CN108060165B

CN108060165B - Alpha-subunit-combined F1F0-ATP synthetase RNA inhibitor and application thereof

Info

Publication number: CN108060165B
Application number: CN201711337054.7A
Authority: CN
Inventors: 罗月球; 姚克; 刘思雨
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2017-12-14
Filing date: 2017-12-14
Publication date: 2021-03-02
Anticipated expiration: 2037-12-14
Also published as: CN108060165A

Abstract

The invention relates to a medicine for resisting F1F0-ATP synthetase activity. The inhibitor is combined with an alpha subunit of F1F0-ATP synthetase to release the alpha subunit from a mitochondrial membrane into cytoplasm, so that F1F0-ATP synthetase is depolymerized, the ATP synthetase activity is reduced, intracellular ATP synthesis is inhibited, and the inhibitor has the characteristics of high inhibition efficiency, convenience in synthesis, low cost and the like. The technical scheme is as follows: coaxial cable fatigue detection equipment, its characterized in that: an F1F0-ATP synthase RNA inhibitor (designated dsRNA-184-U), characterized in that the sequence of the inhibitor is as follows: 5'-UGGAUGGAGAACUGAUAAGGGU-3' are provided.

Description

Alpha-subunit-combined F1F0-ATP synthetase RNA inhibitor and application thereof

Technical Field

The invention relates to a medicine for resisting the activity of F1F0-ATP synthetase, in particular to an RNA inhibitor for inhibiting the activity of ATP synthetase by combining with F1F0-ATP synthetase alpha subunit.

Background

F1F0-ATP synthase is a multi-subunit containing transmembrane protein complex. F1 is located in the cytoplasm and comprises 3 α,3 β, ε, γ and δ subunits. F1F0-ATP synthase can synthesize and hydrolyze ATP (adenosine triphosphate) and regulate energy supply of living bodies. Abnormal expression of ATP is the basis of the occurrence of various diseases, such as the abnormal expression of ATP is closely related to abnormal proliferation of fundus blood vessels, myocardial ischemia, malignancy of tumors, invasiveness and poor prognosis; tumor cell metastasis and proliferation require more ATP, and therefore, by inhibiting intracellular ATP levels, tumor cells can be selectively killed and tumor angiogenesis inhibited. F1F0-ATP synthase is a key enzyme for synthesizing ATP in mitochondria, and inhibition of the synthase can inhibit excessive abnormal expression of ATP. In myocardial cells, the excessive consumption of ATP by F1F0-ATP synthetase leads to the occurrence of severe myocardial ischemia, and the reduction of the activity of F1F0-ATP synthetase regulates the occurrence of myocardial ischemia.

The F1F0-ATP synthetase inhibitor can be used for treating tumor. It was found that drug-resistant tumor cells show increased sensitivity to chemotherapy in the presence of ATP depletion, whereas drug-sensitive tumor cells show increased resistance to chemotherapy in the presence of exogenous ATP supplementation. The destruction of ATP metabolism by tumor cells is therefore of great importance for tumor therapy.

The tumor molecule targeted therapy is more effective and has less side effects than the current chemotherapy, and is a very promising tumor therapy method. For example, the epidermal growth factor receptor tyrosine kinase (EGFR-TK) inhibitor gefitinib is mainly used for treating lung cancer, rituximab is mainly used for treating non-Hodgkin lymphoma, and trastuzumab is a signal transduction inhibitor and is used for treating breast cancer. For the treatment of chronic myelogenous leukemia and gastrointestinal stromal cell tumors is imatinib mesylate. However, with the clinical application of targeted therapy, more and more patients have drug resistance, and the clinical effect of targeted therapy drugs is not satisfactory. Therefore, patients undergoing chemoradiotherapy and targeted therapy, if combined with ATP inhibitors, can produce the most effective therapeutic effect, such as GLUT-1 (glucose transporter 1) inhibitors, including the irreversible inhibitors WZB117 and Phloretin Phloretin, diclofenac (diclofenac), apigenin, fasentin, STF-31, and the clinical approved drug ritonavir (ritonavir). WZB117 and phroretin are irreversible inhibitors of GLUT1 and can significantly reduce glucose uptake and intracellular ATP levels, thereby inhibiting glycolysis and cell growth. Supplementation with exogenous ATP can alleviate WZB 117-induced cytotoxicity of tumor cells, suggesting that GLUT inhibitors such as WZB117 inhibit tumor cell growth by reducing intracellular ATP levels.

