WO2018090851A1 - Use of fbp aldolase in preparation of drug activating ampk - Google Patents

Use of fbp aldolase in preparation of drug activating ampk Download PDF

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WO2018090851A1
WO2018090851A1 PCT/CN2017/109647 CN2017109647W WO2018090851A1 WO 2018090851 A1 WO2018090851 A1 WO 2018090851A1 CN 2017109647 W CN2017109647 W CN 2017109647W WO 2018090851 A1 WO2018090851 A1 WO 2018090851A1
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aldolase
drug
drugs
inhibits
ampk
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Chinese (zh)
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林圣彩
张宸崧
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厦门大学
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Definitions

  • the invention belongs to the field of biomedicine and relates to the use of FBP aldolase in the preparation of a medicament for activating AMPK.
  • the present invention also relates to a medicament for inhibiting cholesterol synthesis, a medicament for reducing fatty acid synthesis, a medicament for preventing and/or treating diabetes, a medicament for preventing and/or treating a tumor, a medicament for preventing and/or treating Parkinson's disease, prevention and/or Or use in the treatment of drugs for Alzheimer's disease or drugs that prolong the lifespan of mammals.
  • AMPK 5'-adenosine monophosphate-activated protein kinase
  • AMPK is an important molecule that regulates the energy balance between cells and the body.
  • AMPK is composed of three different subunits, each of which has several isoforms: ⁇ subunit ( ⁇ 1 or ⁇ 2); ⁇ subunit ( ⁇ 1 or ⁇ 2); and ⁇ subunit ( ⁇ 1, ⁇ 2 or ⁇ 3)
  • ⁇ subunit ⁇ 1 or ⁇ 2
  • ⁇ subunit ⁇ subunit
  • ⁇ subunit ⁇ 1, ⁇ 2 or ⁇ 3
  • AMPK activation is mediated by its allosteric activator, AMP/ADP and its analogs.
  • AMP/ADP binds to the gamma subunit of AMPK, causing structural changes in the holoenzyme, making it more susceptible to phosphorylation of its threonine site 172 (p-AMPK ⁇ ) on its alpha subunit by its upstream kinase.
  • p-AMPK ⁇ threonine site 172
  • the beta subunit of AMPK can also be bound by and regulated by metabolites such as glycogen.
  • Activated AMPK is capable of phosphorylating a variety of substrates.
  • Classical AMPK substrates include 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase 1 and acetyl-CoA carboxylase (ACC) 2 . respectively inhibit cholesterol biosynthesis and reduce fatty acid synthesis, leading to lipid synthesis pathway is inhibited, lipolysis pathways are strengthened, and the formation and development of this function and suppress diabetes have important links 3.
  • HMG-CoA 3-hydroxy-3-methylglutaryl coenzyme A
  • ACC acetyl-CoA carboxylase
  • AMPK also promotes the transfer of glucose transporter 4 (GLUT4) to the cell membrane, which can significantly promote the absorption and assimilation of glucose in the blood, lowering blood sugar, which is parallel to the insulin pathway, and thus in diabetic patients with insulin resistance.
  • GLUT4 glucose transporter 4
  • AMPK has an important role in the treatment of 4-5 .
  • AMPK above for the inhibition of protein synthesis and lipid can significantly inhibit the growth of tumor cells, thereby inhibiting tumor (e.g., melanoma, pancreatic cancer, ovarian cancer or breast cancer) and 6 development.
  • tumor e.g., melanoma, pancreatic cancer, ovarian cancer or breast cancer
  • AMPK is able to up-regulate the ratio of NAD + /NADH in vivo through molecules such as PGC1 and SIRT, which is thought to be closely related to longevity 7-9 .
  • Caenorhabditis elegans (C.elegans) fruit flies and mice have demonstrated the activation of AMPK significantly prolonged the life of these organisms 10-12.
  • AMPK is also an important regulator of autophagy.
  • AMPK can significantly promote the occurrence of macroautophagy by activating ULK1 (Unc-51Like Autophagy Activating Kinase 1), which is one of the basic processes for maintaining the balance of energy and material metabolism in living organisms. From a physiological point of view, it has been shown to be closely related to the occurrence of major diseases such as diabetes and cancer 13-14 . In particular, this function of AMPK is also closely related to the browning of adipose tissue 15-16 , which is considered to be an important process for relieving diabetes and reshaping healthy adipose tissue. Activated AMPK is indeed inhibited by promoting fat browning. The development of diabetes conditions 17 .
  • ULK1 Unc-51Like Autophagy Activating Kinase 1
  • AMPK is also possible by activating MFF (mitochondrial fission factor), mitochondria promote autophagy (mitophagy) 18, the latter disorders and Parkinson's disease, Alzheimer's psychosis important neurological diseases have important links 19.
  • MFF mitochondria fission factor
  • mitochondria promote autophagy mitochondria promote autophagy 18
  • Parkinson's disease Alzheimer's psychosis important neurological diseases have important links 19.
  • AMPK has a versatile effect on carbohydrate, fat and cholesterol metabolism and biosynthesis
  • AMPK is one of the most attractive drug targets for the treatment of major diseases.
  • AMPK has used a variety of screening methods, using AMPK as a target to obtain a large number of AMPK activators, and conducted a lot of research.
  • AMPK as a direct target for drugs has many drawbacks, such as insufficient efficacy or low specificity.
  • the most mature AMPK activators found so far are mostly used in the treatment of diabetes.
  • metformin the drug has minimal side effects on the body.
  • By activating AMPK it can significantly lower blood sugar and fatty liver levels, thereby alleviating diabetes. 20 Therefore, it has great advantages in many AMPK activators.
  • due to the poor permeability of the metformin molecule to the cell membrane a corresponding transporter is required to transport it into the cell, and these transport proteins are only distributed in a few tissues such as the liver, thereby greatly limiting the efficacy of the drug.
  • A-769662 which binds to the beta subunit directly and thus activates AMPK 22 in an allosteric manner.
  • A-769662 has a good cell membrane permeability, can enter most tissues, and has good continuity, it can play a role in 23 long.
  • A-769662 is not suitable for oral administration 23 , which greatly limits its range of applications. More trouble is, with regard to specific A-769662, in recent years has been reported that, in addition it also has a plurality of AMPK targets 24. Further, as far as the inventors are aware, none of the other drugs have entered the clinical experimental stage 22 .
  • Aldehydease (fructose-1, 6-bisphosphate aldolase, abbreviated as FBP aldolase, also referred to as aldolase in the present invention) includes aldolase A, aldolase B and aldolase C, which are important metabolism in sugar metabolism.
  • Enzyme in the glycolytic pathway, catalyzes the production of three-carbon fructose-1,6-diphosphate (FBP) to produce three-carbon glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), the latter further Pyruvate is produced by multiple enzymatic reactions; at the same time, it can catalyze the reverse process of this reaction in the gluconeogenesis pathway.
  • FBP three-carbon fructose-1,6-diphosphate
  • G3P three-carbon glyceraldehyde-3-phosphate
  • DHAP dihydroxyacetone phosphate
  • This process catalyzed by Aldolase cannot be compensated by other metabolic enzymes.
  • the function of the known aldolase is only limited to the nature of its metabolic enzyme itself.
  • this inhibitor was widely used as a classical inhibitor of GSK3, another kinase in the cell, that is, the inhibitor has a clear target of non-aldolase, and its IC 50 for aldolase and The IC 50 of GSK3 is close.
  • the preliminary results of the present inventors indicate that TDZD-8 is unable to inhibit the action of aldolase in MEF cells, which at least indicates that the inhibitor does not have universality for inhibition of aldolase.
  • One aspect of the invention relates to the use of any one of items (1) to (6) below for the preparation of a medicament for activating AMPK or for the preparation of a model for screening for a drug that activates AMPK:
  • nucleic acid construct comprising a polynucleotide for completely knocking out or partially knocking out the Aldolase gene; preferably, the polynucleotide is an siRNA such as shRNA, or a guide RNA for a CRISPR/Cas9 system;
  • a host cell wherein the polynucleotide encoding Aldolase is completely knocked out or partially knocked out; preferably, it comprises the nucleic acid construct of item (3);
  • the use, wherein the drug that inhibits or blocks Aldolase activity is an anti-Aldolase antibody or TDZD-8; preferably, the antibody is a monoclonal antibody.
  • the use, wherein the drug that inhibits or reduces the expression level of an Aldolase gene is selected from the group consisting of siRNAs such as shRNAs, and guide RNAs for the CRISPR-Cas9 system.
  • the present invention relates to the use of Aldolase as a drug target for the preparation of a medicament for activating AMPK.
  • Example 2 of the present invention AMPK was activated by inhibiting aldolase.
  • lentiviral-mediated shRNA infection was used to inhibit the expression of ALDOA-C (ie, ALDOA, ALDOB, and ALDOC) genes, and AMPK was found to be activated by detection of AMPK phosphorylation.
  • ALDOA-C ie, ALDOA, ALDOB, and ALDOC
  • shRNA refers to an RNA sequence of short hairpin RNA (short hairpin RNA) which is a short hairpin structure that can be expressed via a plasmid and interfere with target gene expression.
  • the shRNA targets ALDOA, ALDOB and ALDOC. In one embodiment of the invention, the shRNA comprises:
  • the model may be a mammalian cell (such as a human or mouse cell) or a mammal (such as a human or a mouse). If the test drug is capable of inhibiting or reducing the Aldolase gene expression level in the model, or inhibiting or blocking the level of Aldolase activity in the model, it can be used as a drug candidate.
  • Another aspect of the invention relates to any one of the following items (1) to (6) for inhibiting cholesterol production
  • a drug for alcohol synthesis a drug for reducing fatty acid synthesis, a drug for preventing obesity (for example, preventing obesity or losing weight), a drug for preventing and/or treating diabetes, a drug for preventing and/or treating a tumor, preventing and/or treating Parkinson's disease.
  • nucleic acid construct comprising a polynucleotide for completely knocking out or partially knocking out the Aldolase gene; preferably, the polynucleotide is an siRNA such as shRNA, or a guide RNA for a CRISPR/Cas9 system;
  • a host cell wherein the polynucleotide encoding Aldolase is completely knocked out or partially knocked out; preferably, it comprises the nucleic acid construct of item (3);
  • the tumor is any one or more selected from the group consisting of melanoma, pancreatic cancer, ovarian cancer, and breast cancer.
  • the use, wherein the drug that inhibits or blocks Aldolase activity is an anti-Aldolase antibody or TDZD-8; preferably, the antibody is a monoclonal antibody.
  • the use, wherein the drug that inhibits or reduces the expression level of an Aldolase gene is selected from the group consisting of siRNAs such as shRNAs, and guide RNAs for the CRISPR-Cas9 system.
  • the shRNA targets ALDOA, ALDOB and ALDOC. In one embodiment of the invention, the shRNA comprises:
  • a further aspect of the invention relates to a method of activating AMPK in vivo or in vitro, comprising the steps of inhibiting Aldolase activity or downregulating Aldolase gene expression levels, for example, including inhibiting the activity of Aldolase in a subject or in a cell or A step of downregulating the expression level of the Aldolase gene.
  • a further aspect of the invention relates to a method of screening for a drug selected from the group consisting of: adding a drug to be tested, and detecting Aldolase activity or detecting the expression level of the Aldolase gene:
  • the tumor is any one or more selected from the group consisting of melanoma, pancreatic cancer, ovarian cancer, and breast cancer.
  • test drug can inhibit or reduce the Aldolase gene expression level, or inhibit or block the Aldolase activity level, it can be used as a drug candidate.
  • test drug can inhibit or reduce the Aldolase gene expression level, or inhibit or block the Aldolase activity level, it can be used as a drug candidate.
  • the test drug is added to cells of an isolated mammal, such as a human or mouse, with the cells without the test drug as a control.
  • the test drug is administered to a mammal, such as a human or a mouse, to observe or detect whether the target symptom or indicator is improved.
  • a further aspect of the invention relates to a recombinant vector comprising an siRNA such as shRNA which downregulates the expression level of an Aldolase gene, or a guide RNA for the CRISPR-Cas9 system; preferably, the recombinant vector is a recombinant lentiviral vector.
  • an siRNA such as shRNA which downregulates the expression level of an Aldolase gene, or a guide RNA for the CRISPR-Cas9 system
  • the recombinant vector is a recombinant lentiviral vector.
