CN108079315B

CN108079315B - Application of FBP aldose in preparation of AMPK activating medicine

Info

Publication number: CN108079315B
Application number: CN201611041240.1A
Authority: CN
Inventors: 林圣彩; 张宸崧
Original assignee: Xiamen Huachuo Biomedical Technology Co ltd
Current assignee: XIAMEN HUACHUO BIOMEDICAL TECHNOLOGY Co.,Ltd.
Priority date: 2016-11-21
Filing date: 2016-11-21
Publication date: 2021-09-24
Anticipated expiration: 2036-11-21
Also published as: TW201818959A; CN108079315A; WO2018090851A1

Abstract

The invention belongs to the field of biomedicine, and relates to application of FBP aldolase in preparation of a medicine for activating AMPK. The invention also relates to application of the compound in preparing drugs for inhibiting cholesterol synthesis, drugs for reducing fatty acid synthesis, 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 preventing and/or treating Alzheimer's disease or drugs for prolonging the life of organisms. The invention realizes that the FBP aldose can be used as a target point to develop a medicine for activating the AMPK, overcomes the difficulty in the prior art that the AMPK is directly used as the medicine target point, and has good application prospect.

Description

Application of FBP aldose in preparation of AMPK activating medicine

Technical Field

The invention belongs to the field of biomedicine, and relates to application of FBP aldolase in preparation of a medicine for activating AMPK. The invention also relates to application of the compound in preparing medicaments for inhibiting cholesterol synthesis, reducing fatty acid synthesis, preventing and/or treating diabetes, preventing and/or treating tumors, preventing and/or treating Parkinson's disease, preventing and/or treating Alzheimer's disease or prolonging the life of mammals.

Background

5' -adenosine monophosphate activated protein kinase (AMPK) is an important molecule that regulates the energy balance of cells and the body. AMPK consists of 3 different subunits, each having several isoforms: an alpha subunit (alpha 1 or alpha 2); a β subunit (β 1 or β 2); and gamma subunits (gamma 1, gamma 2 or gamma 3); there are a total of 12 possible isoforms of heterotrimers, each with its specific distribution of tissue types, which together make up the AMPK complex in all tissues widely distributed throughout the body. In addition, the functions of the 12 heterotrimers of AMPK are similar or no different functions have been reported so far, and the difference is only in tissue-specific distribution, and activation of any one isoform of AMPK can cause activation of the same downstream protein.

Traditionally, it has been thought that activation of AMPK is mediated by its allosteric activator, AMP/ADP and its analogues. AMP/ADP is capable of binding to the gamma subunit of AMPK, causing structural changes in the holoenzyme, making it more susceptible to activation by phosphorylation at threonine 172 (p-AMPK. alpha.) on its alpha subunit by its upstream kinases. In addition to allosteric activation by the gamma subunit, the beta subunit of AMPK is also 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) reductase¹And acetyl-CoA carboxylase (ACC)²Which inhibit cholesterol biosynthesis and decrease fatty acid synthesis, respectively, resulting in the inhibition of lipid anabolic pathways and the enhancement of lipid catabolic pathways, which is important in connection with the inhibition of the development and progression of diabetes³. AMPK is also capable of promoting the transfer of glucose transporter 4(GLUT4) to cell membrane, which can significantly promote the absorption and assimilation of glucose in blood, and lower blood glucose, parallel to the insulin pathway, and thus has an important role in the treatment of diabetic patients with insulin resistance^4-5. The AMPK can inhibit lipid and protein synthesis, and inhibit tumor (such as melanoma, pancreatic cancer, ovarian cancer or breast cancer) generation and development⁶. In addition, AMPK is able to upregulate in vivo NAD via molecules such as PGC1 and SIRT⁺The ratio of NADH, which is considered to be closely related to longevityCorrelation^7-9. Activation of AMPK has been demonstrated to significantly extend the lifespan of these organisms in nematodes (c.elegans), drosophila and mice^10-12. In addition, AMPK is also an important regulatory factor for autophagy. For example, AMPK can significantly promote the occurrence of macroautophagy (macroautophagy), which is one of the basic processes of a living body in maintaining energy and substance metabolic balance, by Activating ULK1(Unc-51Like Autophagy Activating Kinase 1), which has been proved to be closely related to the occurrence of serious diseases such as diabetes, tumor, and the Like from the physiological function viewpoint^13-14. In particular, this function of AMPK is also closely related to the browning of adipose tissue^15-16The latter is believed to be an important process for relieving diabetes and remodeling healthy adipose tissue, and activated AMPK also does inhibit the development of diabetic conditions by promoting lipopalmation¹⁷. AMPK can also promote mitophagy by activating MFF (mitochondrial fire factor)¹⁸The latter disorder has important connection with important nervous system diseases such as Parkinson's disease and Alzheimer's disease¹⁹. AMPK is one of the most attractive drug targets for the treatment of significant diseases due to its multifunctional role in carbohydrate, fat and cholesterol metabolism and biosynthesis. In the last two decades, various screening methods have been applied by academia, and AMPK as a target has resulted in a large number of activators of AMPK and has been studied in a large amount.

However, the results of these studies indicate that AMPK as a direct target for drugs has many drawbacks, such as insufficient drug efficacy or low specificity. The most mature AMPK activators discovered to date are mostly used in the field of diabetes therapy. Taking widely applied AMPK activator metformin as an example, the medicine has little side effect on the organism, and can obviously reduce the blood sugar and the fatty liver level by activating AMPK, thereby relieving the diabetic condition²⁰And thus has great advantages in many AMPK activators. However, due to the poor permeability of the metformin molecule to the cell membrane, corresponding transporters are required for its transport into the cell, and these transporters are distributed in only a few tissues, such as the liverThereby greatly limiting the exertion of the drug effect of the drug and the exertion of the AMPK function²¹. In addition to metformin, the drug currently most widely used in clinical trials is A-769662, a compound capable of direct binding to the beta subunit for allosteric activation of AMPK²². Compared with metformin, A-769662 has good cell membrane permeability, can enter most tissues, has good persistence, and can exert effect for a long time²³. However, A-769662 is not suitable for oral administration²³This greatly limits the range of applications. To be more troublesome, with regard to the specificity of A-769662, it has been reported in recent years that it has multiple targets other than AMPK²⁴. In addition, to the knowledge of the present inventors, other drugs were not in clinical trials²²。

Aldolases (fructose-1,6-bisphosphate aldolase, FBP aldolase for short, also abbreviated as aldolase in the present invention) including aldolase a, aldolase B and aldolase C, are important metabolic enzymes in sugar metabolism, and in glycolysis pathway, it catalyzes fructose-1,6-bisphosphate (FBP) at six carbons to generate glyceraldehyde-3-phosphate at three carbons (G3P) and dihydroxyacetone phosphate (DHAP), which further undergoes enzymatic reactions for many times to generate pyruvate; at the same time, in the gluconeogenic pathway, it is able to catalyze the reverse of this reaction. This process catalyzed by Aldolase is not compensated by other metabolic enzymes. At present, the function of known aldolase is also limited only to the nature of its metabolic enzymes themselves. It has been reported that some mutants of aldolase may be associated with fructose intolerance, but the specific mechanism is not clear. It is worth mentioning that the expression level of Aldolase is significantly increased in tumor tissues, which may promote the Warburg effect and the development of tumor cells, where knocking down Aldolase directly causes the growth of tumor cells to stop²⁵。

As early as 1970, researchers designed numerous analogues of fructose-1,6-bisphosphate that could not be catalyzed by aldolase, and in vitro experiments performed to compete for the binding of fructose-1,6-bisphosphate to aldolase to achieve the effect of inhibiting aldolase. However, these inhibitors are allCannot permeate cell membranes and enter cells to play a role, and the application of the method only stays at the level of in vitro biochemical experiments. At the physiological level, the only inhibitor of aldolase reported to date was TDZD-8 (reported in 2016, CAS #327036-89-5, using MDA-MB-231 cells as a physiological model)²⁶. However, previously the inhibitor was widely used as a classical inhibitor of GSK3, another kinase in cells, i.e. the inhibitor had a well-defined non-aldolase target and its IC for aldolase₅₀And IC for GSK3₅₀Are close. In addition, preliminary experimental results of the present inventors showed that TDZD-8 cannot function to inhibit aldolase in MEF cells, which at least indicates that the inhibitor is not universal for aldolase inhibition.

