficient heterozygous mice (Ube3a m+/p- ) (obtained from Duke University) with male Ptpa-deficient heterozygous mice (Ptpa +/- ) (Institute of Neuroscience, Shanghai Academy of Biological Sciences, Chinese Academy of Sciences, obtained by transforming Ptpa with CRISPR/Cas9 technology) mating, which produces four offspring: wild-type (WT), maternal Ube3a-deficient AS mice, and Ptpa-deficient heterozygous mice (Ptpa +/- ), and mice with double knockout of Ube3a and Ptpa (AS; Ptpa +/- ).
- mice with the four genes were respectively subjected to Golgi staining to observe the dendritic spines of Purkinje cells in the mouse cerebellum.
- the dendritic spines are divided into three types according to their morphology: mushroom, stubby and thin. Count the proportions of three kinds of dendritic spines in different mice.
- Example 8 The shRNA targeting the PTPA gene and specifically inhibiting the expression of the PTPA gene can alleviate the abnormal movement of the angel syndrome model mice
- shRNA is artificially designed in the order of sense-loop-antisense, sense refers to the sense strand of the target sequence, antisense refers to the antisense strand of the target sequence, loop refers to the sequence that forms a loop, and TTCAAGAGA is used here. Then add the sequences recognized by the restriction enzymes BglII and HindIII at the two ends to obtain two shRNA sequences for one target as shown below: sense strand: 5'-GATCCCC-sense-TTCAAGAGA-antisense-TTTTTA-3' ; Antisense strand: 5'-AGCTTAAAAA-sense-TCTCTTGAA-antisense-GGG-3'.
- the sense chain of sh1# (sh1#-F): 5’-GATCCCCCAGTTCCAGATATGGGCAAATGGAATTCAAGAGAttccatttgcccatatctggaactgTTTTTA-3’ (SEQ ID NO: 4);
- Antisense strand of sh1# (sh1#-R): 5’-AGCTTAAAAACAGTTCCAGATATGGGCAAATGGAATCTCTTGAAttccatttgcccatatctggaactgGGG-3’ (SEQ ID NO: 5);
- the sense chain of sh3# (sh1#-F): 5’-GATCCCCCCTGGTATGCCAAACTTGATCAGGATTCAAGAGAtcctgatcaagtttggcataccaggTTTTTA-3’ (SEQ ID NO: 6);
- sh3# 5’-AGCTTAAAAACCTGGTATGCCAAACTTGATCAGGATCTCTTGAAtcctgatcaagtttggcataccaggGGG-3’ (SEQ ID NO: 7);
- the sense chain of sh4# (sh1#-F): 5’-GATCCCCTCAAGGTGTTTGATAGGTATCTTGATTCAAGAGAtcaagatacctatcaaacaccttgaTTTTTA-3’ (SEQ ID NO: 8);
- sh4# 5'-AGCTTAAAAATCAAGGTGTTTGATAGGTATCTTGATCTCTTGAAtcaagatacctatcaaacaccttgaGGG-3' (SEQ ID NO: 9);
- DNA Oligo is dissolved in ddH 2 O at a concentration of 3 mg/ml. Take 2ul DNA oligo of the sense and antisense strands of shRNA and 5ul of 10 ⁇ annealing buffer (composition of 100mM Tris-HCl (pH 7.5), 10mM EDTA, 1mM NaCl) and mix them in a water bath at 95°C for 12min. Cool to room temperature at room temperature. At 25°C, use T4 ligase (produced by NEB, USA) to ligate the complementary double strand with the pSuper basic vector digested with BglII and HindIII at 16°C overnight. The system was prepared according to the instructions (DNA 4 ⁇ l, vector 2 ⁇ l, 10 ⁇ T4 buffer 2 ⁇ l, T4 ligase 2 ⁇ l, ddH 2 O 10 ⁇ l).
- T7primer 5’-AATACGACTCACTATAG-3’ (SEQ ID NO: 16);
- T3primer 5'-TTAACCCTCACTAAAGG-3' (SEQ ID NO: 17).
- the primers used for sequencing are: T7primer: 5'-AATACGACTCACTATAG-3' (SEQ ID NO.: 16).
- T7primer 5'-AATACGACTCACTATAG-3' (SEQ ID NO.: 16).