  • the shRNA targets ALDOA, ALDOB and ALDOC. In one embodiment of the invention, the shRNA comprises:
  • a further aspect of the invention relates to a host cell comprising a recombinant vector of the invention, or wherein the polynucleotide encoding Aldolase is completely knocked out or partially knocked out.
  • a further aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a recombinant vector of the invention or a host cell of the invention, optionally further comprising a pharmaceutically acceptable excipient.
  • the pharmaceutical composition for activating AMPK inhibiting cholesterol synthesis, reducing fatty acid synthesis, preventing obesity, preventing and/or treating diabetes, preventing and/or treating tumors, prevention and/or Or treating Parkinson's disease, preventing and/or treating Alzheimer's disease, anti-aging or for prolonging mammalian life Life.
  • the tumor is any one or more selected from the group consisting of melanoma, pancreatic cancer, ovarian cancer, and breast cancer.
  • the invention also relates to a method of treating and/or preventing hypercholesterolemia, diabetes, tumor, Parkinson's disease or Alzheimer's disease or an anti-obesity (for example to prevent obesity or weight loss), anti-aging or prolonging the life of a mammal
  • a method comprising the steps of inhibiting Aldolase activity in a subject or downregulating an Aldolase gene expression level in a subject; for example comprising the step of administering to the subject an effective amount of a host cell or composition of the invention; for example comprising administering a need
  • the tumor is any one or more selected from the group consisting of melanoma, pancreatic cancer, ovarian cancer, and breast cancer.
  • the drug that inhibits or blocks Aldolase activity is an antibody against Aldolase or TDZD-8; preferably, the antibody is a monoclonal antibody.
  • the drug that inhibits or reduces the expression level of the Aldolase gene is selected from the group consisting of siRNAs such as shRNA, and guide RNA for the CRISPR-Cas9 system.
  • Inhibiting the level of Aldolase activity in a subject or downregulating the level of Aldolase gene expression in a subject depends on a number of factors, such as the severity of the condition being treated, the sex, age, weight and individual response of the patient or animal, and the condition to be treated The patient's condition and past medical history were chosen. It is common practice in the art to gradually increase the dosage from a level below that required to achieve the desired therapeutic effect and/or prophylactic effect until the desired effect is achieved.
  • the present invention also relates to any one of the following items (1) to (6) for activating AMPK or for preparing a drug for activating AMPK or for preparing a drug for screening for activating AMPK:
  • nucleic acid construct comprising a polynucleotide for completely knocking out or partially knocking out the Aldolase gene; preferably, the polynucleotide is an siRNA such as shRNA, or a guide RNA for a CRISPR/Cas9 system;
  • the present invention also relates to any one of the following items (1) to (6) for use in the preparation of a medicament for inhibiting cholesterol synthesis, a medicament for reducing fatty acid synthesis, an anti-obesity drug, and for preventing and/or treating diabetes.
  • nucleic acid construct comprising a polynucleotide for completely knocking out or partially knocking out the Aldolase gene; preferably, the polynucleotide is an siRNA such as shRNA, or a guide RNA for a CRISPR/Cas9 system;
  • a host cell wherein the polynucleotide encoding Aldolase is completely knocked out or partially knocked out; preferably, it comprises the nucleic acid construct of item (3);
  • the tumor is any one or more selected from the group consisting of melanoma, pancreatic cancer, ovarian cancer, and breast cancer.
  • fructose-1 (6-bisphosphate aldolase, abbreviated as FBP aldolase) is also referred to as aldolase in the present invention, and includes three isomers ALDOA, ALDOB and ALDOC.
  • ALDOA amino acid sequence of Aldolase or Aldolase
  • Aldolase includes the full length of the protein of Aldolase, and also includes its fusion protein.
  • mutations or variations may be naturally occurring or artificially introduced in the amino acid sequence of Aldolase without affecting its biological function.
  • the Aldolase is human Aldolase.
  • the Aldolase is any one, two or three selected from the group consisting of ALDOA, ALDOB and ALDOC.
  • Aldolase is ALDOA, ALDOB and ALDOC, it is also expressed as "ALDOA-C".
  • ADOA human Aldolase A
  • ADOB human Aldolase B
  • amino acid sequence of human Aldolase C (ALDOC) is as follows: (364 AA)
  • the aldolase gene when referring to the aldolase gene, it comprises not only the nucleic acid sequence encoding the aldolase but also the degenerate sequence thereof; further, it may further comprise a regulatory sequence other than the reading frame.
  • the aldolase gene is a human aldolase gene.
  • CDS nucleic acid sequence encoding ALDOA is as follows: (1095 BP)
  • CDS The nucleic acid sequence (CDS) encoding ALDOB is as follows: (1095 BP)
  • CDS The nucleic acid sequence (CDS) encoding ALDOC is as follows: (1095 BP)
  • nucleic acid construct is a single- or double-stranded nucleic acid molecule, preferably an artificially constructed nucleic acid molecule.
  • the nucleic acid construct further comprises one or more regulatory sequences operably linked.
  • operably linked refers to a spatial arrangement of the functionality of two or more nucleotide regions or nucleic acid sequences.
  • the "operably linked” can be achieved by means of genetic recombination.
  • vector refers to a nucleic acid delivery vehicle into which a polynucleotide inhibiting a certain protein can be inserted.
  • vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast Artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1 derived artificial chromosome (PAC); phage such as ⁇ phage or M13 phage and animal virus.
  • the animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papilloma polyves Virus (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as lentiviruses
  • adeno-associated viruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papillomaviruses such as papilloma polyves Virus (such as SV40).
  • a vector may contain a variety of elements that control expression.
  • the term "host cell” refers to a cell into which a vector is introduced, including many cell types such as prokaryotic cells such as Escherichia coli or Bacillus subtilis, such as fungal cells such as yeast cells or Aspergillus, such as S2 Drosophila cells or Insect cells such as Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • S2 Drosophila cells such as S2 Drosophila cells or Insect cells
  • Sf9 fibroblasts
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • the term "effective amount” refers to a dose that can achieve a treatment, prevention, alleviation, and/or alleviation of a disease or condition described herein in a subject.
  • disease and/or condition refers to a physical state of the subject that is associated with the disease and/or condition described herein.
  • subject can refer to a patient or other animal that receives the pharmaceutical composition of the invention to treat, prevent, ameliorate and/or alleviate the disease or condition of the invention, particularly a mammal, such as a human, a dog, a monkey, or a cow. , horses, etc.
  • knockdown of DNA or RNA includes, but is not limited to, complete knockout and partial knockout.
  • Complete knockout refers to reducing the level of target DNA or target RNA or the level of expressed protein to an almost undetectable level (in fact, it is generally difficult to knock out the target DNA or target RNA 100%).
  • Partial knockout means that the degree of knockout is greater than zero, less than the case of complete knockout.
  • the shRNA and protein mutants of the present invention using aldolase can significantly activate AMPK directly, thereby realizing the use of aldolase as a target to develop drugs for activating AMPK, overcoming the direct AMPK in the prior art. Difficulties in the drug target.
  • FIG. 1 Immunoblot results of shRNA inhibition of ALDOA-C gene expression.
  • #1 and #2 represent one of two shRNAs targeting ALDOA, ALDOB or ALDOC, respectively.
  • ALDOA #1 stands for SEQ ID NO: 7
  • #2 represents SEQ ID NO: 8.
  • ALDOB #1 represents SEQ ID NO: 9
  • #2 represents SEQ ID NO: 10.
  • ALDOC #1 represents SEQ ID NO: 11, and #2 represents SEQ ID NO: 12.
  • Figure 2 Detection map of the effect of shRNA on inhibition of AMPK after ALDOA-C. Among them, siGFP was a control group.
  • Example 1 shRNA inhibits expression of ALDOA-C gene
  • mouse fibroblast MEF which is an immortalized MEF, which is obtained by isolating SV40T antigen from mouse embryos and immortalizing the cells. Construction methods can be found in the literature Lei Y, Methods Mol Biol. 2013; 1031: 59-64. Generation and culture of mouse embryonic fibroblasts.
  • Human embryonic kidney cell HEK293T (cat. CRL-3216), purchased from ATCC.
  • the vector pLVX-IRES (cat. #631849) was purchased from Clontech.
  • Transfection reagent Lipofectamine 2000 (cat. 11668-027), purchased from Invitrogen.
  • Dulbecco's modified Eagle's medium (DMEM, Gibco, cat. 11965), available from Thermofisher.
  • anti-ALDOB catalog.18065-1-AP, purchased from Ptoteintech;
  • anti-ALDOC catalog.AM2215b, purchased from Abgent
  • Anti- ⁇ -tubulin (cat. #2128), available from Cell Signaling Technology.
  • HRP-conjugated goat anti-mouse IgG (cat. 115-035-003) and HRP-conjugated goat anti-rabbit IgG (cat. 111-035-003) were purchased from Jackson ImmunoResearch.
  • the expression plasmid (pLVX-IRES-ALDOA-C) of ALDOA-C induced expression by Doxycycline (Dox) was constructed. Using the conventional cloning method, see the Guide to Molecular Cloning, the steps are as follows:
  • CDS fragments of ALDOA, ALDOB and ALDOC were amplified by PCR, respectively, while restriction endonuclease treatment (EcoRI and BamHI double digestion) vector pLVX-IRES, and finally CDS fragments and enzymes The cleaved vectors were ligated to obtain pLVX-IRES-ALDOA-C.
  • pLVX-IRES-ALDOA-C was packaged into a lentivirus in HEK293T cells, and MEF cells were infected with the virus for more than 24 hours.
  • the steps of introducing a lentivirus into an expression plasmid are as follows:
  • MEFs mouse fibroblasts
  • the cells were cultured in DMEM medium containing 100 ng/ml of Dox, and thus constructed to induce MEF cell lines expressing ALDOA-C.
  • each of the two shRNAs targeting ALDOA, ALDOB or ALDOC was introduced into the above MEF cell strain by lentivirus for more than 24 hours, respectively, such that endogenously expressed ALDOA, ALDOB or ALDOC were blocked.
  • the specific steps for introducing a lentivirus into shRNA are as follows:
  • MEFs mouse fibroblasts
  • the cells were cultured in DMEM medium without Dox for 12 hours, so that the induced expression of exogenous ALDOA-C disappeared, and then the cells were collected for the following Western blotting experiments.
  • Example 2 shRNA targeting ALDOA-C is capable of activating AMPK
  • anti-AMPK ⁇ catalog#2532, 1:1000 for IB, purchased from Cell Signaling Technology;
  • anti-phospho-ACC-Ser79 catalog. #3661, 1:1000 for IB, purchased from Cell Signaling Technology;
  • Glucose-free DMEM (Gibco, cat. 11966) was purchased from Thermofisher.
  • the MEF cells simultaneously knocked down by ALDOA-C as shown in Fig. 1 were prepared by referring to the method as described in Example 1.
  • shRNA targeting GFP Green Fluorescent Protein
  • non-targeting control group shRNA sequence 5'-GGCACAAGCTGGAGTACAA-3' (SEQ ID NO: 13) targeting GFP was constructed. Refer to Example 1.
  • Cool B Zinker B, Chiou W, Kifle L, Cao N, Perham M, Dickinson R, Adler A, Gagne G, Iyengar R, Zhao G, Marsh K, Kym P, Jung P, Camp HS, Frevert E. Identification and characterization of a small molecule AMPK activator that treats key components of type 2diabetes and the metabolic syndrome. Cell Metab.2006Jun;3(6):403-16.
  • AMP-activated protein kinase activator A-769662 is an inhibitor of the Na(+)-K(+)-ATPase.Am J Physiol Cell Physiol.2009Dec;297( 6): C1554-66.

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Abstract

Disclosed is the use of FBP aldolase in the preparation of a drug activating AMPK. Also disclosed are the uses of FBP aldolase in the preparation of a drug for inhibiting cholesterol synthesis, a drug for reducing fatty acid synthesis, a drug for preventing and/or treating diabetes, a drug for preventing and/or treating tumours, a drug for preventing and/or treating Parkinson's disease, a drug for preventing and/or treating Alzheimer's disease, or a drug for prolonging the lifespan of an organism. The use of FBP aldolase as a target to develop drugs to activate AMPK overcomes the difficulties of directly using AMPK as a drug target in the prior art and has good application prospects.