At present, there is a high necessity to develop a new drug or technical means for activating AMPK.

Disclosure of Invention

Through intensive research and creative work, the inventor discovers a novel AMPK regulation factor, namely aldolase (aldolase), which can directly regulate the activation of AMPK and can be further researched and applied as an important target point for regulating AMPK in the future; and the inventor also surprisingly found that downregulation of the expression level of the Aldolase gene, or inhibition of Aldolase, can significantly activate AMPK, with potential for application to diseases associated with low AMPK levels. The following invention is thus provided:

one aspect of the present invention relates to use of any one selected from the following items (1) to (6) for preparing an AMPK-activating drug or for preparing a model for screening AMPK-activating drugs:

(1)Aldolase；

(2) an Aldolase-encoding polynucleotide;

(3) a nucleic acid construct comprising a polynucleotide for full knock-out or partial knock-out of an Aldolase gene; preferably, the polynucleotide is an siRNA such as shRNA, or a guide RNA for CRISPR/Cas9 system;

(4) a host cell in which an Aldolase-encoding polynucleotide has been fully or partially knocked out; preferably, it contains the nucleic acid construct of item (3);

(5) (ii) an agent that inhibits or blocks Aldolase activity;

(6) a drug that inhibits or reduces the level of expression of an Aldolase gene.

In one embodiment of the present invention, the use, wherein the agent that inhibits or blocks Aldolase activity is an anti-Aldolase antibody or TDZD-8; preferably, the antibody is a monoclonal antibody.

In one embodiment of the invention, the use, wherein the medicament for inhibiting or reducing the expression level of an Aldolase gene is selected from the group consisting of siRNA such as shRNA, and guide RNA for CRISPR-Cas9 system.

The invention relates to application of Aldolase as a drug target in preparing drugs for activating AMPK.

In example 2 of the present invention, AMPK was activated by inhibiting aldolase. Specifically, the expression of ALDOA-C (namely ALDOA, ALDOB and ALDOC) genes is inhibited by using a lentivirus-mediated shRNA infection method, and then AMPK is detected to be activated through the detection of AMPK phosphorylation. The term "shRNA" refers to RNA (Short Hairpin RNA, abbreviated as "shRNA") with a small Hairpin structure, which is an RNA sequence with a Short Hairpin structure, can be expressed by a plasmid, and interferes with the expression of a target gene.

In one embodiment of the invention, the shRNA targets ALDOA, ALDOB and ALDOC. In one embodiment of the invention, the shRNA comprises:

at least one selected from the group consisting of the sequences shown in SEQ ID NO. 7 and SEQ ID NO. 8,

at least one selected from the group consisting of the sequences shown in SEQ ID NO 9 and SEQ ID NO 10, and

at least one selected from the group consisting of the sequences shown in SEQ ID NO. 11 and SEQ ID NO. 12.

In one embodiment of the invention, the model may be a mammalian cell (e.g., a human or mouse cell) or a mammal (e.g., a human or mouse). The test agent may be a candidate agent if it inhibits or reduces the level of Aldolase gene expression in the model, or inhibits or blocks the level of Aldolase activity in the model.

Another aspect of the present invention relates to the use of any one selected from the following items (1) to (6) for the preparation of a medicament for inhibiting cholesterol synthesis, a medicament for reducing fatty acid synthesis, an anti-obesity (e.g., preventing obesity or losing weight), a medicament for preventing and/or treating diabetes, a medicament for preventing and/or treating tumors, a medicament for preventing and/or treating parkinson's disease, a medicament for preventing and/or treating alzheimer's disease, an anti-aging medicament, or a medicament for prolonging the lifespan of a mammal:

(1)Aldolase；

(2) an Aldolase-encoding polynucleotide;

(5) (ii) an agent that inhibits or blocks Aldolase activity;

Preferably, the tumor is any one or more selected from melanoma, pancreatic cancer, ovarian cancer and breast cancer.

Yet another aspect of the invention relates to a method of activating AMPK in vivo or in vitro comprising the step of inhibiting Aldolase activity or down-regulating the level of Aldolase gene expression.

Yet another aspect of the invention relates to a method of screening for a drug selected from the group consisting of the addition of a test drug, and the step of detecting Aldolase activity or detecting the level of Aldolase gene expression:

a drug that activates AMPK, a drug that inhibits cholesterol synthesis, a drug that reduces fatty acid synthesis, an anti-obesity drug, a drug that prevents and/or treats diabetes, a drug that prevents and/or treats tumors, a drug that prevents and/or treats parkinson's disease, a drug that prevents and/or treats alzheimer's disease, an anti-aging drug, or a drug for prolonging the lifespan of a mammal. Preferably, the tumor is any one or more selected from melanoma, pancreatic cancer, ovarian cancer and breast cancer.

If the test agent is capable of inhibiting or reducing the level of Aldolase gene expression, or inhibiting or blocking the level of Aldolase activity, then it is a candidate agent. For example:

in one embodiment of the invention, the test agent is added to isolated mammalian cells, e.g., human or mouse cells, with the cells without the test agent as a control.

In one embodiment of the invention, the test agent is administered to a mammal, such as a human or mouse, and the target condition or indication is observed or examined for improvement.

Yet another aspect of the invention relates to a recombinant vector comprising an siRNA such as shRNA that down-regulates the expression level of an Aldolase gene, or a guide RNA for use in a CRISPR-Cas9 system; preferably, the recombinant vector is a recombinant lentiviral vector.

Yet another aspect of the invention relates to a host cell comprising a recombinant vector of the invention, or wherein the Aldolase-encoding polynucleotide is fully or partially knocked out.

Yet another aspect of the invention relates to a pharmaceutical composition comprising a recombinant vector of the invention or a host cell of the invention, optionally together with pharmaceutically acceptable adjuvants.

In one embodiment of the present invention, the pharmaceutical composition for activating AMPK, inhibiting cholesterol synthesis, reducing fatty acid synthesis, anti-obesity, preventing and/or treating diabetes, preventing and/or treating tumor, preventing and/or treating parkinson's disease, preventing and/or treating alzheimer's disease, anti-aging or for prolonging the lifespan of a mammal. Preferably, the tumor is any one or more selected from melanoma, pancreatic cancer, ovarian cancer and breast cancer.

The present invention also relates to a method of treating and/or preventing hypercholesterolemia, diabetes, tumors, parkinson's disease or alzheimer's disease or a method of combating obesity (e.g. preventing obesity or weight loss), delaying aging or prolonging the longevity of a mammal comprising the step of inhibiting Aldolase activity or down-regulating the level of Aldolase gene expression in the subject; for example, comprising the step of administering to the subject an effective amount of a host cell or composition of the invention. Preferably, the tumor is any one or more selected from melanoma, pancreatic cancer, ovarian cancer and breast cancer.

The level of inhibiting Aldolase activity or down-regulating the level of Aldolase gene expression in a subject is selected depending 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 and prior medical history of the patient being treated. It is common practice in the art to increase the dosage from a level below that required to achieve the desired therapeutic and/or prophylactic effect until the desired effect is achieved.