- T7primer 5'-AATACGACTCACTATAG-3' (SEQ ID NO.: 16).
- T7primer 5'-AATACGACTCACTATAG-3' (SEQ ID NO.: 16).
- the plasmid is extracted from the kit (produced by Kaijie Biotechnology Co., Ltd.), and the plasmid extraction is performed according to the Plasmid Maxi Kit instructions.
- a platinum medium (95% MEM, Invitrogen, USA; 5% FBS (v/v), Invitrogen, USA; 0.4% Glucose (w/v), Sigma, USA) was added to terminate the digestion of Trypsin.
- the cell suspension was diluted 10 times and counted with a hemocytometer. All petri dishes and glass slides were coated with polylysine (PDL) before use (0.1 mg/ml glass slide, 0.01 mg/ml petri dish).
- PDL polylysine
- the cells were cultured in a 5% CO 2 , 37°C cell incubator. When the cells adhere to the wall (about 1-2 hours), use Neruobasal cell culture medium (97% Neurobasal medium (v/v), Invitrogen, USA; 2% B-27 supplement (v/v), Invitrogen, USA; 1% GlutaMAX (v/v), Invitrogen, USA) change the fluid.
- the electro-transfer fluid is composed of liquid A and liquid B. Before use, mix the two in a ratio of 1:50 before use.
- Electroporation solution A 2g ATP-disodium salt, sigma A2383; 1.2g MgCl 2 ⁇ 6H 2 O, sigma M2393; after distilled water to 10ml, filter and aliquot with a 0.22 micron filter and store at -20°C.
- Electron to B solution 6g KH 2 PO 4 , sigma P5655; 0.6g NaHCO 3 , sigma S5761; 0.2g Glucose, sigma G6152, after dissolving, adjust the pH to 7.4, dilute to 500ml, filter and aliquot with a 0.22 micron filter, Store at -20°C.
- PTPA interferes with shRNA to relieve the motor phenotype of Angel Syndrome
- the sh4# sequence of PTPA was sent to Shanghai Taiertu Biotechnology Co., Ltd. to construct the AAV virus, and the sequence was inserted into the p-CMV-bGlobin-Egfp-H1 (Shanghai Taiertu Biotechnology Co., Ltd.) vector to obtain the serotype AAV9 type empty vector control virus and virus particles that interfere with PTPA shRNA.
- the constructed two viruses in the first day of birth using 10ul microsyringe (Shanghai high pigeon) takes 1ul (1 ⁇ 10 12 VG / ml) and AS intracerebroventricular injection of WT mice, respectively. After four weeks, the mice's rotating rod behavior was tested. See Example 6 for the operation steps of this behavior.
- PP2A is the screening target
- Test group HEK 293T cells (which express PP2A), and given candidate substances;
- Control group HEK 293T cells (which express PP2A), no candidate substance is given.
- the expression of PP2A in the test group and the control group were detected and compared. If the expression of PP2A in the test group is statistically lower (for example, 30% or lower) than the control group, it indicates that the candidate is a potential substance for relieving or treating Angel's syndrome.
- PTPA is the screening target
- Test group HEK 293T cells (which express PTPA), and given candidate substances;
- Control group HEK 293T cells (which express PTPA), no candidate substance is given.
- PP2A is the screening target, PTPA, Ube3a participate
- Test group HEK 293T cells (in which Ube3a, PTPA, PP2A are expressed at the same time), and candidate substances are given;
- Control group HEK 293T cells (where Ube3a, PTPA, and PP2A are expressed at the same time), no candidate substance is given.
- Ube3a and PP2A in the test group and the control group were detected and compared. If the expression of Ube3a in the test group is statistically higher (for example, 30% or higher) than the control group, accompanied by degradation of PTPA and a significant reduction of PP2A (for example, a reduction of 30% or lower), it indicates the candidate It is a potential substance for relieving or curing angel syndrome.
- Ube3a sequence mouse Ube3a sequence (NM_011668), obtain its coding sequence from NCBI, design PCR primers, and amplify them.
