TW201818959A - Use of FBP aldolase in preparation of drug activating AMPK - Google Patents

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

Use of FBP ALDOLASE in preparing AMPK-initiating medicine

The invention belongs to the field of biomedicine and relates to the use of FBP aldolase in the preparation of a medicament for starting AMPK. The present invention also relates to 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 tumors, a drug for preventing and / or treating Parkinson's disease, prevention and And / or use in a drug for treating Alzheimer's disease or a drug for extending the life of a mammal.

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 of which has several isotypes: α subunit (α1 or α2); β subunit (β1 or β2); and γ subunit (γ1, γ2, or γ3) ); There are a total of 12 possible heterotrimeric isoforms, each isotype has its specific distribution of tissue types, and various isoforms together form AMPK that is widely distributed in all tissues in the body Complex. In addition, the functions of the 12 heterotrimers of AMPK are similar, or no different functions have been reported at present. The difference is only in the tissue-specific distribution. The activation of any kind of AMPK can cause the same Of downstream proteins.

The traditional view is that the activation of AMPK is mediated by its allosteric activation factor, AMP / ADP and its analogs. AMP / ADP can bind to the γ subunit of AMPK, causing structural changes of the whole enzyme, making it more likely to be activated by its upstream kinase to phosphorylate the threonine 172 site (p-AMPKα) on its α subunit and thus be activated. In addition to the allosteric activation caused by the gamma subunit, the beta subunit of AMPK can also be bound by metabolites such as glycogen and regulated by it.

Activated AMPK can phosphorylate a variety of substrates. Classic AMPK substrates include 3-hydroxy-3-methylglutaridine coenzyme A (HMG-CoA) reductase ¹ and acetamidine coenzyme A carboxylase (ACC) ² , It inhibits cholesterol biosynthesis and decreases fatty acid synthesis, respectively, leading to the inhibition of lipid anabolic pathways and enhanced lipid breakdown pathways, and this function is important to inhibit the formation and development of diabetes ³ . AMPK can also promote the transfer of glucose transporter 4 (GLUT4) to the cell membrane, which can significantly promote the absorption and assimilation of glucose in the blood and reduce blood sugar. This effect is parallel to the insulin pathway, and therefore in patients with diabetes with insulin resistance Has an important role in treatment ^4-5 . The aforementioned inhibitory effect of AMPK on lipid and protein synthesis can significantly inhibit the growth of tumor cells, thereby inhibiting the occurrence and development of tumors (such as melanoma, pancreatic cancer, ovarian cancer or breast cancer) ⁶ . In addition, AMPK can up-regulate the ratio of NAD ⁺ / NADH in the body through molecules such as PGC1 and SIRT. This function is considered to be closely related to longevity ^7-9 . C. elegans , Drosophila and mice have been shown to activate AMPK to significantly extend the lifespan of these organisms by ^10-12 . In addition, AMPK is an important regulator of autophagy. For example, AMPK can significantly promote the occurrence of macroautophagy through the activation of ULK1 (Unc-51 Like Autophagy Activating Kinase 1), which is one of the basic processes for a living body to maintain energy and material metabolism balance. From the perspective of physiological functions, it has been shown to be closely related to the occurrence of major diseases such as diabetes and tumors ^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 remodeling healthy adipose tissue. The initiated AMPK does indeed inhibit diabetes by promoting fat browning Development of the condition ¹⁷ . AMPK can also promote mitochondrial fission factor (MFF) to promote mitophagy ¹⁸ , which is imbalanced with important neurological diseases such as Parkinson's disease and Alzheimer's disease ¹⁹ . Because AMPK has multifunctional effects 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, a variety of screening methods have been used in the academic community, and a large number of AMPK initiators have been obtained with AMPK as a target, and a lot of research has been carried out.

However, the results of these studies indicate that there are many shortcomings in using AMPK as a direct target for drugs, such as insufficient efficacy or low specificity. The most mature AMPK initiators found so far are mostly used in the treatment of diabetes. AMPK agent to start Metformin is widely used as an example, the drug with minimal side effects on the body, can significantly reduce the level of blood sugar and fatty liver by activating AMPK, thus alleviating diabetes condition ^20, which has a great advantage in many AMPK start agent. However, due to the poor permeability of metformin molecules to cell membranes, corresponding transporters are required to transport them into cells, and these transporters are only distributed in a few tissues such as the liver, which greatly limits the efficacy of the drug. Exercise and play of AMPK functions ²¹ . In addition to metformin, the drug that has gone the farthest in clinical trials is A-769662, which can directly bind to the β subunit to allosterically activate AMPK ²² . 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 applications. More trouble is, with regard to the specific A-769662, has been reported in recent years showed that in addition to also having a plurality of AMPK targets ^24. Further, according to the present inventors' knowledge, none of the other drugs in addition to ²² to enter clinical trials.

Fructose-1,6-bisphosphate aldolase (referred to as FBP aldolase, also referred to as aldolase in the present invention) includes aldolase A, aldolase B and aldolase C, which are important metabolisms in sugar metabolism. An enzyme, in the glycolytic pathway, it catalyzes six carbon fructose-1,6-diphosphate (FBP) to produce three carbon glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), the latter further Multiple enzymatic reactions produce pyruvate; at the same time, in the gluconeogenesis pathway, it can catalyze the reverse process of this reaction. This process catalyzed by Aldolase cannot be compensated by other metabolic enzymes. At present, the function of known aldolase is limited to the nature of its metabolic enzymes. It has been reported that some mutants of aldolase may be related to 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, and knocking down aldolase in tumor cells will directly cause tumor cell growth. Stop ²⁵ .