The 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 model for screening drugs for activating AMPK:

(1)Aldolase；

(2) an Aldolase-encoding polynucleotide;

(5) (ii) an agent that inhibits or blocks Aldolase activity;

The present invention also relates to any one selected from 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 medicament, a medicament for preventing and/or treating diabetes, a medicament for preventing and/or treating tumor, a medicament for preventing and/or treating parkinson's disease, a medicament for preventing and/or treating alzheimer's disease, an anti-aging medicament, or a medicament for prolonging the life of a mammal:

(1)Aldolase；

(2) an Aldolase-encoding polynucleotide;

(5) (ii) an agent that inhibits or blocks Aldolase activity;

In the present invention, aldolase (FBP) is also referred to herein simply as aldolase, including 3 isomers ALDOA, ALDOB and ALDOC. When referring to the amino acid sequence of Aldolase or Aldolase, it includes the full length protein of Aldolase, as well as fusion proteins thereof. However, it is understood by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and/or additions) can be naturally occurring or artificially introduced into 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 three of ALDOA, ALDOB and ALDOC, it is also referred to as "ALDOA-C".

The amino acid sequence of human Aldolase a (ALDOA) is as follows: (364 AA)

MPYQYPALTPEQKKELSDIAHRIVAPGKGILAADESTGSIAKRLQSIGTENTEENRRFYRQLLLTADDRVNPCIGGVILFHETLYQKADDGRPFPQVIKSKGGVVGIKVDKGVVPLAGTNGETTTQGLDGLSERCAQYKKDGADFAKWRCVLKIGEHTPSALAIMENANVLARYASICQQNGIVPIVEPEILPDGDHDLKRCQYVTEKVLAAVYKALSDHHIYLEGTLLKPNMVTPGHACTQKFSHEEIAMATVTALRRTVPPAVTGITFLSGGQSEEEASINLNAINKCPLLKPWALTFSYGRALQASALKAWGGKKENLKAAQEEYVKRALANSLACQGKYTPSGQAGAAASESLFVSNHAY(SEQ ID NO:1)

The amino acid sequence of human Aldolase B (ALDOB) is as follows: (364 AA)

MAHRFPALTQEQKKELSEIAQSIVANGKGILAADESVGTMGNRLQRIKVENTEENRRQFREILFSVDSSINQSIGGVILFHETLYQKDSQGKLFRNILKEKGIVVGIKLDQGGAPLAGTNKETTIQGLDGLSERCAQYKKDGVDFGKWRAVLRIADQCPSSLAIQENANALARYASICQQNGLVPIVEPEVIPDGDHDLEHCQYVTEKVLAAVYKALNDHHVYLEGTLLKPNMVTAGHACTKKYTPEQVAMATVTALHRTVPAAVPGICFLSGGMSEEDATLNLNAINLCPLPKPWKLSFSYGRALQASALAAWGGKAANKEATQEAFMKRAMANCQAAKGQYVHTGSSGAASTQSLFTACYTY(SEQ ID NO:2)

The amino acid sequence of human Aldolase C (ALDOC) is as follows: (364 AA)

MPHSYPALSAEQKKELSDIALRIVAPGKGILAADESVGSMAKRLSQIGVENTEENRRLYRQVLFSADDRVKKCIGGVIFFHETLYQKDDNGVPFVRTIQDKGIVVGIKVDKGVVPLAGTDGETTTQGLDGLSERCAQYKKDGADFAKWRCVLKISERTPSALAILENANVLARYASICQQNGIVPIVEPEILPDGDHDLKRCQYVTEKVLAAVYKALSDHHVYLEGTLLKPNMVTPGHACPIKYTPEEIAMATVTALRRTVPPAVPGVTFLSGGQSEEEASFNLNAINRCPLPRPWALTFSYGRALQASALNAWRGQRDNAGAATEEFIKRAEVNGLAAQGKYEGSGEDGGAAAQSLYIANHAY(SEQ ID NO:3)

In the present invention, when referring to the aldolase gene, it encompasses not only the nucleic acid sequence encoding aldolase but also degenerate sequences thereof; further, regulatory sequences other than in-frame may be included. In one embodiment of the invention, the aldolase gene is a human aldolase gene.

The nucleic acid sequence (CDS) encoding ALDOA is as follows: (1095 BP)

ATGCCCTACCAATATCCAGCACTGACCCCGGAGCAGAAGAAGGAGCTGTCTGACATCGCTCACCGCATCGTGGCACCTGGCAAGGGCATCCTGGCTGCAGATGAGTCCACTGGGAGCATTGCCAAGCGGCTGCAGTCCATTGGCACCGAGAACACCGAGGAGAACCGGCGCTTCTACCGCCAGCTGCTGCTGACAGCTGACGACCGCGTGAACCCCTGCATTGGGGGTGTCATCCTCTTCCATGAGACACTCTACCAGAAGGCGGATGATGGGCGTCCCTTCCCCCAAGTTATCAAATCCAAGGGCGGTGTTGTGGGCATCAAGGTAGACAAGGGCGTGGTCCCCCTGGCAGGGACAAATGGCGAGACTACCACCCAAGGGTTGGATGGGCTGTCTGAGCGCTGTGCCCAGTACAAGAAGGACGGAGCTGACTTCGCCAAGTGGCGTTGTGTGCTGAAGATTGGGGAACACACCCCCTCAGCCCTCGCCATCATGGAAAATGCCAATGTTCTGGCCCGTTATGCCAGTATCTGCCAGCAGAATGGCATTGTGCCCATCGTGGAGCCTGAGATCCTCCCTGATGGGGACCATGACTTGAAGCGCTGCCAGTATGTGACCGAGAAGGTGCTGGCTGCTGTCTACAAGGCTCTGAGTGACCACCACATCTACCTGGAAGGCACCTTGCTGAAGCCCAACATGGTCACCCCAGGCCATGCTTGCACTCAGAAGTTTTCTCATGAGGAGATTGCCATGGCGACCGTCACAGCGCTGCGCCGCACAGTGCCCCCCGCTGTCACTGGGATCACCTTCCTGTCTGGAGGCCAGAGTGAGGAGGAGGCGTCCATCAACCTCAATGCCATTAACAAGTGCCCCCTGCTGAAGCCCTGGGCCCTGACCTTCTCCTACGGCCGAGCCCTGCAGGCCTCTGCCCTGAAGGCCTGGGGCGGGAAGAAGGAGAACCTGAAGGCTGCGCAGGAGGAGTATGTCAAGCGAGCCCTGGCCAACAGCCTTGCCTGTCAAGGAAAGTACACTCCGAGCGGTCAGGCTGGGGCTGCTGCCAGCGAGTCCCTCTTCGTCTCTAACCACGCCTATTAA(SEQ ID NO:4)

The nucleic acid sequence (CDS) encoding ALDOB is as follows: (1095 BP)