- the primer sequence is as follows:
- Ube3a-F 5’-atgaagcgagcagctgcaagc-3’ (SEQ ID NO: 10);
- Ube3a-R 5’-ttacagcatgccaaatcctttggc-3’ (SEQ ID NO: 11);
- PCR fragment obtained the first time as a template Use the PCR fragment obtained the first time as a template, perform PCR again, and then use the vector pCAG-tag2B empty vector (purchased from Stratagene) after digestion with BamHI and HindIII, and use the recombinase (ClonExpress II One Step Cloning Kit, vazyme) After recombination at 37°C for 30 minutes, add the ligation product to a 1.5ml centrifuge tube containing E. coli DH5 ⁇ competent cells to mix, place on ice for 30 minutes, then put it in a 42°C water bath for 90 seconds, and then put it back on ice. After -2 minutes, add 800 ⁇ l of antibiotic-free medium, and place it in a bacteria incubator at 37°C and shake for 1 hour. The bacteria collected at 4,500 revolutions per centrifugation were coated on a solid LB medium plate and cultured in a bacteria incubator at 37°C overnight until colonies grew.
- pCAG-tag2B-F 5’-ggcaaagaagctagcg-3’ (SEQ ID NO: 14);
- pCAG-tag2B-R 5’-gggcgatcgagtgaattgtaatac-3’ (SEQ ID NO: 15);
- the primers used for sequencing are: pCAG-tag2B-F: ggcaaagaagctagcg.
- pCAG-tag2B-F ggcaaagaagctagcg.
- Culture HEK 293T cells (hereinafter referred to as 293T, purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences) in a 37°C 5% CO 2 cell incubator.
- the medium used DMEM supplemented with 10% fetal bovine serum (Gibco, New York, USA) The company produces) culture medium.
- Subculture 293T cells in a petri dish with a diameter of 6 cm. When the cells grow to a confluence of about 70%, follow the instructions of Lipofectamine 2000 (manufactured by Invitrogen, USA).
- mice 30 days after birth were taken out, wild-type (WT) and littermate AS mice were anesthetized, and brain tissues were taken for western blot detection.
- WT wild-type
- brain tissues were taken for western blot detection.
- the specific method was the same as in Example 1.
- Example 12 PP2A is used as a screening target, and ERK1/2 is used as an index for screening
- the primary cultured rat cortex and hippocampal neurons were derived from Sprague-Dawley (SD) rats within 24 hours of birth.
- the specific steps are roughly as follows: soak the dissecting instrument in 75% alcohol in advance, and irradiate it with UV for 30 minutes in a sterile operating table. Spray the rat with 75% sterile alcohol, carefully tear off the scalp and skull with scissors and tweezers, peel off the brain and place it in the dissection solution (Hank's buffer; NaHCO 3 0.35g/L; HEPES 10mM, pH 7.3; Glucose 33.3mM) )in.
- the entire solution was filtered through a filter with a pore size of 70 ⁇ m to remove undigested tissue mass.
- the filtered product was centrifuged at 500 g for 5 minutes to obtain cells, and the supernatant was removed. Resuspend the cells in culture medium, centrifuge, and remove the supernatant.
- the obtained cells were resuspended in culture medium, counted with a hemocytometer, and plated in a well plate of cells according to the required density. All petri dishes and glass slides are coated with polylysine (PDL) before use (slides 0.1 mg/mL, petri dishes 0.01 mg/mL). Put the cells in a 5% CO2, 37 degrees Celsius cell culture incubator. After 2-4 hours, change the medium to Nuerobasal medium for culture.
- PDL polylysine
- Dissecting fluid Hank's buffer; NaHCO 3 0.35g/L; HEPES 10mM, pH 7.3; Glucose 33.3mM.
- Plating medium 450mL MEM, 50mL HBS, 2g Glucose.
- Nuerobasal culture medium 500mL Neurobasal A medium, 5mL B-27 supplement, 5mL GlutaMAX.