As early as 1970, researchers have designed many analogs of fructose-1,6-diphosphate that cannot be catalyzed by aldolase. They compete for the combination of fructose-1,6-diphosphate and aldolase in in vitro experiments. To achieve the effect of inhibiting aldolase. However, none of these inhibitors can penetrate the cell membrane and enter the cells to play a role, and their application is only at the level of in vitro biochemical experiments. At physiological level, the only reported inhibitor of aldolase 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 another classic intracellular GSK3 inhibitor, that is, the inhibitor has a clear non-aldolase target, and its IC ₅₀ for aldolase and for GSK3 The IC ₅₀ is close. In addition, the preliminary experimental results of the inventors show that TDZD-8 cannot play a role in inhibiting aldolase in MEF cells, which at least indicates that the inhibitor does not have universal inhibition of aldolase.

At present, there is an urgent need to develop new drugs or technical means to start AMPK.

After intensive research and creative labor, the inventor discovered a new regulation factor of AMPK-aldolase, which can directly regulate the activation of AMPK, and will be an important target for regulating AMPK in the future. The point has been further studied and applied; and the inventors have also surprisingly found that down-regulating the level of Aldolase gene expression, or inhibiting Aldolase, can significantly activate AMPK and has the potential to apply to diseases associated with low AMPK levels. The following inventions are thus provided:

One aspect of the present invention relates to the use of any one selected from the following items (1) to (6) in the preparation of a drug that initiates AMPK or in the model for the screening of a drug that initiates AMPK: (1) Aldolase; (2) ) A polynucleotide encoding Aldolase; (3) a nucleic acid construct containing a polynucleotide for completely or partially knocking out the Aldolase gene; preferably, the polynucleotide is an siRNA such as shRNA, or Guide RNA of the CRISPR / Cas9 system; (4) a host cell in which the polynucleotide encoding Aldolase is completely or partially deleted; preferably, it contains the nucleic acid construct according to item (3); ( 5) Drugs that inhibit or block Aldolase activity; (6) Drugs that inhibit or decrease Aldolase gene expression level.

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.

In one embodiment of the present invention, the use, wherein the drug that inhibits or reduces the expression level of the Aldolase gene is selected from siRNAs such as shRNA, and guide RNA for the CRISPR-Cas9 system.

The present invention relates to the use of Aldolase as a drug target in the preparation of a AMPK-initiating medicament.

In Example 2 of the present invention, AMPK is activated by inhibiting aldolase. Specifically, the lentivirus-mediated shRNA infection method was used to suppress the expression of ALDOA-C (ie, ALDOA, ALDOB, and ALDOC) genes, and then AMPK was found to be activated by detection of AMPK phosphorylation. The term "shRNA" refers to a short hairpin RNA (Short Hairpin RNA), which is a short hairpin RNA sequence that can be expressed through 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 present invention, the shRNA includes: at least one selected from the sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8, and selected from the sequences shown by SEQ ID NO: 9 and SEQ ID NO: 10 And at least one selected from 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 (such as a human or mouse cell) or a mammal (such as a human or mouse). If the test drug can inhibit or reduce the level of Aldolase gene expression in the model, or inhibit or block the level of Aldolase activity in the model, it can be a candidate drug.

Another aspect of the present invention relates to a drug selected from any one of the following items (1) to (6) for preparing a drug that inhibits cholesterol synthesis, a drug that reduces fatty acid synthesis, an anti-obesity (such as preventing obesity or losing weight), Drugs for the prevention and / or treatment of diabetes, 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 for Uses in medicines to extend the life of mammals: (1) Aldolase; (2) a polynucleotide encoding Aldolase; (3) a nucleic acid construct containing a polynucleotide for completely or partially knocking out the Aldolase gene; Preferably, the polynucleotide is an siRNA such as shRNA, or guide RNA used in the CRISPR / Cas9 system; (4) a host cell in which the polynucleotide encoding Aldolase is completely or partially deleted; preferably It contains the nucleic acid construct according to item (3); (5) a drug that inhibits or blocks Aldolase activity; (6) inhibits or reduces the level of Aldolase gene expression Drug.

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

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.

In one embodiment of the present invention, the use, wherein the drug that inhibits or reduces the expression level of the Aldolase gene is selected from siRNAs such as shRNA, and guide RNA for the CRISPR-Cas9 system.

In one embodiment of the invention, the shRNA targets ALDOA, ALDOB, and ALDOC. In one embodiment of the present invention, the shRNA includes: at least one selected from the sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8, and selected from the sequences shown by SEQ ID NO: 9 and SEQ ID NO: 10 And at least one selected from the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12.

Yet another aspect of the present invention relates to a method for initiating AMPK in vivo or in vitro, comprising the steps of inhibiting Aldolase activity or down-regulating Aldolase gene expression level, for example, including inhibiting Aldolase activity or down-regulating in a subject in need or in a cell Aldolase gene expression level steps.