ATGGCCCACCGATTTCCAGCCCTCACCCAGGAGCAGAAGAAGGAGCTCTCAGAAATTGCCCAGAGCATTGTTGCCAATGGAAAGGGGATCCTGGCTGCAGATGAATCTGTAGGTACCATGGGGAACCGCCTGCAGAGGATCAAGGTGGAAAACACTGAAGAGAACCGCCGGCAGTTCCGAGAAATCCTCTTCTCTGTGGACAGTTCCATCAACCAGAGCATCGGGGGTGTGATCCTTTTCCACGAGACCCTCTACCAGAAGGACAGCCAGGGAAAGCTGTTCAGAAACATCCTCAAGGAAAAGGGGATCGTGGTGGGAATCAAGTTAGACCAAGGAGGTGCTCCTCTTGCAGGAACAAACAAAGAAACCACCATTCAAGGGCTTGATGGCCTCTCAGAGCGCTGTGCTCAGTACAAGAAAGATGGTGTTGACTTTGGGAAGTGGCGTGCTGTGCTGAGGATTGCCGACCAGTGTCCATCCAGCCTCGCTATCCAGGAAAACGCCAACGCCCTGGCTCGCTACGCCAGCATCTGTCAGCAGAATGGACTGGTACCTATTGTTGAACCAGAGGTAATTCCTGATGGAGACCATGACCTGGAACACTGCCAGTATGTTACTGAGAAGGTCCTGGCTGCTGTCTACAAGGCCCTGAATGACCATCATGTTTACCTGGAGGGCACCCTGCTAAAGCCCAACATGGTGACTGCTGGACATGCCTGCACCAAGAAGTATACTCCAGAACAAGTAGCTATGGCCACCGTAACAGCTCTCCACCGTACTGTTCCTGCAGCTGTTCCTGGCATCTGCTTTTTGTCTGGTGGCATGAGTGAAGAGGATGCCACTCTCAACCTCAATGCTATCAACCTTTGCCCTCTACCAAAGCCCTGGAAACTAAGTTTCTCTTATGGACGGGCCCTGCAGGCCAGTGCACTGGCTGCCTGGGGTGGCAAGGCTGCAAACAAGGAGGCAACCCAGGAGGCTTTTATGAAGCGGGCCATGGCTAACTGCCAGGCGGCCAAAGGACAGTATGTTCACACGGGTTCTTCTGGGGCTGCTTCCACCCAGTCGCTCTTCACAGCCTGCTATACCTACTAG(SEQ ID NO:5)

The nucleic acid sequence (CDS) encoding ALDOC is as follows: (1095 BP)

ATGCCTCACTCGTACCCAGCCCTTTCTGCTGAGCAGAAGAAGGAGTTGTCTGACATTGCCCTGCGGATTGTAGCCCCGGGCAAAGGCATTCTGGCTGCGGATGAGTCTGTAGGCAGCATGGCCAAGCGGCTGAGCCAAATTGGGGTGGAAAACACAGAGGAGAACCGCCGGCTGTACCGCCAGGTCCTGTTCAGTGCTGATGACCGTGTGAAAAAGTGCATTGGAGGCGTCATTTTCTTCCATGAGACCCTCTACCAGAAAGATGATAATGGTGTTCCCTTCGTCCGAACCATCCAGGATAAGGGCATCGTCGTGGGCATCAAGGTTGACAAGGGTGTGGTGCCTCTAGCTGGGACTGATGGAGAAACCACCACTCAAGGGCTGGATGGGCTCTCAGAACGCTGTGCCCAATACAAGAAGGATGGTGCTGACTTTGCCAAGTGGCGCTGTGTGCTGAAAATCAGTGAGCGTACACCCTCTGCACTTGCCATTCTGGAGAACGCCAACGTGCTGGCCCGTTATGCCAGTATCTGCCAGCAGAATGGCATTGTGCCTATTGTGGAACCTGAAATATTGCCTGATGGAGACCACGACCTCAAACGTTGTCAGTATGTTACAGAGAAGGTCTTGGCTGCTGTGTACAAGGCCCTGAGTGACCATCATGTATACCTGGAGGGGACCCTGCTCAAGCCCAACATGGTGACCCCGGGCCATGCCTGTCCCATCAAGTATACCCCAGAGGAGATTGCCATGGCAACTGTCACTGCCCTGCGTCGCACTGTGCCCCCAGCTGTCCCAGGAGTGACCTTCCTGTCTGGGGGTCAGAGCGAAGAAGAGGCATCATTCAACCTCAATGCCATCAACCGCTGCCCCCTTCCCCGACCCTGGGCGCTTACCTTCTCCTATGGGCGTGCCCTGCAAGCCTCTGCACTCAATGCCTGGCGAGGGCAACGGGACAATGCTGGGGCTGCCACTGAGGAGTTCATCAAGCGGGCTGAGGTGAATGGGCTTGCAGCCCAGGGCAAGTATGAAGGCAGTGGAGAAGATGGTGGAGCAGCAGCACAGTCACTCTACATTGCCAACCATGCCTACTGA(SEQ ID NO:6)

In the present invention,

the term "nucleic acid construct", defined herein as a single-or double-stranded nucleic acid molecule, preferably refers to an artificially constructed nucleic acid molecule. Optionally, the nucleic acid construct further comprises 1 or more regulatory sequences operably linked.

In the present invention, the term "operably linked" refers to a functional spatial arrangement of two or more nucleotide regions or nucleic acid sequences. The "operably linked" may be achieved by means of genetic recombination.

In the present invention, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide inhibiting a protein is inserted. By way of example, the carrier includes: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal virus species used as vectors are retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus vacuolatum (e.g., SV 40). A vector may contain a variety of elements that control expression.

In the present invention, the term "host cell" refers to a cell into which a vector is introduced, and includes many cell types such as prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

The term "effective amount" refers to a dose that achieves treatment, prevention, alleviation and/or amelioration of a disease or disorder described herein in a subject.

The term "disease and/or disorder" refers to a physical condition of the subject that is associated with the disease and/or disorder of the present invention.

The term "subject" can refer to a patient or other animal, particularly a mammal, e.g., a human, dog, monkey, cow, horse, etc., that receives a pharmaceutical composition of the invention to treat, prevent, ameliorate, and/or alleviate a disease or disorder described herein.

In the present invention, knockdown of DNA or RNA includes, but is not limited to, full knockdown and partial knockdown. A complete knock-out refers to a reduction of the level of a target DNA or target RNA or of a protein expressed therefrom to a level that is barely detectable (in fact, in general, it is difficult to knock out the target DNA or target RNA 100%). Partial knockouts are those where the degree of knockout is greater than zero and less than full knockout.

Advantageous effects of the invention

Compared with the prior art, the invention can obviously and directly activate AMPK by applying shRNA and protein mutant of the aldolase, thereby realizing that the aldolase can be used as a target point to develop a medicament for activating the AMPK, and overcoming the difficulty in the prior art that the AMPK is directly used as a medicament target point.

Drawings

FIG. 1: immunoblotting results of shRNA inhibition of ALDOA-C gene expression. Wherein #1 and #2 represent one of two shrnas targeting ALDOA, ALDOB, or ALDOC, respectively. For ALDOA: #1 represents SEQ ID NO. 7 and #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.

FIG. 2: graph of AMPK Effect after inhibition of ALDOA-C by shRNA. Among them, siGFP was used as a control group.

Detailed Description

Embodiments of the present invention will be described in detail with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruker et al, Huang Petang et al) or according to the product instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1: shRNA (short hairpin ribonucleic acid) for inhibiting expression of ALDOA-C gene

1. Experimental materials and Primary reagents

Cell lines: mouse fibroblast MEF, is an immortalized MEF, i.e. after isolation of fibroblasts from mouse embryos, cells are immortalized by transfection with SV 40T antigen. The construction method can be found in Lei Y, Methods Mol biol.2013; 1031:59-64 Generation and culture of mouse organizing fibrates.

Human embryonic kidney cells HEK293T (cat. crl-3216), purchased from ATCC.

Vector pLVX-IRES (cat. #631849), purchased from Clontech.

Transfection reagent: lipofectamine 2000(cat.11668-027), available from Invitrogen.

Doxycycline (cat. S4163) from Selleckchem.

Dulbecco's modified Eagle's medium (DMEM, Gibco, cat.11965) available from Thermofisiher.

Primary antibody for Western:

anti-ALDOA (cat. #8060), available from Cell Signaling Technology;

anti-ALDOB (cat.18065-1-AP), available from Ptoeintech;

anti-ALDOC (cat. AM2215b), available from Abgent;

anti- β -tubulin (cat. #2128), available from Cell Signaling Technology.

Secondary antibody for Western:

HRP-conjugated goat anti-mouse IgG (cat.115-035-.