The F1F0-ATP synthetase inhibitor can also be used for treating tuberculosis, autoimmune deficiency, myocardial ischemia, etc., and is also used in pesticide. For example, the compound acts with ATP synthase C subunit to quickly and effectively kill tubercle bacillus, such as antitubercular medicaments TMC207 of antituberculous ATP synthase inhibitor diaryl quinoline; an autoimmune disease-resistant ATP synthase inhibitor Bz-423 for treating type I lupus erythematosus immune diseases; anti-tumor synthetase inhibitors, such as polyene alpha-pyrones; an ATP inhibitor is mainly used for inhibiting GLUT1, different cells have a plurality of GLUT isomers, and the same or different functions are performed, such as monosaccharide transportation, hexose transportation, bidirectional transportation and the like; and multiple types of GLUT are distributed on each cell, so that even if the function of GLUT1 is inhibited, other GLUTs will compensate for its function. Taking GLUT3 as an example, the GLUT is mainly distributed in neurons and placenta tissues, and when GLUT1 on neurons of a brain tumor patient is inhibited, the cells can still take glucose by relying on GLUT3, so that cancer cells can continue to grow. Meanwhile, GLUT1 is also easily induced, i.e. low glucose level, it is highly expressed, and high glucose level, it is lowly expressed, which makes the activity of GLUT1 inhibitors difficult to suppress or easily induce drug resistance. The GLUT1 inhibitor can be used for a long time to make brain tissue lack energy supply and make brain development be disordered. These ATP synthase inhibitors are all chemical compounds, and have the disadvantages of high cytotoxicity and low specificity.

Disclosure of Invention

The invention aims to overcome the defects of the background technology and provide an alpha subunit-combined F1F0-ATP synthetase RNA inhibitor, which enables alpha subunit to be released into cytoplasm from a mitochondrial membrane by combining with the alpha subunit of F1F0-ATP synthetase to cause F1F0-ATP synthetase to be depolymerized, thereby reducing ATP synthetase activity and inhibiting intracellular ATP synthesis.

The technical scheme provided by the invention is as follows: an F1F0-ATP synthase RNA inhibitor (designated dsRNA-184-U), characterized in that the sequence of the inhibitor is as follows:

5’-UGGAUGGAGAACUGAUAAGGGU-3’。

the complementary sequence of the inhibitor is: 5'-ACCCUUAUCAGUUCUCCAUCCA-3' are provided.

The preparation method comprises the following steps:

1) synthesizing single chains:

the sequence of the F1F0-ATP synthetase RNA inhibitor obtained by an ABI394RNA automatic synthesizer is as follows: 5'-UGGAUGGAGAACUGAUAAGGGU-3', and complementary sequences

5'-ACCCUUAUCAGUUCUCCAUCCA-3', and respectively carrying out purification and separation;

2) and (3) double strand synthesis:

adding the same amount of DEPC water into each single-stranded RNA sample with the same mole number, then adding the mixture into an annealing buffer solution in a ratio of 1 to 1, denaturing at 90 ℃ for 1 minute, and naturally cooling to room temperature to obtain a product, namely the required double-stranded RNA.

The step 1) of synthesizing the single strand comprises the following steps:

a. removing a protecting group;

b. activating;

c. performing nucleophilic reaction;

d. removing the protecting group;

e. purifying and separating;

f. and (5) centrifugally drying and freezing.

The F1F0-ATP synthetase RNA inhibitor is applied to diseases with abnormally high ATP expression, such as vascular proliferative diseases and tumor treatment.

The invention has the beneficial effects that:

the small fragment RNA sequence (named as dsRNA-184-U) synthesized by the invention can be combined with an intracellular F1F0-ATP synthetase alpha subunit (ATP5A) to release the alpha subunit of the F1F0-ATP synthetase from a cell membrane to cytoplasm, so that the F1F0-ATP synthetase is depolymerized, and the intracellular ATP synthesis is inhibited, and the small fragment RNA sequence has the characteristics of strong specificity and high inhibition efficiency; can be used for treating various diseases, such as fundus blood vessel hyperplasia, tuberculosis, autoimmune deficiency, tumor, myocardial ischemia, etc., and can also be applied in pesticide; the RNA sequence has small fragment, only 22 nucleotide sequences and simple preparation process. Because RNA is used as a genetic information carrier existing in biological cells and is a biological macromolecule originally possessed by the cells, the RNA synthesized by the invention has the characteristics of small side effect, convenient synthesis, low cost and the like when being used as a medicament.

Drawings

FIG. 1 is a 2.0% PAGE running electrophoresis of RNA duplexes.

FIG. 2 is a diagram showing the cytoplasmic release of the alpha subunit of F1F0-ATP synthase in the mitochondrial membrane in the dsRNA-184-U treated group, which is the control group of transfection reagents in the lip treated group.

FIG. 3 is a graph comparing F1F0-ATP synthase levels in cells from dsRNA-184-U treated groups and lip treated groups.

Detailed Description

The following further description is made with reference to the embodiments shown in the drawings.

An RNA inhibitor of the alpha subunit of F1F0-ATP synthase, the sequence of the inhibitor is:

5‘-UGGAUGGAGAACUGAUAAGGGU-3‘；

the complementary sequences are: 5'-ACCCUUAUCAGUUCUCCAUCCA-3' are provided.