Yet another aspect of the present invention relates to a method for screening a drug selected from the group consisting of adding a test drug, and detecting Aldolase activity or detecting an Aldolase gene expression level: a drug that activates AMPK, a drug that inhibits cholesterol synthesis, and reduces fatty acid synthesis Drugs, 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 preventing and / or treating Alzheimer's disease , Anti-aging drugs or drugs used to extend the life of mammals. Preferably, the tumor is any one or more selected from the group consisting of melanoma, pancreatic cancer, ovarian cancer and breast cancer.

If the drug under test can inhibit or reduce the level of Aldolase gene expression, or inhibit or block the level of Aldolase activity, it can be a candidate drug. For example: In one embodiment of the present invention, the test drug is added to the cells of an isolated mammal such as a human or a mouse, and cells without the test drug are used as a control.

In one embodiment of the present invention, a test drug is administered to a mammal, such as a human or a mouse, to observe or detect whether the target symptom or index is improved.

Another aspect of the present invention relates to a recombinant vector, which contains an siRNA such as shRNA that down-regulates the expression level of the Aldolase gene, or a guide RNA used in the CRISPR-Cas9 system; preferably, the recombinant vector is a recombinant lentiviral vector.

In one embodiment of the invention, the shRNA targets ALDOA, ALDOB, and ALDOC. In one embodiment of the present invention, the shRNA includes: at least one selected from the sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8, and selected from the sequences shown by SEQ ID NO: 9 and SEQ ID NO: 10 And at least one selected from the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12.

Still another aspect of the present invention relates to a host cell containing the recombinant vector of the present invention, or a polynucleotide encoding Aldolase therein is completely or partially deleted.

Yet another aspect of the present invention relates to a pharmaceutical composition comprising a recombinant vector of the present invention or a host cell of the present invention, optionally, further comprising a pharmaceutically acceptable excipient.

In one embodiment of the present invention, the pharmaceutical composition is used to start AMPK, inhibit cholesterol synthesis, reduce fatty acid synthesis, anti-obesity, prevent and / or treat diabetes, prevent and / or treat tumors, prevent and / Or treat Parkinson's disease, prevent and / or treat Alzheimer's disease, anti-aging or use to extend the life of mammals. Preferably, 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, tumors, Parkinson's disease or Alzheimer's disease or an anti-obesity (such as preventing obesity or losing weight), anti-aging or prolonging the life of mammals A method comprising the steps of inhibiting the Aldolase activity of a subject or down-regulating the level of Aldolase gene expression in the subject; for example, the step of administering to the subject an effective amount of a host cell or composition of the present invention; The step of the tester using an effective amount of a drug that inhibits or blocks Aldolase activity or a drug that inhibits or reduces the level of Aldolase gene expression; preferably, the tumor is any one selected from melanoma, pancreatic cancer, ovarian cancer, and breast cancer One or more.

Preferably, the drug that inhibits or blocks Aldolase activity is an anti-Aldolase antibody or TDZD-8; preferably, the antibody is a monoclonal antibody.

Preferably, the drug that inhibits or reduces the expression level of the Aldolase gene is selected from siRNAs such as shRNA, and guide RNA used in the CRISPR-Cas9 system.

The level of inhibition of the subject's Aldolase activity or the level of down-regulation of the subject's Aldolase gene expression depends on many factors, such as the severity of the condition being treated, the sex, age, weight and individual response of the patient or animal, and the treatment The patient's condition and past medical history are selected. It is common practice in the art to start with a dose that is lower than required to obtain the desired therapeutic and / or preventive effect, and gradually increase the dose until the desired effect is obtained.

The present invention also relates to any one selected from the following items (1) to (6), which is used for starting AMPK or for preparing a drug for starting AMPK or for preparing a model for screening a drug for starting AMPK: (1) Aldolase; (2) a polynucleotide encoding Aldolase; (3) a nucleic acid construct containing a polynucleotide for completely or partially knocking out the Aldolase gene; preferably, the polynucleotide is an siRNA such as shRNA, Or guide RNA for the CRISPR / Cas9 system; (4) a host cell in which the polynucleotide encoding Aldolase is completely or partially deleted; preferably, it contains the nucleic acid described in item (3) Constructs; (5) drugs that inhibit or block Aldolase activity; (6) drugs that inhibit or reduce the level of Aldolase gene expression.

The present invention also relates to any one selected from the following items (1) to (6), which is used for preparing a drug for inhibiting cholesterol synthesis, a drug for reducing fatty acid synthesis, an anti-obesity drug, and 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 extending the life of mammals: (1) Aldolase; (2) a polynucleotide encoding Aldolase; (3) a nucleic acid construct containing a polynucleotide for completely or partially knocking out the Aldolase gene; preferably, the polynucleotide is an siRNA For example, shRNA, or guide RNA used in the CRISPR / Cas9 system; (4) a host cell in which the polynucleotide encoding Aldolase is completely or partially knocked out; preferably, it contains the enzyme of item (3) (5) a drug that inhibits or blocks Aldolase activity; (6) a drug that inhibits or reduces Aldolase gene expression level.