2. Experimental methods

It has been reported that knock-down of ALDOA can cause cell death²⁵. Previous experimental results of the present inventors also indicate that the knock-down of ALDOB or ALDOC can cause cell death. In order to avoid the influence of the cell death on the experimental results, the inventor reestablishes a new experimental method:

(1) construction of recombinant expression plasmid for inducible expression of ALDOA-C

An expression plasmid (pLVX-IRES-ALDOA-C) for ALDOA-C induced expression of Doxycycline (Dox) was constructed. The conventional cloning method can be used in molecular cloning experimental guidelines, and comprises the following steps:

CDS fragments (SEQ ID NO: 4-6) of ALDOA, ALDOB and ALDOC are respectively amplified by PCR, meanwhile, the vector pLVX-IRES is processed by restriction endonuclease (EcoRI and BamHI are subjected to double digestion), and finally, the CDS fragment and the digested vector are connected to obtain pLVX-IRES-ALDOA-C.

(2) Construction of MEF cell line for inducible expression of ALDOA-C

pLVX-IRES-ALDOA-C was packaged as lentivirus in HEK293T cells and MEF cells were infected with this virus for more than 24 hours. Wherein, the steps of introducing the lentivirus into the expression plasmid are as follows:

inoculation 1.5X10⁶HEK293T to 35mm cell culture dish for human embryonic kidney cells, adding 2ml DMEM cell culture solution into each dish, and placing in 5% CO₂Cultured in an incubator (culture conditions: 5% CO)₂37 ℃ C.), and overnight, when the cell density reaches more than 80%, preparing a transfection mixture of the transfection reagent and the expression plasmid, placing the mixture at room temperature for 20 minutes, adding the mixture into a cell culture solution, and collecting a supernatant containing the lentiviral particles after culturing for another 48 hours. The supernatant containing the lentiviral particles was added to a 35mm cell culture dish inoculated with mouse fibroblasts (MEFs), and cultured for more than 24 hours, after which the cells were collected for Western blotting.

MEF cell lines inducing the expression of ALDOA-C were constructed by culturing the cells in DMEM containing 100ng/ml Dox.

(3) shRNA sequence design

Targeting of ALDOA:

5'-CCAAGTGGCGCTGTGTGCT-3' (SEQ ID NO:7), or

5’-GCCATGGGCCTTGACTTTC-3’(SEQ ID NO:8)

Targeting of ALDOB:

5'-GCTCTCTGAGCAGATCCAT-3' (SEQ ID NO:9), or

5’-GGCAGTTCCGAGAACTCCT-3’(SEQ ID NO:10)

Targeting ALDOC:

5'-GAGTCTAGAGCTTATGTCT-3' (SEQ ID NO:11), or

5’-CAGTTACCCTTGATGGTAT-3’(SEQ ID NO:12)

(4) Cell transfection and culture

Respectively introducing two shRNAs targeting ALDOA, ALDOB or ALDOC into the MEF cell strain by lentivirus for more than 24 hours, so that the endogenously expressed ALDOA, ALDOB or ALDOC is blocked. The specific steps for introducing the lentivirus into the shRNA are as follows:

inoculation 1.5X10⁶HEK293T to 35mm cell culture dish for human embryonic kidney cells, adding 2ml DMEM cell culture solution into each dish, and placing in 5% CO₂Cultured in an incubator (culture conditions: 5% CO)₂37 ℃ C.), staying overnight, preparing a transfection mixture of the transfection reagent and the plasmid containing shRNA when the cell density reaches more than 80 percent, placing the mixture at room temperature for 20 minutes, adding the mixture into a cell culture solution, continuing to culture for 48 hours, and collecting the supernatant containing the lentiviral particles. The supernatant containing the lentiviral particles was added to a 35mm cell culture dish inoculated with mouse fibroblasts (MEFs) and cultured for over 24 hours.

Finally, the cells were cultured in DMEM medium without Dox for 12 hours to allow the disappearance of exogenous ALDOA-C that induced expression, and then the cells were collected for the following western blotting experiment.

(5) Detection of ALDOA-C protein level by Western blotting experiment

The procedure was carried out according to the conventional Western Blot procedure.

3. Results of the experiment

As shown in fig. 1.

The results show that ALDOA-C protein levels were significantly down-regulated in the shRNA-treated group. The shRNA is proved to remarkably inhibit the expression of the ALDOA-C gene.

Example 2: AlDOA-C-targeted shRNA (short hairpin ribonucleic acid) capable of activating AMPK

1. Experimental materials and Primary reagents

Primary antibody for Western:

rabbit 548 anti-phosphorus-AMPK α -T172(cat #2535), available from Cell Signaling Technology;

anti-AMPK α (cat #2532,1:1000 for IB), available from Cell Signaling Technology;

anti-phospho-ACC-Ser79(cat. #3661,1:1000 for IB), available from Cell Signaling Technology;

anti-ACC (cat. #3662,1:1000 for IB), available from Cell Signaling Technology.

Other primary and secondary antibodies used were as described in example 1.

Glucose-free DMEM (Gibco, cat.11966) was purchased from thermolisiher.

2. Experimental methods

(1) ALDOA-C simultaneously knocked-down MEF cells as shown in FIG. 1 were prepared first, with reference to the method as described in example 1. The construction method is referred to in example 1, wherein the shRNA targeting GFP (Green Fluorescent Protein Green Fluorescent Protein) is used as a control group (non-targeting control group), and the shRNA sequence 5'-GGCACAAGCTGGAGTACAA-3' targeting GFP (SEQ ID NO:13) is used.

(2) The cells were treated with DMEM medium without glucose (Glc) for 2 hours (AMPK activation), while DMEM medium with glucose was used as a control.

(3) The cells were lysed and the lysates were used for immunoblotting to measure the levels of p-AMPK and p-ACC and thus measure the activation of AMPK.

3. Results of the experiment

As shown in fig. 2.

The results show that knocking down ALDOA-C can significantly activate AMPK under conditions that exclude cell death.

Reference documents:

1.Gil G,Sitges M,BovéJ,Hegardt FG.Phosphorylation--dephosphorylation of rat liver 3-hydroxy 3-methylglutaryl coenzyme A reductase associated with changes in activity.FEBS Lett.1980 Feb 11；110(2):195-9.

2.Yeh LA,Lee KH,Kim KH.Regulation of rat liver acetyl-CoA carboxylase.Regulation of phosphorylation and inactivation of acetyl-CoA carboxylase by the adenylate energy charge.J Biol Chem.1980 Mar 25；255(6):2308-14.

3.Hardie DG,Ross FA,Hawley SA.AMPK:a nutrient and energy sensor that maintains energy homeostasis.Nat Rev Mol Cell Biol.2012 Mar 22；13(4):251-62.

4.Chen S,Murphy J,Toth R,Campbell DG,Morrice NA,Mackintosh C.Complementary regulation of TBC1D1and AS160 by growth factors,insulin and AMPK activators.Biochem J.2008 Jan 15；409(2):449-59.

5.

C,Treebak JT,Birk JB,Chen S,Mackintosh C,Hardie DG,Richter EA,Wojtaszewski JF.Genetic disruption of AMPK signaling abolishes both contraction-and insulin-stimulated TBC1D1 phosphorylation and 14-3-3 binding in mouse skeletal muscle.Am J Physiol Endocrinol Metab.2009 Sep；297(3):E665-75.

6.Ji C,Yang B,Yang YL,He SH,Miao DS,He L,Bi ZG.Exogenous cell-permeable C6 ceramide sensitizes multiple cancer cell lines to Doxorubicin-induced apoptosis by promoting AMPK activation and mTORC1 inhibition.Oncogene.2010 Dec 16；29(50):6557-68.