Description

FBP aldolase在制备激活AMPK的药物中的用途Use of FBP aldolase in the preparation of a drug for activating AMPK 技术领域Technical field
本发明属于生物医药领域，涉及FBP aldolase在制备激活AMPK的药物中的用途。本发明还涉及在制备抑制胆固醇合成的药物、降低脂肪酸合成的药物、预防和/或治疗糖尿病的药物、预防和/或治疗肿瘤的药物、预防和/或治疗帕金森症的药物、预防和/或治疗阿尔茨海默症的药物或者延长哺乳动物寿命的药物中的用途。The invention belongs to the field of biomedicine and relates to the use of FBP aldolase in the preparation of a medicament for activating AMPK. The present invention also relates to a medicament for inhibiting cholesterol synthesis, a medicament for reducing fatty acid synthesis, a medicament for preventing and/or treating diabetes, a medicament for preventing and/or treating a tumor, a medicament for preventing and/or treating Parkinson's disease, prevention and/or Or use in the treatment of drugs for Alzheimer's disease or drugs that prolong the lifespan of mammals.
背景技术Background technique
5’-腺苷一磷酸活化的蛋白激酶(AMPK)是调节细胞和机体能量平衡的重要分子。AMPK由3个不同的亚基所组成，各个亚基分别具有数个同功型：α亚基(α1或α2)；β亚基(β1或β2)；及γ亚基(γ1、γ2或γ3)；总共有12种可能的杂三聚体的同功型，每种同功型均有其特异性分布的组织类型，各种同功型共同组成了广泛分布在机体内的所有组织中的AMPK复合体。另外，AMPK的12种杂三聚体的功能是类似的，或者说目前并未见其不同功能的报道，其差别仅仅在组织特异性分布上，任意一种同功型的AMPK激活都能够引起同样的下游蛋白的激活。The 5'-adenosine monophosphate-activated protein kinase (AMPK) is an important molecule that regulates the energy balance between cells and the body. AMPK is composed of three different subunits, each of which has several isoforms: α subunit (α1 or α2); β subunit (β1 or β2); and γ subunit (γ1, γ2 or γ3) There are a total of 12 possible heterotrimer isoforms, each of which has its specific distribution of tissue types, and various isoforms together constitute all tissues widely distributed in the body. AMPK complex. In addition, the functions of the 12 heterotrimers of AMPK are similar, or there are no reports of different functions at present, the difference is only in the tissue-specific distribution, and any isoform of AMPK activation can cause The same downstream protein is activated.
传统的观点认为，AMPK的激活是由其别构激活因子——AMP/ADP及其类似物介导的。AMP/ADP能够结合AMPK的γ亚基，引起全酶的结构变化，使其更易被其上游激酶磷酸化其α亚基上的苏氨酸172位位点(p-AMPKα)从而被激活。除了γ亚基引起的别构激活，AMPK的β亚基也能被糖原等代谢物结合并被其调节活力。Traditionally, AMPK activation is mediated by its allosteric activator, AMP/ADP and its analogs. AMP/ADP binds to the gamma subunit of AMPK, causing structural changes in the holoenzyme, making it more susceptible to phosphorylation of its threonine site 172 (p-AMPKα) on its alpha subunit by its upstream kinase. In addition to allosteric activation by the gamma subunit, the beta subunit of AMPK can also be bound by and regulated by metabolites such as glycogen.
活化的AMPK能够磷酸化多种底物，经典的AMPK底物包括3-羟基-3-甲基戊二酰辅酶A(HMG-CoA)还原酶1和乙酰辅酶A羧化酶(ACC)2，其分别抑制胆固醇生物合成及降低脂肪酸合成，导致脂质合成代谢途径被抑制，脂类分解途径被加强，而这一功能与抑制糖尿病的形成和发展有重要的联系3。AMPK还能够促进葡萄糖转运子4(GLUT4)向细胞膜上的转移，后者能够显著促进血液中葡萄糖的吸收和同化，降低血糖，该作用与胰岛素通路相平行，因而在具有胰岛素抵抗的糖尿病病患的治疗中具有重要的作用4-5。上述AMPK对于脂质和蛋白质合成的抑制作用能够显著抑制肿瘤细胞的生长，从而抑制肿瘤(例如黑色素瘤、胰腺癌、卵巢癌或乳腺癌)的发生和发展6。此外，AMPK能够通过PGC1和SIRT等分子，上调体内NAD+/NADH的比例，该功能被 认为和长寿密切相关7-9。在线虫(C.elegans)、果蝇和小鼠中都已经证明了激活AMPK能够显著延长这些有机体的寿命10-12。此外，AMPK还是自噬作用重要的调节因子。例如，AMPK能够通过激活ULK1(Unc-51Like Autophagy Activating Kinase 1)，显著地促进巨自噬作用(macroautophagy)的发生，巨自噬作用是生命体维持能量和物质代谢平衡的基本过程之一，从生理功能的角度来看，它已被证明和糖尿病、肿瘤等重大疾病的发生密切相关13-14。特别地，AMPK的这一功能还和脂肪组织的棕化密切相关15-16，后者被认为是缓解糖尿病，重塑健康脂肪组织的重要过程，激活的AMPK也确实通过促进脂肪棕化抑制了糖尿病病情的发展17。AMPK还能够通过激活MFF(mitochondrial fission factor)，促进线粒体自噬作用(mitophagy)18，而后者的失调和帕金森症、阿尔茨海默症等重要的神经系统疾病有着重要的联系19。由于AMPK对碳水化合物、脂肪和胆固醇代谢及生物合成具有多功能性的作用，所以AMPK是治疗重大疾病最吸引人的药物靶标之一。近二十年来，学术界已经运用了多种筛选方法，以AMPK作为靶标得到了大量AMPK的激活剂，并展开了大量研究。Activated AMPK is capable of phosphorylating a variety of substrates. Classical AMPK substrates include 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase 1 and acetyl-CoA carboxylase (ACC) 2 . respectively inhibit cholesterol biosynthesis and reduce fatty acid synthesis, leading to lipid synthesis pathway is inhibited, lipolysis pathways are strengthened, and the formation and development of this function and suppress diabetes have important links 3. AMPK also promotes the transfer of glucose transporter 4 (GLUT4) to the cell membrane, which can significantly promote the absorption and assimilation of glucose in the blood, lowering blood sugar, which is parallel to the insulin pathway, and thus in diabetic patients with insulin resistance. Has an important role in the treatment of 4-5 . AMPK above for the inhibition of protein synthesis and lipid can significantly inhibit the growth of tumor cells, thereby inhibiting tumor (e.g., melanoma, pancreatic cancer, ovarian cancer or breast cancer) and 6 development. In addition, AMPK is able to up-regulate the ratio of NAD + /NADH in vivo through molecules such as PGC1 and SIRT, which is thought to be closely related to longevity 7-9 . Caenorhabditis elegans (C.elegans), fruit flies and mice have demonstrated the activation of AMPK significantly prolonged the life of these organisms 10-12. In addition, AMPK is also an important regulator of autophagy. For example, AMPK can significantly promote the occurrence of macroautophagy by activating ULK1 (Unc-51Like Autophagy Activating Kinase 1), which is one of the basic processes for maintaining the balance of energy and material metabolism in living organisms. From a physiological point of view, it has been shown to be closely related to the occurrence of major diseases such as diabetes and cancer 13-14 . In particular, this function of AMPK is also closely related to the browning of adipose tissue 15-16 , which is considered to be an important process for relieving diabetes and reshaping healthy adipose tissue. Activated AMPK is indeed inhibited by promoting fat browning. The development of diabetes conditions 17 . AMPK is also possible by activating MFF (mitochondrial fission factor), mitochondria promote autophagy (mitophagy) 18, the latter disorders and Parkinson's disease, Alzheimer's psychosis important neurological diseases have important links 19. Because AMPK has a versatile effect on carbohydrate, fat and cholesterol metabolism and biosynthesis, AMPK is one of the most attractive drug targets for the treatment of major diseases. In the past two decades, academia has used a variety of screening methods, using AMPK as a target to obtain a large number of AMPK activators, and conducted a lot of research.
然而，这些研究的结果表明，以AMPK作为药物的直接靶标有诸多的缺陷，例如药效不足或特异性较低。目前发现的最为成熟的AMPK激活剂大多应用于糖尿病的治疗领域。以广泛应用的AMPK激活剂二甲双胍为例，该药物对机体副作用极小，通过激活AMPK能够显著降低血糖和脂肪肝水平，从而缓解糖尿病病情20，因而在众多AMPK激活剂中具有很大的优势。但是，由于二甲双胍分子对细胞膜的通透性较差，需要相应的转运蛋白将其转运进细胞中，而这些转运蛋白仅仅分布于少数几个组织如肝脏中，从而极大地限制了该药物药效的行使和AMPK功能的发挥21。除二甲双胍之外，目前在临床实验中走得最远的药物是A-769662，该化合物能够直接结合β亚基从而别构激活AMPK22。与二甲双胍相比，A-769662具有良好的细胞膜通透性，能够进入大部分组织中，并具有良好的持续性，能够长时间发挥作用23。然而，A-769662不适合口服23，这大大限制了其应用范围。更为麻烦的是，关于A-769662的特异性，近年来已经有报道表明，其还具有多个除了AMPK之外的靶点24。另外，据本发明人所知，除此之外的其它药物均未进入临床实验阶段22。However, the results of these studies indicate that AMPK as a direct target for drugs has many drawbacks, such as insufficient efficacy or low specificity. The most mature AMPK activators found so far are mostly used in the treatment of diabetes. Taking the widely used AMPK activator metformin as an example, the drug has minimal side effects on the body. By activating AMPK, it can significantly lower blood sugar and fatty liver levels, thereby alleviating diabetes. 20 Therefore, it has great advantages in many AMPK activators. However, due to the poor permeability of the metformin molecule to the cell membrane, a corresponding transporter is required to transport it into the cell, and these transport proteins are only distributed in a few tissues such as the liver, thereby greatly limiting the efficacy of the drug. The exercise and play of the AMPK function 21 . In addition to metformin, the drug that is currently the most distantly used in clinical trials is A-769662, which binds to the beta subunit directly and thus activates AMPK 22 in an allosteric manner. Compared with metformin, A-769662 has a good cell membrane permeability, can enter most tissues, and has good continuity, it can play a role in 23 long. However, A-769662 is not suitable for oral administration 23 , which greatly limits its range of applications. More trouble is, with regard to specific A-769662, in recent years has been reported that, in addition it also has a plurality of AMPK targets 24. Further, as far as the inventors are aware, none of the other drugs have entered the clinical experimental stage 22 .
醛缩酶(fructose-1,6-bisphosphate aldolase，简称为FBP aldolase，本发明中亦简称为aldolase)包括醛缩酶A、醛缩酶B以及醛缩酶C，是糖代谢中的重要的代谢酶，在糖酵解途径中，它催化六碳的果糖-1,6-二磷酸(FBP)生成三碳的甘油醛 -3-磷酸(G3P)和磷酸二羟丙酮(DHAP)，后者进一步经过多次酶促反应生成丙酮酸；同时，在糖异生途径中，它能够催化这一反应的逆过程。Aldolase所催化的这一过程是不能被其它代谢酶补偿的。目前，已知的aldolase的功能还仅仅局限在其代谢酶本身的性质上。目前已经报道，aldolase的某些突变体可能与果糖不耐受有关，但具体机制还不明确。值得一提的是，在肿瘤组织中，Aldolase的表达水平明显升高，这可能促进了瓦氏效应(Warburg effect)和肿瘤细胞的发展，而在肿瘤细胞中敲低aldolase会直接引起肿瘤细胞生长的停止25。Aldehydease (fructose-1, 6-bisphosphate aldolase, abbreviated as FBP aldolase, also referred to as aldolase in the present invention) includes aldolase A, aldolase B and aldolase C, which are important metabolism in sugar metabolism. Enzyme, in the glycolytic pathway, catalyzes the production of three-carbon fructose-1,6-diphosphate (FBP) to produce three-carbon glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), the latter further Pyruvate is produced by multiple enzymatic reactions; at the same time, it can catalyze the reverse process of this reaction in the gluconeogenesis pathway. This process catalyzed by Aldolase cannot be compensated by other metabolic enzymes. Currently, the function of the known aldolase is only limited to the nature of its metabolic enzyme itself. It has been reported that certain mutants of aldolase may be involved in fructose intolerance, but the specific mechanism is not clear. It is worth mentioning that in tumor tissues, the expression level of Aldolase is significantly increased, which may promote the Warburg effect and the development of tumor cells, and knocking down aldolase in tumor cells will directly cause tumor cell growth. Stop 25 .