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

In the present invention, fructose-1,6-bisphosphate aldolase (abbreviated as FBP aldolase) is also abbreviated 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 protein of Aldolase and also includes its fusion protein. However, those skilled in the art understand that in the amino acid sequence of Aldolase, mutations or mutations (including but not limited to substitutions, deletions and / or additions) can be naturally generated or artificially introduced without affecting its biological function. In one embodiment of the invention, Aldolase is human Aldolase. Preferably, Aldolase is any one, two or three selected from ALDOA, ALDOB and ALDOC. When Aldolase is ALDOA, ALDOB, and ALDOC, it is also expressed as "ALDOA-C".

Human Aldolase A (ALDOA) an amino acid sequence as follows: (364 AA) MPYQYPALTPEQKKELSDIAHRIVAPGKGILAADESTGSIAKRLQSIGTENTEENRRFYRQLLLTADDRVNPCIGGVILFHETLYQKADDGRPFPQVIKSKGGVVGIKVDKGVVPLAGTNGETTTQGLDGLSERCAQYKKDGADFAKWRCVLKIGEHTPSALAIMENANVLARYASICQQNGIVPIVEPEILPDGDHDLKRCQYVTEKVLAAVYKALSDHHIYLEGTLLKPNMVTPGHACTQKFSHEEIAMATVTALRRTVPPAVTGITFLSGGQSEEEASINLNAINKCPLLKPWALTFSYGRALQASALKAWGGKKENLKAAQEEYVKRALANSLACQGKYTPSGQAGAAASESLFVSNHAY (SEQ ID NO: 1)

Human Aldolase B (ALDOB) an amino acid sequence as follows: (364 AA) MAHRFPALTQEQKKELSEIAQSIVANGKGILAADESVGTMGNRLQRIKVENTEENRRQFREILFSVDSSINQSIGGVILFHETLYQKDSQGKLFRNILKEKGIVVGIKLDQGGAPLAGTNKETTIQGLDGLSERCAQYKKDGVDFGKWRAVLRIADQCPSSLAIQENANALARYASICQQNGLVPIVEPEVIPDGDHDLEHCQYVTEKVLAAVYKALNDHHVYLEGTLLKPNMVTAGHACTKKYTPEQVAMATVTALHRTVPAAVPGICFLSGGMSEEDATLNLNAINLCPLPKPWKLSFSYGRALQASALAAWGGKAANKEATQEAFMKRAMANCQAAKGQYVHTGSSGAASTQSLFTACYTY (SEQ ID NO: 2)

Human Aldolase C (ALDOC) an amino acid sequence as follows: (364 AA) MPHSYPALSAEQKKELSDIALRIVAPGKGILAADESVGSMAKRLSQIGVENTEENRRLYRQVLFSADDRVKKCIGGVIFFHETLYQKDDNGVPFVRTIQDKGIVVGIKVDKGVVPLAGTDGETTTQGLDGLSERCAQYKKDGADFAKWRCVLKISERTPSALAILENANVLARYASICQQNGIVPIVEPEILPDGDHDLKRCQYVTEKVLAAVYKALSDHHVYLEGTLLKPNMVTPGHACPIKYTPEEIAMATVTALRRTVPPAVPGVTFLSGGQSEEEASFNLNAINRCPLPRPWALTFSYGRALQASALNAWRGQRDNAGAATEEFIKRAEVNGLAAQGKYEGSGEDGGAAAQSLYIANHAY (SEQ ID NO: 3)

In the present invention, when referring to the aldolase gene, it includes not only a nucleic acid sequence encoding the aldolase, but also a degenerate sequence thereof; further, it may also include a regulatory sequence outside the reading frame. In one embodiment of the present invention, the aldolase gene is a human aldolase gene.

The nucleic acid sequence encoding ALDOA (CDS) as follows: (1095 BP) ATGCCCTACCAATATCCAGCACTGACCCCGGAGCAGAAGAAGGAGCTGTCTGACATCGCTCACCGCATCGTGGCACCTGGCAAGGGCATCCTGGCTGCAGATGAGTCCACTGGGAGCATTGCCAAGCGGCTGCAGTCCATTGGCACCGAGAACACCGAGGAGAACCGGCGCTTCTACCGCCAGCTGCTGCTGACAGCTGACGACCGCGTGAACCCCTGCATTGGGGGTGTCATCCTCTTCCATGAGACACTCTACCAGAAGGCGGATGATGGGCGTCCCTTCCCCCAAGTTATCAAATCCAAGGGCGGTGTTGTGGGCATCAAGGTAGACAAGGGCGTGGTCCCCCTGGCAGGGACAAATGGCGAGACTACCACCCAAGGGTTGGATGGGCTGTCTGAGCGCTGTGCCCAGTACAAGAAGGACGGAGCTGACTTCGCCAAGTGGCGTTGTGTGCTGAAGATTGGGGAACACACCCCCTCAGCCCTCGCCATCATGGAAAATGCCAATGTTCTGGCCCGTTATGCCAGTATCTGCCAGCAGAATGGCATTGTGCCCATCGTGGAGCCTGAGATCCTCCCTGATGGGGACCATGACTTGAAGCGCTGCCAGTATGTGACCGAGAAGGTGCTGGCTGCTGTCTACAAGGCTCTGAGTGACCACCACATCTACCTGGAAGGCACCTTGCTGAAGCCCAACATGGTCACCCCAGGCCATGCTTGCACTCAGAAGTTTTCTCATGAGGAGATTGCCATGGCGACCGTCACAGCGCTGCGCCGCACAGTGCCCCCCGCTGTCACTGGGATCACCTTCCTGTCTGGAGGCCAGAGTGAGGAGGAGGCGTCCATCAACCTCAATGCCATTAACAAGTGCCCCCTGCTGAAGCCCTGGGCCCTGACCTTCTCCTACGGCCGAGCCCTGCAGGCCTCTGCCCTGAAGGCCTGG GGCGGGAAGAAGGAGAACCTGAAGGCTGCGCAGGAGGAGTATGTCAAGCGAGCCCTGGCCAACAGCCTTGCCTGTCAAGGAAAGTACACTCCGAGCGGTCAGGCTGGGGCTGCTGCCAGCGAGTCCCTCTTCGTCTCTAACCACGCCTATTAA (SEQ ID NO: 4)