7.Wood JG,Rogina B,Lavu S,Howitz K,Helfand SL,Tatar M,Sinclair D.Sirtuin activators mimic caloric restriction and delay ageing in metazoans.Nature.2004 Aug 5；430(7000):686-9.

8.CantóC,Gerhart-Hines Z,Feige JN,Lagouge M,Noriega L,Milne JC,Elliott PJ,Puigserver P,Auwerx J.AMPK regulates energy expenditure by modulating NAD+metabolism and SIRT1 activity.Nature.2009 Apr 23；458(7241):1056-60.

9.Rera M,Bahadorani S,Cho J,Koehler CL,Ulgherait M,Hur JH,Ansari WS,Lo T Jr,Jones DL,Walker DW.Modulation of longevity and tissue homeostasis by the Drosophila PGC-1 homolog.Cell Metab.2011 Nov 2；14(5):623-34.

10.Greer EL,Dowlatshahi D,Banko MR,Villen J,Hoang K,Blanchard D,Gygi SP,Brunet A.An AMPK-FOXO pathway mediates longevity induced by a novel method of dietary restriction in C.elegans.Curr Biol.2007 Oct 9；17(19):1646-56.

11.Stenesen D,Suh JM,Seo J,Yu K,Lee KS,Kim JS,Min KJ,Graff JM.Adenosine nucleotide biosynthesis and AMPK regulate adult life span and mediate the longevity benefit of caloric restriction in flies.Cell Metab.2013 Jan 8；17(1):101-12.

12.Martin-Montalvo A,Mercken EM,Mitchell SJ,Palacios HH,Mote PL,Scheibye-Knudsen M,Gomes AP,Ward TM,Minor RK,Blouin MJ,Schwab M,Pollak M,Zhang Y,Yu Y,Becker KG,Bohr VA,Ingram DK,Sinclair DA,Wolf NS,Spindler SR,Bernier M,de Cabo R.Metformin improves healthspan and lifespan in mice.Nat Commun.2013；4:2192.

13.Mihaylova MM,Shaw RJ.The AMPK signalling pathway coordinates cell growth,autophagy and metabolism.Nat Cell Biol.2011 Sep 2；13(9):1016-23.

14.He C,Klionsky DJ.Regulation mechanisms and signaling pathways of autophagy.Annu Rev Genet.2009；43:67-93.

15.Mottillo EP,Desjardins EM,Crane JD,Smith BK,Green AE,Ducommun S,Henriksen TI,Rebalka IA,Razi A,Sakamoto K,Scheele C,Kemp BE,Hawke TJ,Ortega J,Granneman JG,Steinberg GR.Lack of Adipocyte AMPK Exacerbates Insulin Resistance and Hepatic Steatosis through Brown and Beige Adipose Tissue Function.Cell Metab.2016 Jul 12；24(1):118-29.

16.Martínez de Morentin PB,González-García I,Martins L,Lage R,Fernández-Mallo D,Martínez-Sánchez N,Ruíz-Pino F,Liu J,Morgan DA,Pinilla L,Gallego R,Saha AK,Kalsbeek A,Fliers E,Bisschop PH,Diéguez C,Nogueiras R,Rahmouni K,Tena-Sempere M,López M.Estradiol regulates brown adipose tissue thermogenesis via hypothalamic AMPK.Cell Metab.2014 Jul 1；20(1):41-53.

17.Bartelt A,Heeren J.Adipose tissue browning and metabolic health.Nat Rev Endocrinol.2014 Jan；10(1):24-36.

18.Toyama EQ,Herzig S,Courchet J,Lewis TL Jr,Losón OC,Hellberg K,Young NP,Chen H,Polleux F,Chan DC,Shaw RJ.Metabolism.AMP-activated protein kinase mediates mitochondrial fission in response to energy stress.Science.2016 Jan 15；351(6270):275-81.

19.Mishra P,Chan DC.Mitochondrial dynamics and inheritance during cell division,development and disease.Nat Rev Mol Cell Biol.2014 Oct；15(10):634-46.

20.Fullerton MD,Galic S,Marcinko K,Sikkema S,Pulinilkunnil T,Chen ZP,O'Neill HM,Ford RJ,Palanivel R,O'Brien M,Hardie DG,Macaulay SL,Schertzer JD,Dyck JR,van Denderen BJ,Kemp BE,Steinberg GR.Single phosphorylation sites in Acc1 and Acc2 regulate lipid homeostasis and the insulin-sensitizing effects of metformin.Nat Med.2013 Dec；19(12):1649-54.

21.Foretz M,Guigas B,Bertrand L,Pollak M,Viollet B.Metformin:from mechanisms of action to therapies.Cell Metab.2014 Dec 2；20(6):953-66.

22.Grahame Hardie D.Regulation of AMP-activated protein kinase by natural and synthetic activators.Acta Pharm Sin B.2016 Jan；6(1):1-19.

23.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 2 diabetes and the metabolic syndrome.Cell Metab.2006 Jun；3(6):403-16.

24.Benziane B,

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.2009 Dec；297(6):C1554-66.

25.Ritterson Lew C,Tolan DR.Targeting of several glycolytic enzymes using RNA interference reveals aldolase affects cancer cell proliferation through a non-glycolytic mechanism.J Biol Chem.2012 Dec 14；287(51):42554-63.

26.Grandjean G,de Jong PR,James BP,Koh MY,Lemos R,Kingston J,Aleshin A,Bankston LA,Miller CP,Cho EJ,Edupuganti R,Devkota A,Stancu G,Liddington RC,Dalby KN,Powis G.Definition of a Novel Feed-Forward Mechanism for Glycolysis-HIF1αSignaling in Hypoxic Tumors Highlights Aldolase A as a Therapeutic Target.Cancer Res.2016 Jul 15；76(14):4259-69.

although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

SEQUENCE LISTING

<110> university of mansion

Application of <120> Aldolase in preparation of AMPK activating medicine

<130> IDC160146

<160> 13

<170> PatentIn version 3.2

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<211> 364

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<213> Homo sapiens

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Met Pro Tyr Gln Tyr Pro Ala Leu Thr Pro Glu Gln Lys Lys Glu Leu

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Ser Asp Ile Ala His Arg Ile Val Ala Pro Gly Lys Gly Ile Leu Ala

20 25 30

Ala Asp Glu Ser Thr Gly Ser Ile Ala Lys Arg Leu Gln Ser Ile Gly

35 40 45

Thr Glu Asn Thr Glu Glu Asn Arg Arg Phe Tyr Arg Gln Leu Leu Leu

50 55 60

Thr Ala Asp Asp Arg Val Asn Pro Cys Ile Gly Gly Val Ile Leu Phe

65 70 75 80

His Glu Thr Leu Tyr Gln Lys Ala Asp Asp Gly Arg Pro Phe Pro Gln

85 90 95

Val Ile Lys Ser Lys Gly Gly Val Val Gly Ile Lys Val Asp Lys Gly

100 105 110

Val Val Pro Leu Ala Gly Thr Asn Gly Glu Thr Thr Thr Gln Gly Leu

115 120 125

Asp Gly Leu Ser Glu Arg Cys Ala Gln Tyr Lys Lys Asp Gly Ala Asp

130 135 140

Phe Ala Lys Trp Arg Cys Val Leu Lys Ile Gly Glu His Thr Pro Ser

145 150 155 160

Ala Leu Ala Ile Met Glu Asn Ala Asn Val Leu Ala Arg Tyr Ala Ser

165 170 175

Ile Cys Gln Gln Asn Gly Ile Val Pro Ile Val Glu Pro Glu Ile Leu

180 185 190

Pro Asp Gly Asp His Asp Leu Lys Arg Cys Gln Tyr Val Thr Glu Lys

195 200 205

Val Leu Ala Ala Val Tyr Lys Ala Leu Ser Asp His His Ile Tyr Leu

210 215 220

Glu Gly Thr Leu Leu Lys Pro Asn Met Val Thr Pro Gly His Ala Cys

225 230 235 240

Thr Gln Lys Phe Ser His Glu Glu Ile Ala Met Ala Thr Val Thr Ala

245 250 255

Leu Arg Arg Thr Val Pro Pro Ala Val Thr Gly Ile Thr Phe Leu Ser

260 265 270

Gly Gly Gln Ser Glu Glu Glu Ala Ser Ile Asn Leu Asn Ala Ile Asn

275 280 285

Lys Cys Pro Leu Leu Lys Pro Trp Ala Leu Thr Phe Ser Tyr Gly Arg

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Ala Leu Gln Ala Ser Ala Leu Lys Ala Trp Gly Gly Lys Lys Glu Asn