早在1970年以前，研究人员就设计了众多不能被aldolase催化的果糖-1,6-二磷酸的类似物，在体外实验中起到竞争果糖-1,6-二磷酸和aldolase结合的作用来达到抑制aldolase的效果。然而，这些抑制剂都不能透过细胞膜、进入细胞发挥作用，其应用也只停留在体外的生化实验水平上。在生理水平上，目前唯一报道的aldolase的抑制剂是TDZD-8(报道于2016年，CAS#327036-89-5，使用MDA-MB-231细胞作为生理模型)26。但是，早先该抑制剂是作为细胞内的另一种激酶——GSK3的经典抑制剂被广泛应用，也就是说，该抑制剂有明确的非aldolase的靶标，且其对于aldolase的IC50和对于GSK3的IC50是接近的。除此之外，本发明人初步的实验结果表明，TDZD-8无法在MEF细胞中起到抑制aldolase的作用，这至少说明该抑制剂对aldolase的抑制不具有普遍性。As early as 1970, the researchers designed a number of analogues of fructose-1,6-diphosphate that could not be catalyzed by aldolase, which competed in vitro for the binding of fructose-1,6-diphosphate and aldolase. Achieve the effect of inhibiting aldolase. However, these inhibitors can not penetrate the cell membrane and enter the cell, and their application only stays at the level of biochemical experiments in vitro. At the physiological level, the only inhibitor of aldolase currently reported is TDZD-8 (reported in 2016, CAS#327036-89-5, using MDA-MB-231 cells as a physiological model) 26 . However, earlier this inhibitor was widely used as a classical inhibitor of GSK3, another kinase in the cell, that is, the inhibitor has a clear target of non-aldolase, and its IC 50 for aldolase and The IC 50 of GSK3 is close. In addition, the preliminary results of the present inventors indicate that TDZD-8 is unable to inhibit the action of aldolase in MEF cells, which at least indicates that the inhibitor does not have universality for inhibition of aldolase.
目前，亟需开发新的激活AMPK的药物或者技术手段。At present, there is an urgent need to develop new drugs or technical means to activate AMPK.
发明内容Summary of the invention
本发明人经过深入的研究和创造性的劳动，发现了一个新的AMPK的调节因子——醛缩酶(aldolase)，该因子能够直接调节AMPK的激活，并将在将来作为一个调节AMPK的重要靶点被进一步研究与应用；并且本发明人还惊奇地发现，下调Aldolase基因表达水平，或者抑制Aldolase，能够显著地激活AMPK，具有应用于低AMPK水平相关疾病的潜力。由此提供了下述发明：After intensive research and creative labor, the inventors discovered a new AMPK regulatory factor, aldolase, which directly regulates the activation of AMPK and will serve as an important target for the regulation of AMPK in the future. The point was further studied and applied; and the inventors have also surprisingly found that down-regulating Aldolase gene expression levels, or inhibiting Aldolase, can significantly activate AMPK with potential for application to diseases associated with low AMPK levels. The following invention is thus provided:
本发明的一个方面涉及选自如下的(1)－(6)项中的任意一项在制备激活AMPK的药物或者在制备筛选激活AMPK的药物的模型中的用途：One aspect of the invention relates to the use of any one of items (1) to (6) below for the preparation of a medicament for activating AMPK or for the preparation of a model for screening for a drug that activates AMPK:
(1)Aldolase； (1) Aldolase;
(2)编码Aldolase的多核苷酸；(2) a polynucleotide encoding an Aldolase;
(3)核酸构建体，其含有用于完全敲除或者部分敲除Aldolase基因的多核苷酸；优选地，所述多核苷酸为siRNA例如shRNA，或者为用于CRISPR/Cas9系统的guide RNA；(3) a nucleic acid construct comprising a polynucleotide for completely knocking out or partially knocking out the Aldolase gene; preferably, the polynucleotide is an siRNA such as shRNA, or a guide RNA for a CRISPR/Cas9 system;
(4)宿主细胞，其中的编码Aldolase的多核苷酸被完全敲除或部分敲除；优选地，其含有第(3)项所述的核酸构建体；(4) a host cell, wherein the polynucleotide encoding Aldolase is completely knocked out or partially knocked out; preferably, it comprises the nucleic acid construct of item (3);
(5)抑制或阻断Aldolase活性的药物；(5) a drug that inhibits or blocks the activity of Aldolase;
(6)抑制或降低Aldolase基因表达水平的药物。(6) A drug that inhibits or reduces the expression level of an Aldolase gene.
在本发明的一个实施方案中，所述的用途，其中，所述抑制或阻断Aldolase活性的药物为抗Aldolase的抗体或TDZD-8；优选地，所述抗体为单克隆抗体。In one embodiment of the present invention, the use, wherein the drug that inhibits or blocks Aldolase activity is an anti-Aldolase antibody or TDZD-8; preferably, the antibody is a monoclonal antibody.
在本发明的一个实施方案中，所述的用途，其中，所述抑制或降低Aldolase基因表达水平的药物选自siRNA例如shRNA，以及用于CRISPR-Cas9系统的guide RNA。In one embodiment of the present invention, the use, wherein the drug that inhibits or reduces the expression level of an Aldolase gene is selected from the group consisting of siRNAs such as shRNAs, and guide RNAs for the CRISPR-Cas9 system.
本发明涉及Aldolase作为药物靶点在制备激活AMPK的药物中的用途。The present invention relates to the use of Aldolase as a drug target for the preparation of a medicament for activating AMPK.
本发明的实施例2中，通过抑制aldolase来激活AMPK。具体而言，利用慢病毒介导shRNA感染方法，抑制ALDOA-C(即ALDOA、ALDOB和ALDOC)基因的表达，然后通过对AMPK磷酸化的检测发现AMPK被激活。其中，术语“shRNA”是指小发夹结构RNA(Short Hairpin RNA，简称“shRNA”)是一种短发夹结构的RNA序列，可以经由质粒表达而来，并干扰靶基因表达。In Example 2 of the present invention, AMPK was activated by inhibiting aldolase. Specifically, lentiviral-mediated shRNA infection was used to inhibit the expression of ALDOA-C (ie, ALDOA, ALDOB, and ALDOC) genes, and AMPK was found to be activated by detection of AMPK phosphorylation. The term "shRNA" refers to an RNA sequence of short hairpin RNA (short hairpin RNA) which is a short hairpin structure that can be expressed via a plasmid and interfere with target gene expression.
在本发明的一个实施方案中，所述shRNA靶向ALDOA、ALDOB和ALDOC。在本发明的一个实施方案中，所述shRNA包括：In one embodiment of the invention, the shRNA targets ALDOA, ALDOB and ALDOC. In one embodiment of the invention, the shRNA comprises:
选自SEQ ID NO:7和SEQ ID NO:8所示序列中的至少一条，At least one selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 8,
选自SEQ ID NO:9和SEQ ID NO:10所示序列中的至少一条，以及At least one selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 10, and
选自SEQ ID NO:11和SEQ ID NO:12所示序列中的至少一条。At least one selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12.
在本发明的一个实施方案中，所述模型可以是哺乳动物细胞(例如人或小鼠的细胞)或者哺乳动物(例如人或小鼠)。如果该待测药物能够抑制或降低模型中的Aldolase基因表达水平，或者抑制或阻断模型中的Aldolase活性水平，则可以作为候选药物。In one embodiment of the invention, the model may be a mammalian cell (such as a human or mouse cell) or a mammal (such as a human or a mouse). If the test drug is capable of inhibiting or reducing the Aldolase gene expression level in the model, or inhibiting or blocking the level of Aldolase activity in the model, it can be used as a drug candidate.
本发明的另一方面涉及选自如下的(1)－(6)项中的任意一项在制备抑制胆固 醇合成的药物、降低脂肪酸合成的药物、抗肥胖(例如预防肥胖或者减肥)的药物、预防和/或治疗糖尿病的药物、预防和/或治疗肿瘤的药物、预防和/或治疗帕金森症的药物、预防和/或治疗阿尔茨海默症的药物、抗衰老的药物或者用于延长哺乳动物寿命的药物中的用途：Another aspect of the invention relates to any one of the following items (1) to (6) for inhibiting cholesterol production A drug for alcohol synthesis, a drug for reducing fatty acid synthesis, a drug for preventing obesity (for example, preventing obesity or losing weight), a drug for preventing and/or treating diabetes, a drug for preventing and/or treating a tumor, preventing and/or treating Parkinson's disease. Use of drugs, drugs for the prevention and/or treatment of Alzheimer's disease, anti-aging drugs or drugs for prolonging the lifespan of mammals:
(1)Aldolase；(1) Aldolase;
(2)编码Aldolase的多核苷酸；(2) a polynucleotide encoding an Aldolase;
(3)核酸构建体，其含有用于完全敲除或者部分敲除Aldolase基因的多核苷酸；优选地，所述多核苷酸为siRNA例如shRNA，或者为用于CRISPR/Cas9系统的guide RNA；(3) a nucleic acid construct comprising a polynucleotide for completely knocking out or partially knocking out the Aldolase gene; preferably, the polynucleotide is an siRNA such as shRNA, or a guide RNA for a CRISPR/Cas9 system;
(4)宿主细胞，其中的编码Aldolase的多核苷酸被完全敲除或部分敲除；优选地，其含有第(3)项所述的核酸构建体；(4) a host cell, wherein the polynucleotide encoding Aldolase is completely knocked out or partially knocked out; preferably, it comprises the nucleic acid construct of item (3);
(5)抑制或阻断Aldolase活性的药物；(5) a drug that inhibits or blocks the activity of Aldolase;
(6)抑制或降低Aldolase基因表达水平的药物。(6) A drug that inhibits or reduces the expression level of an Aldolase gene.
优选地，所述肿瘤为选自黑色素瘤、胰腺癌、卵巢癌和乳腺癌中的任意一种或几种。Preferably, the tumor is any one or more selected from the group consisting of melanoma, pancreatic cancer, ovarian cancer, and breast cancer.
在本发明的一个实施方案中，所述的用途，其中，所述抑制或阻断Aldolase活性的药物为抗Aldolase的抗体或TDZD-8；优选地，所述抗体为单克隆抗体。In one embodiment of the present invention, the use, wherein the drug that inhibits or blocks Aldolase activity is an anti-Aldolase antibody or TDZD-8; preferably, the antibody is a monoclonal antibody.
在本发明的一个实施方案中，所述的用途，其中，所述抑制或降低Aldolase基因表达水平的药物选自siRNA例如shRNA，以及用于CRISPR-Cas9系统的guide RNA。In one embodiment of the present invention, the use, wherein the drug that inhibits or reduces the expression level of an Aldolase gene is selected from the group consisting of siRNAs such as shRNAs, and guide RNAs for the CRISPR-Cas9 system.
在本发明的一个实施方案中，所述shRNA靶向ALDOA、ALDOB和ALDOC。在本发明的一个实施方案中，所述shRNA包括：In one embodiment of the invention, the shRNA targets ALDOA, ALDOB and ALDOC. In one embodiment of the invention, the shRNA comprises:
选自SEQ ID NO:7和SEQ ID NO:8所示序列中的至少一条，At least one selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 8,
选自SEQ ID NO:9和SEQ ID NO:10所示序列中的至少一条，以及At least one selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 10, and
选自SEQ ID NO:11和SEQ ID NO:12所示序列中的至少一条。At least one selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12.
本发明的再一方面涉及一种在体内或体外激活AMPK的方法，包括抑制Aldolase活性或者下调Aldolase基因表达水平的步骤，例如，包括抑制有需求的受试者中或者细胞中的Aldolase的活性或者下调Aldolase基因的表达水平的步骤。 A further aspect of the invention relates to a method of activating AMPK in vivo or in vitro, comprising the steps of inhibiting Aldolase activity or downregulating Aldolase gene expression levels, for example, including inhibiting the activity of Aldolase in a subject or in a cell or A step of downregulating the expression level of the Aldolase gene.