The nucleic acid sequence encoding ALDOB (CDS) as follows: (1095 BP) ATGGCCCACCGATTTCCAGCCCTCACCCAGGAGCAGAAGAAGGAGCTCTCAGAAATTGCCCAGAGCATTGTTGCCAATGGAAAGGGGATCCTGGCTGCAGATGAATCTGTAGGTACCATGGGGAACCGCCTGCAGAGGATCAAGGTGGAAAACACTGAAGAGAACCGCCGGCAGTTCCGAGAAATCCTCTTCTCTGTGGACAGTTCCATCAACCAGAGCATCGGGGGTGTGATCCTTTTCCACGAGACCCTCTACCAGAAGGACAGCCAGGGAAAGCTGTTCAGAAACATCCTCAAGGAAAAGGGGATCGTGGTGGGAATCAAGTTAGACCAAGGAGGTGCTCCTCTTGCAGGAACAAACAAAGAAACCACCATTCAAGGGCTTGATGGCCTCTCAGAGCGCTGTGCTCAGTACAAGAAAGATGGTGTTGACTTTGGGAAGTGGCGTGCTGTGCTGAGGATTGCCGACCAGTGTCCATCCAGCCTCGCTATCCAGGAAAACGCCAACGCCCTGGCTCGCTACGCCAGCATCTGTCAGCAGAATGGACTGGTACCTATTGTTGAACCAGAGGTAATTCCTGATGGAGACCATGACCTGGAACACTGCCAGTATGTTACTGAGAAGGTCCTGGCTGCTGTCTACAAGGCCCTGAATGACCATCATGTTTACCTGGAGGGCACCCTGCTAAAGCCCAACATGGTGACTGCTGGACATGCCTGCACCAAGAAGTATACTCCAGAACAAGTAGCTATGGCCACCGTAACAGCTCTCCACCGTACTGTTCCTGCAGCTGTTCCTGGCATCTGCTTTTTGTCTGGTGGCATGAGTGAAGAGGATGCCACTCTCAACCTCAATGCTATCAACCTTTGCCCTCTACCAAAGCCCTGGAAACTAAGTTTCTCTTATGGACGGGCCCTGCAGGCCAGTGCACTGGCTGCCTGG GGTGGCAAGGCTGCAAACAAGGAGGCAACCCAGGAGGCTTTTATGAGACGGGCCATGGCTAACTGCCAGGCGGCCAAAGGACAGTATGTTCACACGGGTTCTTCTGGGGCTGCTTCCACCCAGTCGCTCTTCACAGCCTGCTATACCTACTAG (SEQ ID NO: 5)

The nucleic acid sequence encoding ALDOC (CDS) as follows: (1095 BP) ATGCCTCACTCGTACCCAGCCCTTTCTGCTGAGCAGAAGAAGGAGTTGTCTGACATTGCCCTGCGGATTGTAGCCCCGGGCAAAGGCATTCTGGCTGCGGATGAGTCTGTAGGCAGCATGGCCAAGCGGCTGAGCCAAATTGGGGTGGAAAACACAGAGGAGAACCGCCGGCTGTACCGCCAGGTCCTGTTCAGTGCTGATGACCGTGTGAAAAAGTGCATTGGAGGCGTCATTTTCTTCCATGAGACCCTCTACCAGAAAGATGATAATGGTGTTCCCTTCGTCCGAACCATCCAGGATAAGGGCATCGTCGTGGGCATCAAGGTTGACAAGGGTGTGGTGCCTCTAGCTGGGACTGATGGAGAAACCACCACTCAAGGGCTGGATGGGCTCTCAGAACGCTGTGCCCAATACAAGAAGGATGGTGCTGACTTTGCCAAGTGGCGCTGTGTGCTGAAAATCAGTGAGCGTACACCCTCTGCACTTGCCATTCTGGAGAACGCCAACGTGCTGGCCCGTTATGCCAGTATCTGCCAGCAGAATGGCATTGTGCCTATTGTGGAACCTGAAATATTGCCTGATGGAGACCACGACCTCAAACGTTGTCAGTATGTTACAGAGAAGGTCTTGGCTGCTGTGTACAAGGCCCTGAGTGACCATCATGTATACCTGGAGGGGACCCTGCTCAAGCCCAACATGGTGACCCCGGGCCATGCCTGTCCCATCAAGTATACCCCAGAGGAGATTGCCATGGCAACTGTCACTGCCCTGCGTCGCACTGTGCCCCCAGCTGTCCCAGGAGTGACCTTCCTGTCTGGGGGTCAGAGCGAAGAAGAGGCATCATTCAACCTCAATGCCATCAACCGCTGCCCCCTTCCCCGACCCTGGGCGCTTACCTTCTCCTATGGGCGTGCCCTGCAAGCCTCTGCACTCAATGCCTGG CGAGGGCAACGGGACAATGCTGGGGCTGCCACTGAGGAGTTCATCAAGCGGGCTGAGGTGAATGGGCTTGCAGCCCAGGGCAAGTATGAAGGCAGTGGAGAATATTGTGGAGCAGCAGCACAGTCACTCTACATTGCCAACCATGCCTACTGA (SEQ ID NO: 6)