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Leu Lys Ala Ala Gln Glu Glu Tyr Val Lys Arg Ala Leu Ala Asn Ser

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Leu Ala Cys Gln Gly Lys Tyr Thr Pro Ser Gly Gln Ala Gly Ala Ala

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Ala Ser Glu Ser Leu Phe Val Ser Asn His Ala Tyr

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Ser Glu Ile Ala Gln Ser Ile Val Ala Asn Gly Lys Gly Ile Leu Ala

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Ala Asp Glu Ser Val Gly Thr Met Gly Asn Arg Leu Gln Arg Ile Lys

35 40 45

Val Glu Asn Thr Glu Glu Asn Arg Arg Gln Phe Arg Glu Ile Leu Phe

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Ser Val Asp Ser Ser Ile Asn Gln Ser Ile Gly Gly Val Ile Leu Phe

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His Glu Thr Leu Tyr Gln Lys Asp Ser Gln Gly Lys Leu Phe Arg Asn

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Ile Leu Lys Glu Lys Gly Ile Val Val Gly Ile Lys Leu Asp Gln Gly

100 105 110

Gly Ala Pro Leu Ala Gly Thr Asn Lys Glu Thr Thr Ile Gln Gly Leu

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Asp Gly Leu Ser Glu Arg Cys Ala Gln Tyr Lys Lys Asp Gly Val Asp

130 135 140

Phe Gly Lys Trp Arg Ala Val Leu Arg Ile Ala Asp Gln Cys Pro Ser

145 150 155 160

Ser Leu Ala Ile Gln Glu Asn Ala Asn Ala Leu Ala Arg Tyr Ala Ser

165 170 175

Ile Cys Gln Gln Asn Gly Leu Val Pro Ile Val Glu Pro Glu Val Ile

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Pro Asp Gly Asp His Asp Leu Glu His Cys Gln Tyr Val Thr Glu Lys

195 200 205

Val Leu Ala Ala Val Tyr Lys Ala Leu Asn Asp His His Val Tyr Leu

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Glu Gly Thr Leu Leu Lys Pro Asn Met Val Thr Ala Gly His Ala Cys

225 230 235 240

Thr Lys Lys Tyr Thr Pro Glu Gln Val Ala Met Ala Thr Val Thr Ala

245 250 255

Leu His Arg Thr Val Pro Ala Ala Val Pro Gly Ile Cys Phe Leu Ser

260 265 270

Gly Gly Met Ser Glu Glu Asp Ala Thr Leu Asn Leu Asn Ala Ile Asn

275 280 285

Leu Cys Pro Leu Pro Lys Pro Trp Lys Leu Ser Phe Ser Tyr Gly Arg

290 295 300

Ala Leu Gln Ala Ser Ala Leu Ala Ala Trp Gly Gly Lys Ala Ala Asn

305 310 315 320

Lys Glu Ala Thr Gln Glu Ala Phe Met Lys Arg Ala Met Ala Asn Cys

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Gln Ala Ala Lys Gly Gln Tyr Val His Thr Gly Ser Ser Gly Ala Ala

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Ser Thr Gln Ser Leu Phe Thr Ala Cys Tyr Thr Tyr

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Met Pro His Ser Tyr Pro Ala Leu Ser Ala Glu Gln Lys Lys Glu Leu

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Ser Asp Ile Ala Leu Arg Ile Val Ala Pro Gly Lys Gly Ile Leu Ala

20 25 30

Ala Asp Glu Ser Val Gly Ser Met Ala Lys Arg Leu Ser Gln Ile Gly

35 40 45

Val Glu Asn Thr Glu Glu Asn Arg Arg Leu Tyr Arg Gln Val Leu Phe

50 55 60

Ser Ala Asp Asp Arg Val Lys Lys Cys Ile Gly Gly Val Ile Phe Phe

65 70 75 80

His Glu Thr Leu Tyr Gln Lys Asp Asp Asn Gly Val Pro Phe Val Arg

85 90 95

Thr Ile Gln Asp Lys Gly Ile Val Val Gly Ile Lys Val Asp Lys Gly

100 105 110

Val Val Pro Leu Ala Gly Thr Asp Gly Glu Thr Thr Thr Gln Gly Leu

115 120 125

Asp Gly Leu Ser Glu Arg Cys Ala Gln Tyr Lys Lys Asp Gly Ala Asp

130 135 140

Phe Ala Lys Trp Arg Cys Val Leu Lys Ile Ser Glu Arg Thr Pro Ser

145 150 155 160

Ala Leu Ala Ile Leu Glu Asn Ala Asn Val Leu Ala Arg Tyr Ala Ser

165 170 175

Ile Cys Gln Gln Asn Gly Ile Val Pro Ile Val Glu Pro Glu Ile Leu

180 185 190

Pro Asp Gly Asp His Asp Leu Lys Arg Cys Gln Tyr Val Thr Glu Lys

195 200 205

Val Leu Ala Ala Val Tyr Lys Ala Leu Ser Asp His His Val Tyr Leu

210 215 220

Glu Gly Thr Leu Leu Lys Pro Asn Met Val Thr Pro Gly His Ala Cys

225 230 235 240

Pro Ile Lys Tyr Thr Pro Glu Glu Ile Ala Met Ala Thr Val Thr Ala

245 250 255

Leu Arg Arg Thr Val Pro Pro Ala Val Pro Gly Val Thr Phe Leu Ser

260 265 270

Gly Gly Gln Ser Glu Glu Glu Ala Ser Phe Asn Leu Asn Ala Ile Asn

275 280 285

Arg Cys Pro Leu Pro Arg Pro Trp Ala Leu Thr Phe Ser Tyr Gly Arg

290 295 300

Ala Leu Gln Ala Ser Ala Leu Asn Ala Trp Arg Gly Gln Arg Asp Asn

305 310 315 320

Ala Gly Ala Ala Thr Glu Glu Phe Ile Lys Arg Ala Glu Val Asn Gly

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Leu Ala Ala Gln Gly Lys Tyr Glu Gly Ser Gly Glu Asp Gly Gly Ala

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Ala Ala Gln Ser Leu Tyr Ile Ala Asn His Ala Tyr