本发明的再一方面涉及一种筛选选自如下的药物的方法，包括加入待测药物，以及检测Aldolase活性或者检测Aldolase基因表达水平的步骤：A further aspect of the invention relates to a method of screening for a drug selected from the group consisting of: adding a drug to be tested, and detecting Aldolase activity or detecting the expression level of the Aldolase gene:
激活AMPK的药物、抑制胆固醇合成的药物、降低脂肪酸合成的药物、抗肥胖的药物、预防和/或治疗糖尿病的药物、预防和/或治疗肿瘤的药物、预防和/或治疗帕金森症的药物、预防和/或治疗阿尔茨海默症的药物、抗衰老的药物或者用于延长哺乳动物寿命的药物。优选地，所述肿瘤为选自黑色素瘤、胰腺癌、卵巢癌和乳腺癌中的任意一种或几种。Activating AMPK drugs, drugs for inhibiting cholesterol synthesis, drugs for lowering fatty acid synthesis, anti-obesity drugs, drugs for preventing and/or treating diabetes, drugs for preventing and/or treating tumors, drugs for preventing and/or treating Parkinson's disease , drugs for the prevention and/or treatment of Alzheimer's disease, anti-aging drugs or drugs for prolonging the lifespan of mammals. Preferably, the tumor is any one or more selected from the group consisting of melanoma, pancreatic cancer, ovarian cancer, and breast cancer.
如果该待测药物能够抑制或降低Aldolase基因表达水平，或者抑制或阻断Aldolase活性水平，则可以作为候选药物。例如：If the test drug can inhibit or reduce the Aldolase gene expression level, or inhibit or block the Aldolase activity level, it can be used as a drug candidate. E.g:
在本发明的一个实施方案中，将待测药物加入到分离的哺乳动物例如人或小鼠的细胞中，以不加待测药物的细胞作为对照。In one embodiment of the invention, the test drug is added to cells of an isolated mammal, such as a human or mouse, with the cells without the test drug as a control.
在本发明的一个实施方案中，将待测药物施与哺乳动物例如人或小鼠，观察或者检测目标症状或者指标是否有改善。In one embodiment of the invention, the test drug is administered to a mammal, such as a human or a mouse, to observe or detect whether the target symptom or indicator is improved.
本发明的再一方面涉及一种重组载体，其含有下调Aldolase基因表达水平的siRNA例如shRNA，或者用于CRISPR-Cas9系统的guide RNA；优选地，所述重组载体为重组慢病毒载体。A further aspect of the invention relates to a recombinant vector comprising an siRNA such as shRNA which downregulates the expression level of an Aldolase gene, or a guide RNA for the CRISPR-Cas9 system; preferably, the recombinant vector is a recombinant lentiviral vector.
在本发明的一个实施方案中，所述shRNA靶向ALDOA、ALDOB和ALDOC。在本发明的一个实施方案中，所述shRNA包括：In one embodiment of the invention, the shRNA targets ALDOA, ALDOB and ALDOC. In one embodiment of the invention, the shRNA comprises:
选自SEQ ID NO:7和SEQ ID NO:8所示序列中的至少一条，At least one selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 8,
选自SEQ ID NO:9和SEQ ID NO:10所示序列中的至少一条，以及At least one selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 10, and
选自SEQ ID NO:11和SEQ ID NO:12所示序列中的至少一条。At least one selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12.
本发明的再一方面涉及一种宿主细胞，其含有本发明的重组载体，或者其中的编码Aldolase的多核苷酸被完全敲除或部分敲除。A further aspect of the invention relates to a host cell comprising a recombinant vector of the invention, or wherein the polynucleotide encoding Aldolase is completely knocked out or partially knocked out.
本发明的再一方面涉及一种药物组合物，其包含本发明的重组载体或者本发明的宿主细胞，可选地，其还包含药学上可接受的辅料。A further aspect of the invention relates to a pharmaceutical composition comprising a recombinant vector of the invention or a host cell of the invention, optionally further comprising a pharmaceutically acceptable excipient.
在本发明的一个实施方案中，所述的药物组合物，其用于激活AMPK、抑制胆固醇合成、降低脂肪酸合成、抗肥胖、预防和/或治疗糖尿病、预防和/或治疗肿瘤、预防和/或治疗帕金森症、预防和/或治疗阿尔茨海默症、抗衰老或者用于延长哺乳动物寿 命。优选地，所述肿瘤为选自黑色素瘤、胰腺癌、卵巢癌和乳腺癌中的任意一种或几种。In one embodiment of the invention, the pharmaceutical composition for activating AMPK, inhibiting cholesterol synthesis, reducing fatty acid synthesis, preventing obesity, preventing and/or treating diabetes, preventing and/or treating tumors, prevention and/or Or treating Parkinson's disease, preventing and/or treating Alzheimer's disease, anti-aging or for prolonging mammalian life Life. Preferably, the tumor is any one or more selected from the group consisting of melanoma, pancreatic cancer, ovarian cancer, and breast cancer.
本发明还涉及一种治疗和/或预防高胆固醇症、糖尿病、肿瘤、帕金森症或阿尔茨海默症的方法或者一种抗肥胖(例如预防肥胖或者减肥)、抗衰老或延长哺乳动物寿命的方法，包括抑制受试者的Aldolase活性或者下调受试者的Aldolase基因表达水平的步骤；例如包括给予受试者有效量的本发明的宿主细胞或者组合物的步骤；例如包括给予有需求的受试者以有效量的抑制或阻断Aldolase活性的药物或者抑制或降低Aldolase基因表达水平的药物的步骤；The invention also relates to a method of treating and/or preventing hypercholesterolemia, diabetes, tumor, Parkinson's disease or Alzheimer's disease or an anti-obesity (for example to prevent obesity or weight loss), anti-aging or prolonging the life of a mammal A method comprising the steps of inhibiting Aldolase activity in a subject or downregulating an Aldolase gene expression level in a subject; for example comprising the step of administering to the subject an effective amount of a host cell or composition of the invention; for example comprising administering a need A step of administering an effective amount of a drug that inhibits or blocks Aldolase activity or a drug that inhibits or decreases the expression level of an Aldolase gene;
优选地，所述肿瘤为选自黑色素瘤、胰腺癌、卵巢癌和乳腺癌中的任意一种或几种。Preferably, the tumor is any one or more selected from the group consisting of melanoma, pancreatic cancer, ovarian cancer, and breast cancer.
优选地，所述抑制或阻断Aldolase活性的药物为抗Aldolase的抗体或TDZD-8；优选地，所述抗体为单克隆抗体。Preferably, the drug that inhibits or blocks Aldolase activity is an antibody against Aldolase or TDZD-8; preferably, the antibody is a monoclonal antibody.
优选地，所述抑制或降低Aldolase基因表达水平的药物选自siRNA例如shRNA，以及用于CRISPR-Cas9系统的guide RNA。Preferably, the drug that inhibits or reduces the expression level of the Aldolase gene is selected from the group consisting of siRNAs such as shRNA, and guide RNA for the CRISPR-Cas9 system.
抑制受试者的Aldolase活性的水平或者下调受试者的Aldolase基因表达的水平，取决于许多因素，例如所治疗病况的严重程度，患者或动物的性别、年龄、体重及个体反应，以及待治疗患者的病况和既往病史来选定。本领域通常的做法是，从低于为得到所需治疗效果和/或预防效果而要求的水平开始，逐渐增加剂量，直到得到所需的效果。Inhibiting the level of Aldolase activity in a subject or downregulating the level of Aldolase gene expression in a subject depends on a number of factors, such as the severity of the condition being treated, the sex, age, weight and individual response of the patient or animal, and the condition to be treated The patient's condition and past medical history were chosen. It is common practice in the art to gradually increase the dosage from a level below that required to achieve the desired therapeutic effect and/or prophylactic effect until the desired effect is achieved.
本发明还涉及选自如下的(1)－(6)项中的任意一项，其用于激活AMPK或用于制备激活AMPK的药物或者用于制备筛选激活AMPK的药物的模型：The present invention also relates to any one of the following items (1) to (6) for activating AMPK or for preparing a drug for activating AMPK or for preparing a drug for screening for activating AMPK:
(1)Aldolase；(1) Aldolase;
(2)编码Aldolase的多核苷酸；(2) a polynucleotide encoding an Aldolase;
(3)核酸构建体，其含有用于完全敲除或者部分敲除Aldolase基因的多核苷酸；优选地，所述多核苷酸为siRNA例如shRNA，或者为用于CRISPR/Cas9系统的guide RNA；(3) a nucleic acid construct comprising a polynucleotide for completely knocking out or partially knocking out the Aldolase gene; preferably, the polynucleotide is an siRNA such as shRNA, or a guide RNA for a CRISPR/Cas9 system;
(4)宿主细胞，其中的编码Aldolase的多核苷酸被完全敲除或部分敲除；优选 地，其含有第(3)项所述的核酸构建体；(4) a host cell in which the polynucleotide encoding Aldolase is completely knocked out or partially knocked out; The nucleic acid construct of the above (3);
(5)抑制或阻断Aldolase活性的药物；(5) a drug that inhibits or blocks the activity of Aldolase;
(6)抑制或降低Aldolase基因表达水平的药物。(6) A drug that inhibits or reduces the expression level of an Aldolase gene.
本发明还涉及选自如下的(1)－(6)项中的任意一项，其用于制备抑制胆固醇合成的药物、降低脂肪酸合成的药物、抗肥胖的药物、预防和/或治疗糖尿病的药物、预防和/或治疗肿瘤的药物、预防和/或治疗帕金森症的药物、预防和/或治疗阿尔茨海默症的药物、抗衰老的药物或者用于延长哺乳动物寿命的药物：The present invention also relates to any one of the following items (1) to (6) for use in the preparation of a medicament for inhibiting cholesterol synthesis, a medicament for reducing fatty acid synthesis, an anti-obesity drug, and for preventing and/or treating diabetes. Drugs, drugs for the prevention and/or treatment of tumors, drugs for the prevention and/or treatment of Parkinson's disease, drugs for the prevention and/or treatment of Alzheimer's disease, anti-aging drugs or drugs for prolonging the lifespan of mammals:
(1)Aldolase；(1) Aldolase;
(2)编码Aldolase的多核苷酸；(2) a polynucleotide encoding an Aldolase;
(3)核酸构建体，其含有用于完全敲除或者部分敲除Aldolase基因的多核苷酸；优选地，所述多核苷酸为siRNA例如shRNA，或者为用于CRISPR/Cas9系统的guide RNA；(3) a nucleic acid construct comprising a polynucleotide for completely knocking out or partially knocking out the Aldolase gene; preferably, the polynucleotide is an siRNA such as shRNA, or a guide RNA for a CRISPR/Cas9 system;
(4)宿主细胞，其中的编码Aldolase的多核苷酸被完全敲除或部分敲除；优选地，其含有第(3)项所述的核酸构建体；(4) a host cell, wherein the polynucleotide encoding Aldolase is completely knocked out or partially knocked out; preferably, it comprises the nucleic acid construct of item (3);
(5)抑制或阻断Aldolase活性的药物；(5) a drug that inhibits or blocks the activity of Aldolase;
(6)抑制或降低Aldolase基因表达水平的药物。(6) A drug that inhibits or reduces the expression level of an Aldolase gene.
优选地，所述肿瘤为选自黑色素瘤、胰腺癌、卵巢癌和乳腺癌中的任意一种或几种。Preferably, the tumor is any one or more selected from the group consisting of melanoma, pancreatic cancer, ovarian cancer, and breast cancer.
在本发明中，醛缩酶(fructose-1,6-bisphosphate aldolase，简称为FBP aldolase)在本发明中亦简称为aldolase，包括3个异构体ALDOA、ALDOB和ALDOC。当提及Aldolase或Aldolase的氨基酸序列时，其包括Aldolase的蛋白全长，还包括其融合蛋白。然而，本领域技术人员理解，在Aldolase的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。在本发明的一个实施方案中，Aldolase为人Aldolase。优选地，Aldolase为选自ALDOA、ALDOB和ALDOC中的任意一种、两种或三种。当Aldolase为ALDOA、ALDOB和ALDOC三者时，亦表示为“ALDOA-C”。In the present invention, fructose-1 (6-bisphosphate aldolase, abbreviated as FBP aldolase) is also referred to as aldolase in the present invention, and includes three isomers ALDOA, ALDOB and ALDOC. When referring to the amino acid sequence of Aldolase or Aldolase, it includes the full length of the protein of Aldolase, and also includes its fusion protein. However, it will be understood by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and/or additions) may be naturally occurring or artificially introduced in the amino acid sequence of Aldolase without affecting its biological function. In one embodiment of the invention, the Aldolase is human Aldolase. Preferably, the Aldolase is any one, two or three selected from the group consisting of ALDOA, ALDOB and ALDOC. When Aldolase is ALDOA, ALDOB and ALDOC, it is also expressed as "ALDOA-C".