In the present invention, the term "nucleic acid construct" is defined herein as a single-stranded 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.

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

In the present invention, the term "vector" refers to a nucleic acid carrier into which a polynucleotide that inhibits a protein is inserted. For example, vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1; phages such as lambda phage or M13 phage And animal viruses. Animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papilloma Vacuolar virus (such as SV40). A vector may contain multiple elements that control expression.

In the present invention, the term "host cell" refers to a cell introduced into a vector, and includes many cell types such as prokaryotic cells such as E. coli or subtilis, fungal cells such as yeast cells or Aspergillus, such as S2 Drosophila cells or Sf9 and other insect cells, or 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 the treatment, prevention, reduction, and / or alleviation of a disease or condition described herein in a subject.

The term "disease and / or disorder" refers to a physical state of the subject, which is related to the disease and / or disorder described in the present invention.

The term "subject" may refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, or cow, receiving a pharmaceutical composition of the invention to treat, prevent, reduce, and / or alleviate a disease or condition described in the invention. , Horses, etc.

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 proteins to a level that is almost undetectable (in fact, in general, it is difficult to knock out target DNA or target RNA 100% ). Partial knockout refers to the case where the degree of knockout is greater than zero and less than complete knockout. The beneficial effects of the invention

Compared with the prior art, the shRNA and protein mutants using the aldolase of the present invention can significantly directly activate AMPK, thereby realizing that aldolase can be used as a target to develop a drug to start AMPK, which overcomes the direct use of AMPK as a drug in the prior art Target.

The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Those who do not indicate specific techniques or conditions in the examples follow the techniques or conditions described in the literature in the art (for example, refer to the "Molecular Cloning Experiment Guide" translated by J. Sambrook et al., Huang Peitang et al., Third edition, Science Press) or in accordance with the product manual. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that are commercially available. Example 1: shRNA inhibits ALDOA-C gene expression 1. Experimental materials and main reagents

Cell line: Mouse fibroblast MEF is an immortalized MEF, that is, after fibroblasts are isolated from mouse embryos, SV40 T antigen is transfected to make the cells immortal. 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 cells HEK293T (cat. CRL-3216), purchased from ATCC. Vector pLVX-IRES (cat. # 631849) was purchased from Clontech. Transfection reagent: Lipofectamine 2000 (cat. 11668-027), purchased from Invitrogen. Doxycycline (cat. S4163), purchased from Selleckchem. Dulbecco ’s modified Eagle ’s medium (DMEM, Gibco, cat. 11965) was purchased from Thermofisher. Primary antibodies used in Western: anti-ALDOA (cat. # 8060), purchased from Cell Signaling Technology; anti-ALDOB (cat. 18065-1-AP), purchased from Ptoteintech; anti-ALDOC (cat. AM2215b), purchased from Abgent Anti-β-tubulin (cat. # 2128), purchased from Cell Signaling Technology. Secondary antibodies used in Western: 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. Experimental method

It has been reported that knockdown ALDOA can cause cell death ^25. The results of previous experiments by the inventors also show that knockdown of ALDOB or ALDOC can cause cell death. In order to prevent the above-mentioned cell death from affecting the experimental results, the inventors have re-established a new set of experimental methods: (1) Construction of a recombinant expression plasmid that induces the expression of ALDOA-C

Construction of doxycycline (Dox) induced expression plasmid (ALDV-IRES-ALDOA-C) for ALDOA-C. For the conventional cloning method, please refer to the Molecular Cloning Experiment Guide. The steps are as follows:

The CDS fragments (SEQ ID NO: 4-6) of ALDOA, ALDOB, and ALDOC were amplified by PCR, and the restriction enzyme treatment (EcoRI and BamHI double digestion) vector pLVX-IRES was used. Finally, the CDS fragment and enzyme The cut vectors were ligated to obtain pLVX-IRES-ALDOA-C. (2) Construction of MEF cell lines that induce the expression of ALDOA-C

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

Inoculate 1.5 x 10 ⁶ human embryonic kidney cells HEK293T to a 35 mm cell culture dish, add 2 ml of DMEM cell culture solution to each dish, and culture in a 5% CO ₂ incubator (culture conditions: 5% CO ₂ , 37 ° C) Overnight, when the cell density reaches 80% or more, prepare a transfection mixture of the transfection reagent and the expression plasmid, leave it at room temperature for 20 minutes, add it to the cell culture solution, and continue to culture for 48 hours to collect the supernatant containing the lentiviral particles. . The supernatant containing the lentiviral particles was added to a 35 mm cell culture dish inoculated with mouse fibroblasts (MEFs), and the culture was continued for more than 24 hours, and then the cells were collected for Western blot experiments.