- Okadaic acid and Calyculin A are dissolved in DMSO, and FTY720 is dissolved in water.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pharmacology & Pharmacy (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Analytical Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Neurosurgery (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Neurology (AREA)
- Biomedical Technology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Hospice & Palliative Care (AREA)
- Psychiatry (AREA)
- Psychology (AREA)
- Epidemiology (AREA)
- Pathology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
最初的残基 | 代表性的取代 | 优选的取代 |
Ala(A) | Val;Leu;Ile | Val |
Arg(R) | Lys;Gln;Asn | Lys |
Asn(N) | Gln;His;Lys;Arg | Gln |
Asp(D) | Glu | Glu |
Cys(C) | Ser | Ser |
Gln(Q) | Asn | Asn |
Glu(E) | Asp | Asp |
Gly(G) | Pro;Ala | Ala |
His(H) | Asn;Gln;Lys;Arg | Arg |
Ile(I) | Leu;Val;Met;Ala;Phe | Leu |
Leu(L) | Ile;Val;Met;Ala;Phe | Ile |
Lys(K) | Arg;Gln;Asn | Arg |
Met(M) | Leu;Phe;Ile | Leu |
Phe(F) | Leu;Val;Ile;Ala;Tyr | Leu |
Pro(P) | Ala | Ala |
Ser(S) | Thr | Thr |
Thr(T) | Ser | Ser |
Trp(W) | Tyr;Phe | Tyr |
Tyr(Y) | Trp;Phe;Thr;Ser | Phe |
Val(V) | Ile;Leu;Met;Phe;Ala | Leu |
Claims (21)
- 蛋白磷酸酶2A或磷酸酪氨酸磷酸酶活化因子的抑制剂的用途,用于制备缓解或治疗天使综合症的药物组合物。
- 如权利要求1所述的用途,其特征在于,所述的抑制剂选自:特异性抑制蛋白磷酸酶2A或磷酸酪氨酸磷酸酶活化因子的小分子化合物;特异性干扰蛋白磷酸酶2A或磷酸酪氨酸磷酸酶活化因子的基因表达的干扰分子;特异性敲除蛋白磷酸酶2A或磷酸酪氨酸磷酸酶活化因子的基因编辑试剂;或特异性与蛋白磷酸酶2A或磷酸酪氨酸磷酸酶活化因子结合的抗体或配体。
- 如权利要求2所述的用途,其特征在于,所述的抑制剂是特异性抑制蛋白磷酸酶2A活性的小分子化合物,其选自:抑制剂LB-100。
- 如权利要求1所述的用途,其特征在于,所述的抑制剂是特异性上调Ube3a蛋白的试剂,其通过上调Ube3a蛋白而降解磷酸酪氨酸磷酸酶活化因子、进而抑制蛋白磷酸酶2A。
- 如权利要求4所述的用途,其特征在于,所述的特异性上调Ube3a蛋白的试剂为过表达Ube3a的表达构建物。
- 如权利要求2所述的用途,其特征在于,所述的特异性干扰磷酸酪氨酸磷酸酶活化因子的基因表达的干扰分子是shRNA。
- 如权利要求2所述的用途,其特征在于,所述的shRNA靶向于:SEQ ID NO:1、SEQ ID NO:2或SEQ ID NO:3;较佳地,所述的shRNA具有SEQ ID NO:4的正义链和SEQ ID NO:5的反义链,或具有SEQ ID NO:6的正义链和SEQ ID NO:7的反义链;或具有SEQ ID NO:8的正义链和SEQ ID NO:9的反义链。
- 蛋白磷酸酶2A或磷酸酪氨酸磷酸酶活化因子的用途,用于制备预防和/或治疗孤独症或其相关疾病的药物组合物。
- 蛋白磷酸酶2A或磷酸酪氨酸磷酸酶活化因子的用途,用于筛选缓解或治疗天使综合症的药物。
- 一种筛选缓解或治疗天使综合症的潜在物质的方法,所述方法包括:(1)用候选物质处理表达蛋白磷酸酶2A和/或磷酸酪氨酸磷酸酶活化因子的体系;和(2)检测所述体系中蛋白磷酸酶2A和/或磷酸酪氨酸磷酸酶活化因子的表达或活性;其中,若所述候选物质可降低蛋白磷酸酶2A和/或磷酸酪氨酸磷酸酶活化因子的表达或活性,则表明该候选物质是缓解或治疗天使综合症的潜在物质。
- 如权利要求10所述的方法,其特征在于,步骤(1)包括:在测试组中,将候选物质加入到表达蛋白磷酸酶2A和/或磷酸酪氨酸磷酸酶活化因子的体系中;步骤(2)包括:检测测试组的体系中蛋白磷酸酶2A和/或磷酸酪氨酸磷酸酶活化因子的表达或活性,并与对照组比较,其中所述的对照组是不添加所述候选物质的表达蛋白磷酸酶2A和/或磷酸酪氨酸磷酸酶活化因子的体系;如果测试组中蛋白磷酸酶2A和/或磷酸酪氨酸磷酸酶活化因子的表达或活性在统计学上低于对照组,就表明该候选物是缓解或治疗天使综合症的潜在物质。
- 如权利要求10所述的方法,其特征在于,所述的体系中还表达Ube3a蛋白,所述方法还包括:检测所述体系中Ube3a蛋白的表达;其中,若所述候选物质能够通过提高Ube3a的表达而抑制蛋白磷酸酶2A和/或磷酸酪氨酸磷酸酶活化因子,则表明该候选物质是缓解或治疗天使综合症的潜在物质。
- 特异性识别蛋白磷酸酶2A基因或蛋白的试剂的用途,用于制备进行天使综合症诊断或预后评估的试剂或试剂盒。