人Aldolase A(ALDOA)的氨基酸序列如下：(364 AA) The amino acid sequence of human Aldolase A (ALDOA) is as follows: (364 AA)
Figure PCTCN2017109647-appb-000001
Figure PCTCN2017109647-appb-000001
人Aldolase B(ALDOB)的氨基酸序列如下：(364 AA)The amino acid sequence of human Aldolase B (ALDOB) is as follows: (364 AA)
Figure PCTCN2017109647-appb-000002
Figure PCTCN2017109647-appb-000002
人Aldolase C(ALDOC)的氨基酸序列如下：(364 AA)The amino acid sequence of human Aldolase C (ALDOC) is as follows: (364 AA)
Figure PCTCN2017109647-appb-000003
Figure PCTCN2017109647-appb-000003
在本发明中，当提及aldolase基因时，其不仅包含编码aldolase的核酸序列，还包含其简并序列；进一步地，还可以包含读码框之外的调控序列。在本发明的一个实施方案中，aldolase基因为人aldolase基因。In the present invention, when referring to the aldolase gene, it comprises not only the nucleic acid sequence encoding the aldolase but also the degenerate sequence thereof; further, it may further comprise a regulatory sequence other than the reading frame. In one embodiment of the invention, the aldolase gene is a human aldolase gene.
编码ALDOA的核酸序列(CDS)如下：(1095 BP)The nucleic acid sequence (CDS) encoding ALDOA is as follows: (1095 BP)
Figure PCTCN2017109647-appb-000004
Figure PCTCN2017109647-appb-000004
Figure PCTCN2017109647-appb-000005
Figure PCTCN2017109647-appb-000005
编码ALDOB的核酸序列(CDS)如下：(1095 BP)The nucleic acid sequence (CDS) encoding ALDOB is as follows: (1095 BP)
Figure PCTCN2017109647-appb-000006
Figure PCTCN2017109647-appb-000006
Figure PCTCN2017109647-appb-000007
Figure PCTCN2017109647-appb-000007
编码ALDOC的核酸序列(CDS)如下：(1095 BP)The nucleic acid sequence (CDS) encoding ALDOC is as follows: (1095 BP)
Figure PCTCN2017109647-appb-000008
Figure PCTCN2017109647-appb-000008
本发明中，In the present invention,
术语“核酸构建体”，在文中定义为单链或双链核酸分子，优选是指人工构建的核酸分子。可选地，所述核酸构建体还包含有可操作地连接的1个或多个调控序列。The term "nucleic acid construct", as defined herein, is a single- or double-stranded nucleic acid molecule, preferably an artificially constructed nucleic acid molecule. Optionally, the nucleic acid construct further comprises one or more regulatory sequences operably linked.
在本发明中，术语“可操作地连接”是指两个或多个核苷酸区域或核酸序列的功能性的空间排列。所述“可操作地连接”可以通过基因重组的手段实现。In the present invention, the term "operably linked" refers to a spatial arrangement of the functionality of two or more nucleotide regions or nucleic acid sequences. The "operably linked" can be achieved by means of genetic recombination.
在本发明中，术语“载体”指的是，可将抑制某蛋白的多核苷酸插入其中的一种核酸运载工具。举例来说，载体包括：质粒；噬菌粒；柯斯质粒；人工染色体如酵母 人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可能含有多种控制表达的元件。In the present invention, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide inhibiting a certain protein can be inserted. For example, vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast Artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1 derived artificial chromosome (PAC); phage such as λ phage or M13 phage and animal virus. The animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papilloma polyves Virus (such as SV40). A vector may contain a variety of elements that control expression.
在本发明中，术语“宿主细胞”指的是导入载体的细胞，包括如下许多细胞类型，如大肠杆菌或枯草菌等原核细胞，如酵母细胞或曲霉菌等真菌细胞，如S2果蝇细胞或Sf9等昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞的动物细胞。In the present invention, the term "host cell" refers to a cell into which a vector is introduced, including many cell types such as prokaryotic cells such as Escherichia coli or Bacillus subtilis, such as fungal cells such as yeast cells or Aspergillus, such as S2 Drosophila cells or Insect cells such as Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
术语“有效量”是指可在受试者中实现治疗、预防、减轻和/或缓解本发明所述疾病或病症的剂量。The term "effective amount" refers to a dose that can achieve a treatment, prevention, alleviation, and/or alleviation of a disease or condition described herein in a subject.
术语“疾病和/或病症”是指所述受试者的一种身体状态，该身体状态与本发明所述疾病和/或病症有关。The term "disease and/or condition" refers to a physical state of the subject that is associated with the disease and/or condition described herein.
术语“受试者”可以指患者或者其它接受本发明药物组合物以治疗、预防、减轻和/或缓解本发明所述疾病或病症的动物，特别是哺乳动物，例如人、狗、猴、牛、马等。The term "subject" can refer to a patient or other animal that receives the pharmaceutical composition of the invention to treat, prevent, ameliorate and/or alleviate the disease or condition of the invention, particularly a mammal, such as a human, a dog, a monkey, or a cow. , horses, etc.
本发明中，对于DNA或RNA的敲低包括但不限于，完全敲除和部分敲除。完全敲除是指将目标DNA或目标RNA的水平或其表达的蛋白的水平降低至几乎检测不到的水平(事实上，一般而言，很难将目标DNA或目标RNA 100％地敲除掉)。部分敲除是指敲除的程度大于零，小于完全敲除的情况。In the present invention, knockdown of DNA or RNA includes, but is not limited to, complete knockout and partial knockout. Complete knockout refers to reducing the level of target DNA or target RNA or the level of expressed protein to an almost undetectable level (in fact, it is generally difficult to knock out the target DNA or target RNA 100%). ). Partial knockout means that the degree of knockout is greater than zero, less than the case of complete knockout.
发明的有益效果Advantageous effects of the invention
与现有技术相比，本发明应用aldolase的shRNA和蛋白突变体能够显著地直接激活AMPK，从而实现了可以将aldolase作为靶点来开发药物用来激活AMPK，克服了现有技术中直接以AMPK为药物靶点而存在的困难。Compared with the prior art, the shRNA and protein mutants of the present invention using aldolase can significantly activate AMPK directly, thereby realizing the use of aldolase as a target to develop drugs for activating AMPK, overcoming the direct AMPK in the prior art. Difficulties in the drug target.
附图说明DRAWINGS
图1：shRNA抑制ALDOA-C基因表达的免疫印迹结果。其中，#1和#2分别代表靶向ALDOA、ALDOB或ALDOC的两条shRNA中的一条。对于ALDOA：#1代表SEQ ID NO: 7，#2代表SEQ ID NO:8。对于ALDOB：#1代表SEQ ID NO:9，#2代表SEQ ID NO:10。对于ALDOC：#1代表SEQ ID NO:11，#2代表SEQ ID NO:12。Figure 1: Immunoblot results of shRNA inhibition of ALDOA-C gene expression. Among them, #1 and #2 represent one of two shRNAs targeting ALDOA, ALDOB or ALDOC, respectively. For ALDOA: #1 stands for SEQ ID NO: 7, #2 represents SEQ ID NO: 8. For ALDOB: #1 represents SEQ ID NO: 9, and #2 represents SEQ ID NO: 10. For ALDOC: #1 represents SEQ ID NO: 11, and #2 represents SEQ ID NO: 12.
图2：shRNA抑制ALDOA-C后AMPK的效果的检测图。其中，siGFP为对照组。Figure 2: Detection map of the effect of shRNA on inhibition of AMPK after ALDOA-C. Among them, siGFP was a control group.
具体实施方式detailed description
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。Embodiments of the present invention will be described in detail below with reference to the embodiments. Those skilled in the art will appreciate that the following examples are merely illustrative of the invention and are not to be considered as limiting the scope of the invention. In the examples, the specific techniques or conditions are not indicated, according to the techniques or conditions described in the literature in the field (for example, refer to J. Sambrook et al., Huang Peitang et al., Molecular Cloning Experimental Guide, Third Edition, Science Press) or in accordance with the product manual. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially.
实施例1：shRNA抑制ALDOA-C基因的表达Example 1: shRNA inhibits expression of ALDOA-C gene
1.实验材料和主要试剂1. Experimental materials and main reagents
细胞株：小鼠成纤维细胞MEF，是永生化的MEF，即从小鼠胚胎分离出成纤维细胞之后，转染SV40T antigen使细胞永生。构建方法可参见文献Lei Y,Methods Mol Biol.2013；1031:59-64.Generation and culture of mouse embryonic fibroblasts。Cell line: mouse fibroblast MEF, which is an immortalized MEF, which is obtained by isolating SV40T antigen from mouse embryos and immortalizing the cells. Construction methods can be found in the literature Lei Y, Methods Mol Biol. 2013; 1031: 59-64. Generation and culture of mouse embryonic fibroblasts.
人胚胎肾细胞HEK293T(cat.CRL-3216)，购自ATCC。Human embryonic kidney cell HEK293T (cat. CRL-3216), purchased from ATCC.
载体pLVX-IRES(cat.#631849)，购自Clontech。The vector pLVX-IRES (cat. #631849) was purchased from Clontech.
转染试剂：Lipofectamine 2000(cat.11668-027)，购自Invitrogen公司。Transfection reagent: Lipofectamine 2000 (cat. 11668-027), purchased from Invitrogen.
强力霉素Doxycycline(cat.S4163)，购自Selleckchem。Doxycycline (cat. S4163), available from Selleckchem.
Dulbecco’s modified Eagle’s medium(DMEM,Gibco,cat.11965)，购自Thermofisher。Dulbecco's modified Eagle's medium (DMEM, Gibco, cat. 11965), available from Thermofisher.
Western所用一抗：Primary antibody used in Western:
anti-ALDOA(cat.#8060)，购自Cell Signaling Technology；anti-ALDOA (cat. #8060), available from Cell Signaling Technology;
anti-ALDOB(cat.18065-1-AP)，购自Ptoteintech；anti-ALDOB (cat.18065-1-AP), purchased from Ptoteintech;
anti-ALDOC(cat.AM2215b)，购自Abgent；anti-ALDOC (cat.AM2215b), purchased from Abgent;
anti-β-tubulin(cat.#2128)，购自Cell Signaling Technology。Anti-β-tubulin (cat. #2128), available from Cell Signaling Technology.
Western所用二抗： Secondary antibody used in Western:
HRP-conjugated goat anti-mouse IgG(cat.115-035-003)和HRP-conjugated goat anti-rabbit IgG(cat.111-035-003)，均购自Jackson ImmunoResearch。HRP-conjugated goat anti-mouse IgG (cat. 115-035-003) and HRP-conjugated goat anti-rabbit IgG (cat. 111-035-003) were purchased from Jackson ImmunoResearch.
2.实验方法2. Experimental methods
已经有人报道敲低ALDOA能够引起细胞死亡25。本发明人的前期实验结果也表明敲低ALDOB或者ALDOC能够引起细胞死亡。为了避免上述细胞死亡对实验结果造成影响，本发明人重新建立了一套新的实验方法：It has been reported that knocking down ALDOA can cause cell death 25 . The preliminary results of the present inventors also showed that knocking down ALDOB or ALDOC can cause cell death. In order to avoid the above-mentioned cell death affecting the experimental results, the inventors have re-established a new experimental method:
(1)构建诱导表达ALDOA-C的重组表达质粒(1) Construction of a recombinant expression plasmid that induces expression of ALDOA-C
构建强力霉素(Doxycycline，Dox)诱导表达的ALDOA-C的表达质粒(pLVX-IRES-ALDOA-C)。采用常规的克隆方法，可参见《分子克隆实验指南》，其步骤如下：The expression plasmid (pLVX-IRES-ALDOA-C) of ALDOA-C induced expression by Doxycycline (Dox) was constructed. Using the conventional cloning method, see the Guide to Molecular Cloning, the steps are as follows:
通过PCR分别扩增ALDOA、ALDOB和ALDOC的CDS片段(SEQ ID NO:4－6)，同时利用限制性内切酶处理(EcoRI和BamHI双酶切)载体pLVX-IRES，最后将CDS片段和酶切过的载体连接起来，得到pLVX-IRES-ALDOA-C。The CDS fragments of ALDOA, ALDOB and ALDOC (SEQ ID NOS: 4-6) were amplified by PCR, respectively, while restriction endonuclease treatment (EcoRI and BamHI double digestion) vector pLVX-IRES, and finally CDS fragments and enzymes The cleaved vectors were ligated to obtain pLVX-IRES-ALDOA-C.