The cells were cultured in a DMEM medium containing 100 ng / ml Dox, and thus a MEF cell line that induced the expression of ALDOA-C was constructed. (3) shRNA sequence design

Targeting ALDOA: 5'-CCAAGTGGCGCTGTGTGCT-3 '(SEQ ID NO: 7), or 5'-GCCATGGGCCTTGACTTTC-3' (SEQ ID NO: 8) Targeting 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

The two shRNAs targeted to ALDOA, ALDOB or ALDOC were introduced into the above MEF cell line via lentivirus for more than 24 hours, so that the endogenously expressed ALDOA, ALDOB or ALDOC was blocked. The specific steps for introducing lentivirus into shRNA are as follows:

Inoculate 1.5 x 10 ⁶ human embryonic kidney cells HEK293T to a 35 mm cell culture dish, add 2 ml of DMEM cell culture solution to each dish, and culture in a 5% CO ₂ incubator (culture conditions: 5% CO ₂ , 37 ° C) Overnight, when the cell density reaches 80% or more, prepare a transfection mixture of the transfection reagent and the shRNA-containing plasmid, leave it at room temperature for 20 minutes, add it to the cell culture solution, and continue to culture for 48 hours to collect the lentiviral particles. The supernatant. The supernatant containing the lentiviral particles was added to a 35 mm cell culture dish inoculated with mouse fibroblasts (MEFs), and the culture was continued for more than 24 hours.

Finally, the cells were cultured in DMEM medium without Dox for 12 hours to cause exogenous ALDOA-C induced expression to disappear, and then the cells were collected for the following western blot experiments. (5) Western blot detection of ALDOA-C protein level

Follow the conventional Western Blot operation. 3. Experimental results

As shown in Figure 1.

The results showed that the level of ALDOA-C protein was significantly down-regulated in the shRNA-treated group. This indicated that shRNA significantly inhibited the expression of ALDOA-C gene. Example 2: shRNA targeted to ALDOA-C can start AMPK 1. Experimental materials and main reagents

Primary antibodies used in Western: Rabbit 548 anti-phospho-AMPKα-T172 (cat # 2535), purchased from 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), purchased from Cell Signaling Technology; anti-ACC (cat. # 3662, 1: 1000 for IB), purchased from Cell Signaling Technology. The other primary and secondary antibodies used were as described in Example 1. DMEM without glucose (Gibco, cat. 11966) was purchased from Thermofisher. Experimental method

(1) First refer to the method described in Example 1 to prepare MEF cells with simultaneous knockdown of ALDOA-C as shown in Figure 1. Among them, shRNA targeting GFP (Green Fluorescent Protein) was used as a control group (non-targeting control group), and shRNA sequence targeting GFP 5'-GGCACAAGCTGGAGTACAA-3 '(SEQ ID NO: 13) was used to construct For the method, refer to Example 1.

(2) Treat the cells with DMEM medium without glucose (Glc) for 2 hours (activate AMPK), while using DMEM medium with glucose as a control.

(3) Cells were lysed, and the lysate was subjected to immunoblotting to detect the levels of p-AMPK and p-ACC to measure the activation of AMPK. 3. Experimental results

As shown in Figure 2.

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

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Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand. Based on all the teachings that have been disclosed, various modifications and substitutions can be made to those details, and these changes are all within the protection scope of the present invention. The full scope of the invention is given by the scope of the appended claims and any equivalents thereof.

Figure 1: Immunoblot results of shRNA inhibition of ALDOA-C gene expression. Among them, # 1 and # 2 respectively represent one of two shRNAs targeting ALDOA, ALDOB, or ALDOC. 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. Figure 2: Detection of the effect of shRNA on AMPK after ALDOA-C inhibition. Among them, siGFP is the control group.

Claims (18)