- 如权利要求13所述的用途,其特征在于,所述的特异性识别蛋白磷酸酶2A基因或蛋白的试剂选自:特异性扩增蛋白磷酸酶2A基因的引物;特异性识别蛋白磷酸酶2A基因的探针;或特异性结合蛋白磷酸酶2A的抗体或配体。
- 特异性识别磷酸酪氨酸磷酸酶活化因子基因或蛋白的试剂的用途,用于制备进行天使综合症诊断或预后评估的试剂或试剂盒。
- 如权利要求15所述的用途,其特征在于,所述的特异性识别磷酸酪氨酸磷酸酶活化因子基因或蛋白的试剂选自:特异性扩增磷酸酪氨酸磷酸酶活化因子基因的引物;特异性识别磷酸酪氨酸磷酸酶活化因子基因的探针;或特异性结合磷酸酪氨酸磷酸酶活化因子的抗体或配体。
- 一种物质的用途,其特征在于,用于制备预防和/或治疗孤独症或其相关疾病的组合物或制剂,其中,所述物质选自下组:(a)Ube3a基因或其蛋白的抑制剂、(b)PP2A基因、或其蛋白或其促进剂、(c)PTPA基因、或其蛋白或其促进剂、或其组合。
- 一种药物组合物,其特征在于,包括:(a1)用于预防和/或治疗孤独症或其相关疾病的第一活性成分,所述第一活性成分选自下组:(i)Ube3a基因或其蛋白的抑制剂、(ii)PP2A基因、或其蛋白或其促进剂、(iii)PTPA基因、或其蛋白或其促进剂、或其组合;和(b)药学上可接受的载体。
- 一种药盒,其特征在于,包括:(i)第一容器,以及位于该第一容器中的活性成分(a1)Ube3a基因或其蛋白的抑制剂;PP2A基因、或其蛋白或其促进剂;PTPA基因、或其蛋白或其促进剂;或其组合,或含有活性成分(a1)的药物。
- 一种筛选孤独症或其相关疾病的潜在治疗剂的方法,其特征在于,包括:(a)在测试组中,在培养体系中,在测试化合物的存在下,培养表达Ube3a基因的细胞一段时间T1,检测测试组所述培养体系中的Ube3a基因的表达量E1;并且在不存在所述测试化合物且其他条件相同的对照组中,检测对照组所述培养体系中Ube3a基因的表达量E2;和(b)对E1和E2进行比较,如果E1显著低于E2,则表示所述测试化合物是孤独症或其相关疾病的潜在治疗剂。
- 一种筛选孤独症或其相关疾病的潜在治疗剂的方法,其特征在于,包括:(a)在测试组中,在培养体系中,在测试化合物的存在下,培养表达PP2A、和/或PTPA基因的细胞一段时间T1,检测测试组所述培养体系中的PP2A、和/或PTPA基因的表达量E1;并且在不存在所述测试化合物且其他条件相同的对照组中,检测对照组所述培养体系中PP2A、和/或PTPA基因的表达量E2;和(b)对E1和E2进行比较,如果E1显著高于E2,则表示所述测试化合物是孤独症或其相关疾病的潜在治疗剂。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810195972 | 2018-03-09 | ||
CN201910130210.5 | 2019-02-21 | ||
CN201910130210.5A CN110237257B (zh) | 2018-03-09 | 2019-02-21 | Ube3a泛素化PP2A激活因子PTPA在治疗天使综合症和孤独症中的应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2020168850A1 true WO2020168850A1 (zh) | 2020-08-27 |
Family
ID=67882962
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2020/070997 WO2020168850A1 (zh) | 2018-03-09 | 2020-01-08 | Ube3a泛素化PP2A激活因子PTPA在治疗天使综合症和孤独症中的应用 |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN110237257B (zh) |
WO (1) | WO2020168850A1 (zh) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110237257B (zh) * | 2018-03-09 | 2023-01-03 | 中国科学院脑科学与智能技术卓越创新中心 | Ube3a泛素化PP2A激活因子PTPA在治疗天使综合症和孤独症中的应用 |
CN111549033B (zh) * | 2020-06-11 | 2021-02-12 | 南京市江宁医院 | 慢病毒感染人表皮角质细胞株及其构建方法和应用 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2724721A1 (en) * | 2012-10-26 | 2014-04-30 | Matentzoglu, Konstantin | Composition for use in the treatment of Angelman syndrome and/or autism spectrum disorder, the use of such composition and a method for manufacturing a medicament for the treatment of Angelman syndrome and/or autism spectrum disorder |
WO2014066082A1 (en) * | 2012-10-23 | 2014-05-01 | Haas Arthur L | Blocking activities of e6ap ligase |
WO2014172490A1 (en) * | 2013-04-16 | 2014-10-23 | Tufts University | Models for apc related diseases and disorders, methods of diagnosing and treating, and methods for identifying therapeutic agents for treating apc related diseases and disorders |