(2)构建诱导表达ALDOA-C的MEF细胞株(2) Construction of a MEF cell line that induces expression of ALDOA-C
将pLVX-IRES-ALDOA-C在HEK293T细胞中包装成慢病毒，并用此病毒感染MEF细胞24小时以上。其中，慢病毒导入表达质粒的步骤如下：pLVX-IRES-ALDOA-C was packaged into a lentivirus in HEK293T cells, and MEF cells were infected with the virus for more than 24 hours. Among them, the steps of introducing a lentivirus into an expression plasmid are as follows:
接种1.5x 106个人胚胎肾细胞HEK293T至35mm细胞培养皿，每皿加入2ml的DMEM细胞培养液，置于5％CO2培养箱中培养(培养条件为：5％CO2，37℃)，过夜，待细胞密度达到80％以上时，制备转染试剂与表达质粒的转染混合物，室温放置20分钟后加入到细胞培养液中，继续培养48小时后收集含有慢病毒颗粒的上清。将含有慢病毒颗粒的上清加入到已接种小鼠成纤维细胞(MEFs)的35mm细胞培养皿，继续培养24小时以上，后收集细胞进行蛋白质免疫印迹实验。Inoculate 1.5×10 6 human embryonic kidney cells HEK293T to 35 mm cell culture dish, add 2 ml of DMEM cell culture medium per dish, and incubate in a 5% CO 2 incubator (culture conditions: 5% CO 2 , 37 ° C). After overnight, when the cell density reached 80% or more, a transfection mixture of the transfection reagent and the expression plasmid was prepared, and after being allowed to stand at room temperature for 20 minutes, it was added to the cell culture solution, and after 48 hours of further culture, the supernatant containing the lentiviral particles was collected. The supernatant containing the lentiviral particles was added to a 35 mm cell culture dish inoculated with mouse fibroblasts (MEFs), and culture was continued for more than 24 hours, after which the cells were collected for Western blotting.
将细胞培养在含有100ng/ml Dox的DMEM培养液中，如此构建成诱导表达ALDOA-C的MEF细胞株。The cells were cultured in DMEM medium containing 100 ng/ml of Dox, and thus constructed to induce MEF cell lines expressing ALDOA-C.
(3)shRNA序列设计(3) shRNA sequence design
靶向ALDOA：Targeting ALDOA:
5’-CCAAGTGGCGCTGTGTGCT-3’(SEQ ID NO:7)，或5'-CCAAGTGGCGCTGTGTGCT-3' (SEQ ID NO: 7), or
5’-GCCATGGGCCTTGACTTTC-3’(SEQ ID NO:8)5'-GCCATGGGCCTTGACTTTC-3' (SEQ ID NO: 8)
靶向ALDOB： Targeting ALDOB:
5’-GCTCTCTGAGCAGATCCAT-3’(SEQ ID NO:9)，或5'-GCTCTCTGAGCAGATCCAT-3' (SEQ ID NO: 9), or
5’-GGCAGTTCCGAGAACTCCT-3’(SEQ ID NO:10)5'-GGCAGTTCCGAGAACTCCT-3' (SEQ ID NO: 10)
靶向ALDOC：Targeting ALDOC:
5’-GAGTCTAGAGCTTATGTCT-3’(SEQ ID NO:11)，或5'-GAGTCTAGAGCTTATGTCT-3' (SEQ ID NO: 11), or
5’-CAGTTACCCTTGATGGTAT-3’(SEQ ID NO:12)5'-CAGTTACCCTTGATGGTAT-3' (SEQ ID NO: 12)
(4)细胞转染和培养(4) Cell transfection and culture
分别将靶向ALDOA、ALDOB或ALDOC的各两条shRNA通过慢病毒导入上述MEF细胞株24小时以上，使得内源表达的ALDOA、ALDOB或ALDOC被阻断。其中将慢病毒导入shRNA的具体步骤如下：Each of the two shRNAs targeting ALDOA, ALDOB or ALDOC was introduced into the above MEF cell strain by lentivirus for more than 24 hours, respectively, such that endogenously expressed ALDOA, ALDOB or ALDOC were blocked. The specific steps for introducing a lentivirus into shRNA are as follows:
接种1.5x 106个人胚胎肾细胞HEK293T至35mm细胞培养皿，每皿加入2ml的DMEM细胞培养液，置于5％CO2培养箱中培养(培养条件为：5％CO2，37℃)，过夜，待细胞密度达到80％以上时，制备转染试剂与含有shRNA的质粒的转染混合物，室温放置20分钟后加入到细胞培养液中，继续培养48小时后收集含有慢病毒颗粒的上清。将含有慢病毒颗粒的上清加入到已接种小鼠成纤维细胞(MEFs)的35mm细胞培养皿，继续培养24小时以上。Inoculate 1.5×10 6 human embryonic kidney cells HEK293T to 35 mm cell culture dish, add 2 ml of DMEM cell culture medium per dish, and incubate in a 5% CO 2 incubator (culture conditions: 5% CO 2 , 37 ° C). After overnight, when the cell density reached 80% or more, a transfection mixture of the transfection reagent and the shRNA-containing plasmid was prepared, and the mixture was allowed to stand at room temperature for 20 minutes, and then added to the cell culture solution, and the supernatant containing the lentiviral particles was collected after further incubation for 48 hours. . The supernatant containing the lentiviral particles was added to a 35 mm cell culture dish inoculated with mouse fibroblasts (MEFs), and culture was continued for more than 24 hours.
最后，将细胞培养在不含Dox的DMEM培养基12小时，使得诱导表达的外源ALDOA-C消失，然后收集细胞，用于下面的蛋白质免疫印迹实验。Finally, the cells were cultured in DMEM medium without Dox for 12 hours, so that the induced expression of exogenous ALDOA-C disappeared, and then the cells were collected for the following Western blotting experiments.
(5)蛋白质免疫印迹实验检测ALDOA-C蛋白水平(5) Western blotting assay for ALDOA-C protein levels
按照常规的Western Blot操作进行。Follow the usual Western Blot procedure.
3.实验结果3. Experimental results
如图1所示。As shown in Figure 1.
结果显示，shRNA处理组中ALDOA-C蛋白水平显著下调。说明shRNA显著抑制了ALDOA-C基因的表达。The results showed that the level of ALDOA-C protein was significantly down-regulated in the shRNA-treated group. This indicates that shRNA significantly inhibited the expression of the ALDOA-C gene.
实施例2：靶向ALDOA-C的shRNA能够激活AMPKExample 2: shRNA targeting ALDOA-C is capable of activating AMPK
1.实验材料和主要试剂1. Experimental materials and main reagents
Western所用一抗：Primary antibody used in Western:
Rabbit 548 anti-phospho-AMPK α-T172(cat#2535)，购自Cell Signaling Technology； Rabbit 548 anti-phospho-AMPK α-T172 (cat #2535), available from Cell Signaling Technology;
anti-AMPKα(cat#2532,1:1000 for IB)，购自Cell Signaling Technology；anti-AMPKα (cat#2532, 1:1000 for IB), purchased from Cell Signaling Technology;
anti-phospho-ACC-Ser79(cat.#3661,1:1000 for IB)，购自Cell Signaling Technology；anti-phospho-ACC-Ser79 (cat. #3661, 1:1000 for IB), purchased from Cell Signaling Technology;
anti-ACC(cat.#3662,1:1000 for IB)，购自Cell Signaling Technology。anti-ACC (cat. #3662, 1:1000 for IB), available from Cell Signaling Technology.
其它用到的一抗和二抗如实施例1中所述。The other primary and secondary antibodies used were as described in Example 1.
不含葡萄糖的DMEM(Gibco,cat.11966)购自Thermofisher。Glucose-free DMEM (Gibco, cat. 11966) was purchased from Thermofisher.
2.实验方法2. Experimental methods
(1)首先参照如实施例1中所述的方法，制备得到如图1所示的ALDOA-C同时敲低的MEF细胞。其中，利用靶向GFP(Green Fluorescent Protein绿色荧光蛋白)的shRNA作为对照组(non-targeting对照组),靶向GFP的shRNA序列5’-GGCACAAGCTGGAGTACAA-3’(SEQ ID NO:13)，构建方法参考实施例1。(1) First, the MEF cells simultaneously knocked down by ALDOA-C as shown in Fig. 1 were prepared by referring to the method as described in Example 1. Among them, shRNA targeting GFP (Green Fluorescent Protein) was used as a control group (non-targeting control group), and shRNA sequence 5'-GGCACAAGCTGGAGTACAA-3' (SEQ ID NO: 13) targeting GFP was constructed. Refer to Example 1.
(2)用不含葡萄糖(Glc)的DMEM培养液处理该细胞2小时(激活AMPK)，同时用含葡萄糖的DMEM培养基作为对照。(2) The cells were treated with DMEM containing no glucose (Glc) for 2 hours (activated AMPK) while DMEM medium containing glucose was used as a control.
(3)裂解细胞，将裂解液用免疫印迹方法，检测p-AMPK和p-ACC的水平从而衡量AMPK的激活情况。(3) The cells were lysed, and the lysate was detected by immunoblotting to measure the levels of p-AMPK and p-ACC to measure the activation of AMPK.
3.实验结果3. Experimental results
如图2所示。as shown in picture 2.
结果显示，在排除细胞死亡的条件下，敲低ALDOA-C能够显著激活AMPK。The results showed that knocking down ALDOA-C significantly activated AMPK under conditions that ruled out cell death.
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Figure PCTCN2017109647-appb-000009
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Figure PCTCN2017109647-appb-000009
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Figure PCTCN2017109647-appb-000010
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Figure PCTCN2017109647-appb-000010
M, Lantier L, Viollet B, Zierath JR, Chibalin AV. AMP-activated protein kinase activator A-769662 is an inhibitor of the Na(+)-K(+)-ATPase.Am J Physiol Cell Physiol.2009Dec;297( 6): C1554-66.
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尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。 Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and alterations of the details are possible in light of the teachings of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (18)

  1. 选自如下的(1)－(6)项中的任意一项在制备激活AMPK的药物或者在制备筛选激活AMPK的药物的模型中的用途：Use of any of the following items (1) to (6) for the preparation of a drug that activates AMPK or in the preparation of a model for screening for a drug that activates AMPK:
    (1)Aldolase；(1) Aldolase;
    (2)编码Aldolase的多核苷酸；(2) a polynucleotide encoding an Aldolase;
    (3)核酸构建体，其含有用于完全敲除或者部分敲除Aldolase基因的多核苷酸；优选地，所述多核苷酸为siRNA例如shRNA，或者为用于CRISPR/Cas9系统的guide RNA；(3) a nucleic acid construct comprising a polynucleotide for completely knocking out or partially knocking out the Aldolase gene; preferably, the polynucleotide is an siRNA such as shRNA, or a guide RNA for a CRISPR/Cas9 system;
    (4)宿主细胞，其中的编码Aldolase的多核苷酸被完全敲除或部分敲除；优选地，其含有第(3)项所述的核酸构建体；(4) a host cell, wherein the polynucleotide encoding Aldolase is completely knocked out or partially knocked out; preferably, it comprises the nucleic acid construct of item (3);
    (5)抑制或阻断Aldolase活性的药物；(5) a drug that inhibits or blocks the activity of Aldolase;
    (6)抑制或降低Aldolase基因表达水平的药物。(6) A drug that inhibits or reduces the expression level of an Aldolase gene.
  2. 根据权利要求1所述的用途，其中，所述抑制或阻断Aldolase活性的药物为抗Aldolase的抗体或TDZD-8；优选地，所述抗体为单克隆抗体。The use according to claim 1, wherein the drug that inhibits or blocks Aldolase activity is an antibody against Aldolase or TDZD-8; preferably, the antibody is a monoclonal antibody.