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<210> 4

<211> 1095

<212> DNA

<213> Homo sapiens

<400> 4

atgccctacc aatatccagc actgaccccg gagcagaaga aggagctgtc tgacatcgct 60

caccgcatcg tggcacctgg caagggcatc ctggctgcag atgagtccac tgggagcatt 120

gccaagcggc tgcagtccat tggcaccgag aacaccgagg agaaccggcg cttctaccgc 180

cagctgctgc tgacagctga cgaccgcgtg aacccctgca ttgggggtgt catcctcttc 240

catgagacac tctaccagaa ggcggatgat gggcgtccct tcccccaagt tatcaaatcc 300

aagggcggtg ttgtgggcat caaggtagac aagggcgtgg tccccctggc agggacaaat 360

ggcgagacta ccacccaagg gttggatggg ctgtctgagc gctgtgccca gtacaagaag 420

gacggagctg acttcgccaa gtggcgttgt gtgctgaaga ttggggaaca caccccctca 480

gccctcgcca tcatggaaaa tgccaatgtt ctggcccgtt atgccagtat ctgccagcag 540

aatggcattg tgcccatcgt ggagcctgag atcctccctg atggggacca tgacttgaag 600

cgctgccagt atgtgaccga gaaggtgctg gctgctgtct acaaggctct gagtgaccac 660

cacatctacc tggaaggcac cttgctgaag cccaacatgg tcaccccagg ccatgcttgc 720

actcagaagt tttctcatga ggagattgcc atggcgaccg tcacagcgct gcgccgcaca 780

gtgccccccg ctgtcactgg gatcaccttc ctgtctggag gccagagtga ggaggaggcg 840

tccatcaacc tcaatgccat taacaagtgc cccctgctga agccctgggc cctgaccttc 900

tcctacggcc gagccctgca ggcctctgcc ctgaaggcct ggggcgggaa gaaggagaac 960

ctgaaggctg cgcaggagga gtatgtcaag cgagccctgg ccaacagcct tgcctgtcaa 1020

ggaaagtaca ctccgagcgg tcaggctggg gctgctgcca gcgagtccct cttcgtctct 1080

aaccacgcct attaa 1095

<210> 5

<211> 1095

<212> DNA

<213> Homo sapiens

<400> 5

atggcccacc gatttccagc cctcacccag gagcagaaga aggagctctc agaaattgcc 60

cagagcattg ttgccaatgg aaaggggatc ctggctgcag atgaatctgt aggtaccatg 120

gggaaccgcc tgcagaggat caaggtggaa aacactgaag agaaccgccg gcagttccga 180

gaaatcctct tctctgtgga cagttccatc aaccagagca tcgggggtgt gatccttttc 240

cacgagaccc tctaccagaa ggacagccag ggaaagctgt tcagaaacat cctcaaggaa 300

aaggggatcg tggtgggaat caagttagac caaggaggtg ctcctcttgc aggaacaaac 360

aaagaaacca ccattcaagg gcttgatggc ctctcagagc gctgtgctca gtacaagaaa 420

gatggtgttg actttgggaa gtggcgtgct gtgctgagga ttgccgacca gtgtccatcc 480

agcctcgcta tccaggaaaa cgccaacgcc ctggctcgct acgccagcat ctgtcagcag 540

aatggactgg tacctattgt tgaaccagag gtaattcctg atggagacca tgacctggaa 600

cactgccagt atgttactga gaaggtcctg gctgctgtct acaaggccct gaatgaccat 660

catgtttacc tggagggcac cctgctaaag cccaacatgg tgactgctgg acatgcctgc 720

accaagaagt atactccaga acaagtagct atggccaccg taacagctct ccaccgtact 780

gttcctgcag ctgttcctgg catctgcttt ttgtctggtg gcatgagtga agaggatgcc 840

actctcaacc tcaatgctat caacctttgc cctctaccaa agccctggaa actaagtttc 900

tcttatggac gggccctgca ggccagtgca ctggctgcct ggggtggcaa ggctgcaaac 960

aaggaggcaa cccaggaggc ttttatgaag cgggccatgg ctaactgcca ggcggccaaa 1020

ggacagtatg ttcacacggg ttcttctggg gctgcttcca cccagtcgct cttcacagcc 1080

tgctatacct actag 1095

<210> 6

<211> 1095

<212> DNA

<213> Homo sapiens

<400> 6

atgcctcact cgtacccagc cctttctgct gagcagaaga aggagttgtc tgacattgcc 60

ctgcggattg tagccccggg caaaggcatt ctggctgcgg atgagtctgt aggcagcatg 120

gccaagcggc tgagccaaat tggggtggaa aacacagagg agaaccgccg gctgtaccgc 180

caggtcctgt tcagtgctga tgaccgtgtg aaaaagtgca ttggaggcgt cattttcttc 240

catgagaccc tctaccagaa agatgataat ggtgttccct tcgtccgaac catccaggat 300

aagggcatcg tcgtgggcat caaggttgac aagggtgtgg tgcctctagc tgggactgat 360

ggagaaacca ccactcaagg gctggatggg ctctcagaac gctgtgccca atacaagaag 420

gatggtgctg actttgccaa gtggcgctgt gtgctgaaaa tcagtgagcg tacaccctct 480

gcacttgcca ttctggagaa cgccaacgtg ctggcccgtt atgccagtat ctgccagcag 540

aatggcattg tgcctattgt ggaacctgaa atattgcctg atggagacca cgacctcaaa 600

cgttgtcagt atgttacaga gaaggtcttg gctgctgtgt acaaggccct gagtgaccat 660

catgtatacc tggaggggac cctgctcaag cccaacatgg tgaccccggg ccatgcctgt 720

cccatcaagt ataccccaga ggagattgcc atggcaactg tcactgccct gcgtcgcact 780

gtgcccccag ctgtcccagg agtgaccttc ctgtctgggg gtcagagcga agaagaggca 840

tcattcaacc tcaatgccat caaccgctgc ccccttcccc gaccctgggc gcttaccttc 900

tcctatgggc gtgccctgca agcctctgca ctcaatgcct ggcgagggca acgggacaat 960

gctggggctg ccactgagga gttcatcaag cgggctgagg tgaatgggct tgcagcccag 1020

ggcaagtatg aaggcagtgg agaagatggt ggagcagcag cacagtcact ctacattgcc 1080

aaccatgcct actga 1095

<210> 7

<211> 19

<212> DNA

<213> Artificial

<220>

<223> ALDOA-targeted shRNA sequence

<400> 7

ccaagtggcg ctgtgtgct 19

<210> 8

<211> 19

<212> DNA

<213> Artificial

<220>

<223> ALDOA-targeted shRNA sequence

<400> 8

gccatgggcc ttgactttc 19

<210> 9

<211> 19

<212> DNA

<213> Artificial

<220>

<223> ALDOB-targeted shRNA sequence

<400> 9

gctctctgag cagatccat 19

<210> 10

<211> 19

<212> DNA

<213> Artificial

<220>

<223> ALDOB-targeted shRNA sequence

<400> 10

ggcagttccg agaactcct 19

<210> 11

<211> 19

<212> DNA

<213> Artificial

<220>

<223> ALDOC-targeted shRNA sequence

<400> 11

gagtctagag cttatgtct 19

<210> 12

<211> 19

<212> DNA

<213> Artificial

<220>

<223> ALDOC-targeted shRNA sequence

<400> 12

cagttaccct tgatggtat 19

<210> 13

<211> 19

<212> DNA

<213> Artificial

<220>

<223> shRNA sequence targeting GFP

<400> 13

ggcacaagct ggagtacaa 19

Claims

1. Use of any one selected from the following items (1) to (2) for the preparation of a drug that activates AMPK or for the preparation of a model for screening drugs that activate AMPK:

(1) a nucleic acid construct comprising a polynucleotide for full knock-out or partial knock-out of an Aldolase gene;

(2) a medicament that inhibits or reduces the level of expression of an Aldolase gene, wherein the medicament that inhibits or reduces the level of expression of an Aldolase gene is selected from the group consisting of siRNA, shRNA, and guide RNA for use in a CRISPR-Cas9 system.

2. The use of claim 1, wherein the polynucleotide is an siRNA, shRNA or guide RNA for CRISPR/Cas9 system.

3. A method of activating AMPK in vitro comprising the step of inhibiting Aldolase activity or down-regulating the level of Aldolase gene expression.

4. A method of screening for a drug that activates AMPK comprising the steps of adding a test drug and detecting Aldolase activity or detecting the level of Aldolase gene expression.