CN110237257A (zh) * | 2018-03-09 | 2019-09-17 | 中国科学院上海生命科学研究院 | Ube3a泛素化PP2A激活因子PTPA在治疗天使综合症和孤独症中的应用 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3291843B1 (en) * | 2015-05-07 | 2023-03-22 | University of South Florida | Modified ube3a gene for a gene therapy approach for angelman syndrome |
-
2019
- 2019-02-21 CN CN201910130210.5A patent/CN110237257B/zh active Active
-
2020
- 2020-01-08 WO PCT/CN2020/070997 patent/WO2020168850A1/zh active Application Filing
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014066082A1 (en) * | 2012-10-23 | 2014-05-01 | Haas Arthur L | Blocking activities of e6ap ligase |
EP2724721A1 (en) * | 2012-10-26 | 2014-04-30 | Matentzoglu, Konstantin | Composition for use in the treatment of Angelman syndrome and/or autism spectrum disorder, the use of such composition and a method for manufacturing a medicament for the treatment of Angelman syndrome and/or autism spectrum disorder |
WO2014172490A1 (en) * | 2013-04-16 | 2014-10-23 | Tufts University | Models for apc related diseases and disorders, methods of diagnosing and treating, and methods for identifying therapeutic agents for treating apc related diseases and disorders |
CN110237257A (zh) * | 2018-03-09 | 2019-09-17 | 中国科学院上海生命科学研究院 | Ube3a泛素化PP2A激活因子PTPA在治疗天使综合症和孤独症中的应用 |
Non-Patent Citations (4)
Title |
---|
EDWIN J. WEEBER, YONG-HUI JIANG, YPE ELGERSMA, ANDREW W. VARGA, YARIMAR CARRASQUILLO, SARAH E. BROWN, JILL M. CHRISTIAN, BANEFSHEH: "Derangements of Hippocampal Calcium/Calmodulin-Dependent Protein Kinase II in a Mouse Model for Angelman Mental Retardation Syndrome", THE JOURNAL OF NEUROSCIENCE, vol. 23, no. 7, 1 April 2003 (2003-04-01), pages 2634 - 2644, XP055734700, ISSN: 0270-6474, DOI: 10.1523/JNEUROSCI.23-07-02634.2003 * |
REYNHOUT SARA; JANSEN SANDRA; HAESEN DORIEN; VAN BELLE SISKA; DE MUNNIK SONJA A; BONGERS ERNIE M H F; SCHIEVING JOLANDA H; MARCELI: "De Novo Mutations Affecting the Catalytic Ca Subunit of PP2A, PPP2 CA , Cause Syndromic Intellectual Disability Resembling Other PP2A-Related Neurodevelopmental Disorders", THE AMERICAN JOURNAL OF HUMAN GENETICS, vol. 104, no. 1, 3 January 2019 (2019-01-03), pages 139 - 156, XP085573052, ISSN: 0002-9297, DOI: 10.1016/j.ajhg.2018.12.002 * |
WU HONGMEI; WANG XUELAI; GAO JINGQUAN; LIANG SHUANG; HAO YANQIU; SUN CAIHONG; XIA WEI; CAO YONGGANG; WU LIJIE: "Fingolimod (FTY720) attenuates social deficits, learning and memory impairments, neuronal loss and neuroinflammation in the rat model of autism", LIFE SCIENCES, vol. 173, 28 February 2017 (2017-02-28), pages 43 - 54, XP029939086, ISSN: 0024-3205, DOI: 10.1016/j.lfs.2017.01.012 * |