  3. 根据权利要求1所述的用途，其中，所述抑制或降低Aldolase基因表达水平的药物选自siRNA例如shRNA，以及用于CRISPR-Cas9系统的guide RNA。The use according to claim 1, wherein the drug which inhibits or reduces the expression level of the Aldolase gene is selected from the group consisting of siRNA such as shRNA, and guide RNA for the CRISPR-Cas9 system.
  4. 选自如下的(1)－(6)项中的任意一项在制备抑制胆固醇合成的药物、降低脂肪酸合成的药物、抗肥胖的药物、预防和/或治疗糖尿病的药物、预防和/或治疗肿瘤的药物、预防和/或治疗帕金森症的药物、预防和/或治疗阿尔茨海默症的药物、抗衰老的药物或者用于延长哺乳动物寿命的药物中的用途：Any one of the following items (1) to (6) for preparing a drug for inhibiting cholesterol synthesis, a drug for reducing fatty acid synthesis, a drug for preventing obesity, a drug for preventing and/or treating diabetes, prevention and/or treatment Use of tumor drugs, drugs for the prevention and/or treatment of Parkinson's disease, drugs for the prevention and/or treatment of Alzheimer's disease, anti-aging drugs or drugs for prolonging the lifespan of mammals:
    (1)Aldolase；(1) Aldolase;
    (2)编码Aldolase的多核苷酸；(2) a polynucleotide encoding an Aldolase;
    (3)核酸构建体，其含有用于完全敲除或者部分敲除Aldolase基因的多核苷酸；优选地，所述多核苷酸为siRNA例如shRNA，或者为用于CRISPR/Cas9系统的guide RNA； (3) a nucleic acid construct comprising a polynucleotide for completely knocking out or partially knocking out the Aldolase gene; preferably, the polynucleotide is an siRNA such as shRNA, or a guide RNA for a CRISPR/Cas9 system;
    (4)宿主细胞，其中的编码Aldolase的多核苷酸被完全敲除或部分敲除；优选地，其含有第(3)项所述的核酸构建体；(4) a host cell, wherein the polynucleotide encoding Aldolase is completely knocked out or partially knocked out; preferably, it comprises the nucleic acid construct of item (3);
    (5)抑制或阻断Aldolase活性的药物；(5) a drug that inhibits or blocks the activity of Aldolase;
    (6)抑制或降低Aldolase基因表达水平的药物；(6) a drug that inhibits or reduces the expression level of an Aldolase gene;
    优选地，所述肿瘤为选自黑色素瘤、胰腺癌、卵巢癌和乳腺癌中的任意一种或几种。Preferably, the tumor is any one or more selected from the group consisting of melanoma, pancreatic cancer, ovarian cancer, and breast cancer.
  5. 根据权利要求4所述的用途，其中，所述抑制或阻断Aldolase活性的药物为抗Aldolase的抗体或TDZD-8；优选地，所述抗体为单克隆抗体。The use according to claim 4, wherein the drug that inhibits or blocks Aldolase activity is an antibody against Aldolase or TDZD-8; preferably, the antibody is a monoclonal antibody.
  6. 根据权利要求4所述的用途，其中，所述抑制或降低Aldolase基因表达水平的药物选自siRNA例如shRNA，以及用于CRISPR-Cas9系统的guide RNA。The use according to claim 4, wherein the drug which inhibits or reduces the expression level of the Aldolase gene is selected from the group consisting of siRNA such as shRNA, and guide RNA for the CRISPR-Cas9 system.
  7. 一种在体内或体外激活AMPK的方法，包括抑制Aldolase活性或者下调Aldolase基因表达水平的步骤，例如，包括抑制有需求的受试者中或者细胞中的Aldolase的活性或者下调Aldolase基因的表达水平的步骤。A method of activating AMPK in vivo or in vitro, comprising the steps of inhibiting Aldolase activity or downregulating Aldolase gene expression levels, for example, including inhibiting Aldolase activity in a subject or in a cell or downregulating an Aldolase gene expression level. step.
  8. 一种筛选选自如下的药物的方法，包括加入待测药物，以及检测Aldolase活性或者检测Aldolase基因表达水平的步骤：A method of screening a drug selected from the group consisting of adding a drug to be tested, and detecting Aldolase activity or detecting an Aldolase gene expression level:
    激活AMPK的药物、抑制胆固醇合成的药物、降低脂肪酸合成的药物、抗肥胖的药物、预防和/或治疗糖尿病的药物、预防和/或治疗肿瘤的药物、预防和/或治疗帕金森症的药物、预防和/或治疗阿尔茨海默症的药物、抗衰老的药物或者用于延长哺乳动物寿命的药物；Activating AMPK drugs, drugs for inhibiting cholesterol synthesis, drugs for lowering fatty acid synthesis, anti-obesity drugs, drugs for preventing and/or treating diabetes, drugs for preventing and/or treating tumors, drugs for preventing and/or treating Parkinson's disease , drugs for the prevention and/or treatment of Alzheimer's disease, anti-aging drugs or drugs for prolonging the lifespan of mammals;
    优选地，所述肿瘤为选自黑色素瘤、胰腺癌、卵巢癌和乳腺癌中的任意一种或几种。Preferably, the tumor is any one or more selected from the group consisting of melanoma, pancreatic cancer, ovarian cancer, and breast cancer.
  9. 一种重组载体，其含有下调Aldolase基因表达水平的siRNA例如shRNA，或者用于CRISPR-Cas9系统的guide RNA；优选地，所述重组载体为重组慢病毒载体。 A recombinant vector comprising an siRNA such as shRNA that downregulates the expression level of an Aldolase gene, or a guide RNA for a CRISPR-Cas9 system; preferably, the recombinant vector is a recombinant lentiviral vector.
  10. 一种宿主细胞，其含有权利要求9所述的重组载体，或者其中的编码Aldolase的多核苷酸被完全敲除或部分敲除。A host cell comprising the recombinant vector of claim 9, or wherein the polynucleotide encoding Aldolase is completely knocked out or partially knocked out.
  11. 一种药物组合物，其包含权利要求9所述的重组载体或者权利要求10所述的宿主细胞，可选地，其还包含药学上可接受的辅料。A pharmaceutical composition comprising the recombinant vector of claim 9 or the host cell of claim 10, optionally further comprising a pharmaceutically acceptable excipient.
  12. 选自如下的(1)－(4)项中的任意一项，其用于制备激活AMPK的药物、抑制胆固醇合成的药物、降低脂肪酸合成的药物、抗肥胖的药物、预防和/或治疗糖尿病的药物、预防和/或治疗肿瘤的药物、预防和/或治疗帕金森症的药物、预防和/或治疗阿尔茨海默症的药物、抗衰老的药物或者用于延长哺乳动物寿命的药物，或者用于制备筛选激活AMPK的药物的模型：Any one of the following items (1) to (4) for use in the preparation of a drug for activating AMPK, a drug for inhibiting cholesterol synthesis, a drug for lowering fatty acid synthesis, an anti-obesity drug, and for preventing and/or treating diabetes Drugs, drugs for the prevention and/or treatment of tumors, drugs for the prevention and/or treatment of Parkinson's disease, drugs for the prevention and/or treatment of Alzheimer's disease, anti-aging drugs or drugs for prolonging the lifespan of mammals, Or a model for the preparation of drugs that screen for activation of AMPK:
    (1)Aldolase；(1) Aldolase;
    (2)编码Aldolase的多核苷酸；(2) a polynucleotide encoding an Aldolase;
    (3)核酸构建体，其含有用于完全敲除或者部分敲除Aldolase基因的多核苷酸；优选地，所述多核苷酸为siRNA例如shRNA，或者为用于CRISPR/Cas9系统的guide RNA；(3) a nucleic acid construct comprising a polynucleotide for completely knocking out or partially knocking out the Aldolase gene; preferably, the polynucleotide is an siRNA such as shRNA, or a guide RNA for a CRISPR/Cas9 system;
    (4)宿主细胞，其中的编码Aldolase的多核苷酸被完全敲除或部分敲除；优选地，其含有第(3)项所述的核酸构建体；(4) a host cell, wherein the polynucleotide encoding Aldolase is completely knocked out or partially knocked out; preferably, it comprises the nucleic acid construct of item (3);
    优选地，所述肿瘤为选自黑色素瘤、胰腺癌、卵巢癌和乳腺癌中的任意一种或几种。Preferably, the tumor is any one or more selected from the group consisting of melanoma, pancreatic cancer, ovarian cancer, and breast cancer.
  13. 抑制或阻断Aldolase活性的药物，或者抑制或降低Aldolase基因表达水平的药物，其用于激活AMPK、抑制胆固醇合成、降低脂肪酸合成、抗肥胖、预防和/或治疗糖尿病、预防和/或治疗肿瘤、预防和/或治疗帕金森症、预防和/或治疗阿尔茨海默症、抗衰老或者用于延长哺乳动物寿命；A drug that inhibits or blocks Aldolase activity, or a drug that inhibits or reduces the expression level of an Aldolase gene, which is used to activate AMPK, inhibit cholesterol synthesis, reduce fatty acid synthesis, resist obesity, prevent and/or treat diabetes, prevent and/or treat tumors. , prevention and/or treatment of Parkinson's disease, prevention and/or treatment of Alzheimer's disease, anti-aging or for prolonging the lifespan of mammals;
    优选地，所述肿瘤为选自黑色素瘤、胰腺癌、卵巢癌和乳腺癌中的任意一种或几种。Preferably, the tumor is any one or more selected from the group consisting of melanoma, pancreatic cancer, ovarian cancer, and breast cancer.
  14. 根据权利要求13所述的药物，其中，所述抑制或阻断Aldolase活性的药物为抗Aldolase的抗体或TDZD-8；优选地，所述抗体为单克隆抗体。 The drug according to claim 13, wherein the drug that inhibits or blocks Aldolase activity is an antibody against Aldolase or TDZD-8; preferably, the antibody is a monoclonal antibody.
  15. 根据权利要求13所述的药物，其中，所述抑制或降低Aldolase基因表达水平的药物选自siRNA例如shRNA，以及用于CRISPR-Cas9系统的guide RNA。The drug according to claim 13, wherein the drug which inhibits or reduces the expression level of the Aldolase gene is selected from the group consisting of siRNA such as shRNA, and guide RNA for the CRISPR-Cas9 system.
  16. 一种治疗和/或预防高胆固醇症、糖尿病、肿瘤、帕金森症或阿尔茨海默症的方法，或者一种抗肥胖(例如预防肥胖或者减肥)、抗衰老或延长哺乳动物寿命的方法，或者一种抑制胆固醇合成或降低脂肪酸合成的方法，包括抑制有需求的受试者中的Aldolase的活性或者下调有需求的受试者中的Aldolase基因的表达水平的步骤；优选地，包括给予有需求的受试者以有效量的抑制或阻断Aldolase活性的药物或者抑制或降低Aldolase基因表达水平的药物的步骤；A method of treating and/or preventing hypercholesterolemia, diabetes, tumor, Parkinson's disease or Alzheimer's disease, or a method of combating obesity (for example, preventing obesity or losing weight), anti-aging or prolonging the lifespan of a mammal, Or a method of inhibiting cholesterol synthesis or reducing fatty acid synthesis, comprising the steps of inhibiting the activity of Aldolase in a subject in need or downregulating the expression level of an Aldolase gene in a subject in need; preferably, including administering A subject in need of an effective amount of a drug that inhibits or blocks Aldolase activity or a drug that inhibits or reduces the expression level of an Aldolase gene;
    优选地，所述肿瘤为选自黑色素瘤、胰腺癌、卵巢癌和乳腺癌中的任意一种或几种。Preferably, the tumor is any one or more selected from the group consisting of melanoma, pancreatic cancer, ovarian cancer, and breast cancer.
  17. 根据权利要求16所述的方法，其中，所述抑制或阻断Aldolase活性的药物为抗Aldolase的抗体或TDZD-8；优选地，所述抗体为单克隆抗体。The method according to claim 16, wherein the drug that inhibits or blocks Aldolase activity is an antibody against Aldolase or TDZD-8; preferably, the antibody is a monoclonal antibody.
  18. 根据权利要求16所述的方法，其中，所述抑制或降低Aldolase基因表达水平的药物选自siRNA例如shRNA，以及用于CRISPR-Cas9系统的guide RNA。 The method according to claim 16, wherein the drug which inhibits or decreases the expression level of the Aldolase gene is selected from the group consisting of siRNA such as shRNA, and guide RNA for the CRISPR-Cas9 system.
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