Use of any one selected from the following items (1) to (6) in the preparation of a drug that initiates AMPK or in a model for the screening of a drug that initiates AMPK: (1) Aldolase; (2) a polynucleoside encoding an Aldolase (3) a nucleic acid construct containing a polynucleotide for completely or partially knocking out the Aldolase gene; preferably, the polynucleotide is an siRNA such as shRNA, or a guide for the CRISPR / Cas9 system RNA; (4) a host cell in which the polynucleotide encoding Aldolase is completely or partially knocked out; preferably, it contains the nucleic acid construct according to item (3); (5) an inhibition or inhibition Drugs that interrupt Aldolase activity; (6) A drug that inhibits or reduces the level of Aldolase gene expression. The use according to item 1 of the scope of patent application, wherein the drug that inhibits or blocks Aldolase activity is an antibody against Aldolase or TDZD-8; preferably, the antibody is a monoclonal antibody. The use according to item 1 of the scope of patent application, wherein the drug that inhibits or reduces the expression level of the Aldolase gene is selected from siRNA such as shRNA, and guide RNA for the CRISPR-Cas9 system. It is selected from any one of the following items (1) to (6) in the preparation of a drug for inhibiting cholesterol synthesis, a drug for reducing fatty acid synthesis, an anti-obesity drug, a drug for preventing and / or treating diabetes, a preventive and / or treating Use of a medicine for tumor, a medicine for preventing and / or treating Parkinson's disease, a medicine for preventing and / or treating Alzheimer's disease, an anti-aging medicine or a medicine for extending the life of a mammal: (1) Aldolase (2) a polynucleotide encoding Aldolase; (3) a nucleic acid construct containing a polynucleotide for completely or partially knocking out the Aldolase gene; preferably, the polynucleotide is an siRNA such as shRNA, or Is a guide RNA for the CRISPR / Cas9 system; (4) a host cell in which a polynucleotide encoding Aldolase is completely or partially knocked out; preferably, it contains the nucleic acid construct according to item (3) (5) a drug that inhibits or blocks the activity of Aldolase; (6) a drug that inhibits or reduces the level of Aldolase gene expression; preferably, the tumor is selected from black Any one or more of melanoma, pancreatic cancer, ovarian cancer and breast cancer. The use according to item 4 of the scope of patent application, wherein the drug that inhibits or blocks Aldolase activity is an antibody against Aldolase or TDZD-8; preferably, the antibody is a monoclonal antibody. The use according to item 4 of the scope of patent application, wherein the drug that inhibits or reduces the expression level of the Aldolase gene is selected from siRNA such as shRNA, and guide RNA for the CRISPR-Cas9 system. A method for activating AMPK in vivo or in vitro, comprising the steps of inhibiting Aldolase activity or down-regulating Aldolase gene expression level, for example, including the step of inhibiting Aldolase activity or down-regulating Aldolase gene expression level in a subject or cell in need thereof . A method for 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: a drug that activates AMPK, a drug that inhibits cholesterol synthesis, a drug that reduces fatty acid synthesis, and an anti-obesity drug 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, anti-aging drugs or using A drug for extending the life of a mammal; Preferably, the tumor is any one or more selected from the group consisting of melanoma, pancreatic cancer, ovarian cancer, and breast cancer. A recombinant vector containing an siRNA such as shRNA that down-regulates the expression level of the Aldolase gene, or a guide RNA used in the CRISPR-Cas9 system; preferably, the recombinant vector is a recombinant lentiviral vector. A host cell contains the recombinant vector described in item 9 of the patent application scope, or the polynucleotide encoding Aldolase therein is completely or partially knocked out. A pharmaceutical composition comprises the recombinant vector described in claim 9 or the host cell described in claim 10, and optionally, further comprises a pharmaceutically acceptable excipient. Any one selected from the following items (1) to (4), which is used to prepare a drug that initiates AMPK, a drug that inhibits cholesterol synthesis, a drug that reduces fatty acid synthesis, an anti-obesity drug, and prevents and / or treats diabetes Drugs, drugs that prevent and / or treat tumors, drugs that prevent and / or treat Parkinson's disease, drugs that prevent and / or treat Alzheimer's disease, anti-aging drugs, or drugs that extend the life of mammals , Or a model for the preparation of a drug that activates AMPK: (1) Aldolase; (2) a polynucleotide encoding Aldolase; (3) a nucleic acid construct containing a complete or partial knockout of the Aldolase gene Preferably, the polynucleotide is an siRNA such as shRNA, or a guide RNA used in the CRISPR / Cas9 system; (4) a host cell in which the polynucleotide encoding Aldolase is completely knocked out or partially Knockout; preferably, it contains the nucleic acid construct according to item (3); preferably, the tumor is any one selected from the group consisting of melanoma, pancreatic cancer, ovarian cancer, and breast cancer One or several. A drug that inhibits or blocks Aldolase activity, or a drug that inhibits or reduces Aldolase gene expression level, and is used to activate AMPK, inhibit cholesterol synthesis, reduce fatty acid synthesis, anti-obesity, prevent and / or treat diabetes, prevent and / or Treating a tumor, preventing and / or treating Parkinson's disease, preventing and / or treating Alzheimer's disease, anti-aging or for prolonging the life of a mammal; preferably, the tumor is selected from the group consisting of melanoma, pancreatic cancer, Any one or several of ovarian cancer and breast cancer. The drug according to item 13 of the application, wherein 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 according to item 13 of the scope of patent application, wherein the drug that inhibits or reduces the expression level of the Aldolase gene is selected from siRNA such as shRNA, and guide RNA for the CRISPR-Cas9 system. A method of treating and / or preventing hypercholesterolemia, diabetes, tumors, Parkinson's disease, or Alzheimer's disease, or an anti-obesity (such as preventing obesity or losing weight), anti-aging, or extending the life of a mammal, or A method for inhibiting cholesterol synthesis or reducing fatty acid synthesis, comprising the steps of inhibiting the activity of Aldolase in a subject in need or down-regulating the expression level of Aldolase gene in a subject in need; preferably, the method comprises administering Step of the subject with an effective amount of a drug that inhibits or blocks Aldolase activity or a drug that inhibits or reduces the level of Aldolase gene expression; preferably, the tumor is selected from the group consisting of melanoma, pancreatic cancer, ovarian cancer, and breast Any one or more of the cancers. The method according to item 16 of the application, 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 method of claim 16, wherein the drug that inhibits or reduces the expression level of the Aldolase gene is selected from siRNAs such as shRNA, and guide RNA for the CRISPR-Cas9 system.