XU XIANG-QING , WANG KE-WEI: "Research Progress in Medicinal Treatment of Autism Spectrum Disorders", ACTA PHARMACEUTICA SINICA, vol. 53, no. 3, 31 December 2018 (2018-12-31), pages 321 - 327, XP055734711, DOI: : 10.16438/j.0513-4870.2017-0912 * |
Also Published As
Publication number | Publication date |
---|---|
CN110237257B (zh) | 2023-01-03 |
CN110237257A (zh) | 2019-09-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US9902761B2 (en) | Polypeptides, nucleic acids and uses thereof | |
JP4659736B2 (ja) | スクリーニング方法 | |
Chen et al. | Knockdown of circROBO2 attenuates acute myocardial infarction through regulating the miR-1184/TRADD axis | |
WO2020168850A1 (zh) | Ube3a泛素化PP2A激活因子PTPA在治疗天使综合症和孤独症中的应用 | |
KR102194746B1 (ko) | Wnt 억제제와 연관된 마커 | |
JP2005527235A (ja) | デフェンシン:抗ウイルス剤の使用 | |
US20130316958A1 (en) | Highly potent peptides to control cancer and neurodegenerative diseases | |
KR101541668B1 (ko) | 치료 및 진단용의 신규 생성물 및 방법 | |
WO2007136857A2 (en) | Hox compositions and methods | |
JP2023516325A (ja) | 血中コレステロール低下用、心血管代謝疾患の予防又は治療用及び抗炎症用薬学的組成物 | |
WO2011129427A9 (ja) | 癌の診断剤および治療剤 | |
IL179555A (en) | Use of argonionine to prepare a medical preparation for the treatment and / or prevention of autoimmune diseases | |
WO2006129867A2 (en) | ENHANCED EXPRESSION OF LACTOFERRIN mRNA BY LACRITIN | |
WO2016049280A1 (en) | mTORC1 MODULATION | |
TW200831898A (en) | Treatment of insulin resistance | |
JP2010518821A (ja) | 脂肪細胞の分化を制御する分泌型タンパク質Ccdc80 | |
CN112143805B (zh) | Rit1在肝细胞癌的诊断和治疗中的应用 | |
US20090312245A1 (en) | SRA binding protein | |
CN114908158B (zh) | Cdk1在晚期胃肠间质瘤的诊断和治疗中的应用 | |
US20220401515A1 (en) | Application of non-igf1r-binding substance in prevention and/or treatment of inflammatory diseases | |
KR101985577B1 (ko) | Cry1의 당뇨병 치료제 및 이를 표적으로 하는 당뇨병 치료제 스크리닝 용도 | |
Chang et al. | Pathogenic SHQ1 variants result in disruptions to neuronal development and the dopaminergic pathway | |
JP2006525244A (ja) | 糖尿病の治療標的としてのインスリン誘導性遺伝子 | |
TW200530400A (en) | Secreted neural apoptosis inhibiting proteins | |
JPWO2006118289A1 (ja) | 抗うつ作用を有する化合物の同定方法 |
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: 20759857 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: 20759857 Country of ref document: EP Kind code of ref document: A1 |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 20759857 Country of ref document: EP Kind code of ref document: A1 |
|
32PN | Ep: public notification in the ep bulletin as address of the adressee cannot be established |
Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 09.03.2022) |