The preparation method of the inhibitor comprises the following steps:

4. synthesizing single chains:

RNA sequence synthesis using ABI394RNA automated synthesizer, solid phase synthesis of single stranded RNA: first, the RNA sequence 5'-UGGAUGGAGAACUGAUAAG GGU-3' to be synthesized is input into a synthesizer, and chemical synthesis of RNA proceeds from the 3 'end to the 5' end. The synthesis method comprises the following steps:

1) removing a protecting group; removing the protecting group DMT (dimethoxytrityl) of the nucleotide previously attached to CPG with trichloroacetic acid to obtain a free 5' -hydroxyl end;

2) activating; mixing the protonated nucleoside 3 ' -phosphoramidite monomer with a tetrazole activator, and allowing the mixture to enter a synthesis column to form a tetrazole active intermediate of phosphoramidite (the 5 ' end of the tetrazole active intermediate is still protected by DMT and the 3 ' end of the tetrazole active intermediate is activated);

3) mixing the tetrazole active intermediate of phosphoramidite activated in the step 2) with the nucleotide after the deprotection group in the step 1), so that the tetrazole active intermediate of phosphoramidite and the 5' -hydroxyl of the nucleotide are subjected to nucleophilic reaction, and condensation and tetrazole removal are carried out, wherein the synthesized oligosaccharide nucleotide chain is extended by one base forward;

during the oxidative condensation reaction, the nucleotide monomer is connected with the oligosaccharide nucleoside connected on the CPG through a phosphite ester bond, the phosphite ester bond is unstable and is easy to be hydrolyzed by acid and alkali, and then the phosphite amide is converted into the phosphate triester by using a tetrahydrofuran solution of iodine, so that the stable oligosaccharide nucleotide is obtained.

After the above steps, one RNA nucleotide is linked to the nucleotide of CPG; all the steps are automatically completed in a synthesis module of an RNA synthesizer, and one base is added to the carrier-bound nucleotide at a time.

4) The 5'-UGGAUGGAGAACUGAUAAGGGU-3' fragment sample can be obtained by repeating the steps. At this time, the RNA synthesis column was switched to the cleavage module, an ammonium hydroxide solution was added to the column, the oligonucleotide was cleaved from the support, the RNA in the ammonium hydroxide solution was transferred to the deprotection module, and then the nucleotide-containing solution was heated at 65 ℃ for 1 hour to remove the protecting group.

RNA purification and separation the crude product obtained is purified and separated by HPLC (high pressure liquid chromatography), the desired RNA solution molecules are collected and the concentration is determined.

The complementary sequence was synthesized in the same way: 5'-ACCCUUAUCAGUUCUCCAUCCA-3', purification by HPLC.

Synthetic RNA sample preservation: the liquid RNA sample is centrifugally dried on a decompression centrifuge at 37 ℃ and 12000rpm to prepare dry powder which is frozen and stored at-20 ℃ for 6 months.

2. And (3) double strand synthesis:

to each 1OD single-stranded RNA sample, 250. mu.l of DEPC water was added to prepare a 20. mu.M stock solution. The synthesized sequences of 200. mu.M were added to 20. mu.l of annealing buffer (100mM potassium acetate, 30mM HEPES-KOH,2mM magnesium acetate), 100. mu.l of DEPC water was added, denaturation was carried out at 90 ℃ for 1 minute, and then the temperature was naturally lowered to room temperature, and the obtained product was the desired double-stranded RNA.

The synthesized RNA duplex was loaded onto a 2.0% PAGE gel run whose electrophoretogram is shown in FIG. 1 for purity determination.

The results of the related research on the F1F0-ATP synthetase alpha subunit RNA inhibitor are as follows:

first, effect on ATP Down-expression in lens epithelial cells

1. Experimental cells: lens epithelial cells were used as cell models and purchased by ATCC companies abroad.

2. Experimental drugs: a drug synthesized by an RNA chemical synthesizer comprising the following RNA complementary sequences:

5’-UGGAUGGAGAACUGAUAAGGGU-3’

and 5'-ACCCUUAUCAGUUCUCCAUCCA-3'.

3. The experimental method comprises the following steps:

1) inhibition of ATP expression by RNA-184-U complexes

Inoculating normal cultured lens epithelial cells into a 96-well plate at an inoculation density of 2 × 10⁴Each cell was cultured in 100. mu.l medium, 0.108. mu.l 20. mu.M dsRNA-184-U dose and 0.144. mu.l

RNAimax was mixed with 20. mu.l of cell culture medium and added to cultured lens epithelial cells to give a final dsRNA-184-U concentration of 30 nM. At the same time, 0.144. mu.l

RNAimax was mixed directly with 20. mu.l of cell culture medium and added to lens epithelial cells as a control. After further culturing for 0, 12, 24, 48, 72 hours, the amount of ATP synthesized was measured. The experiments were performed in three groups simultaneously and the average was calculated.

2) And statistical treatment: mean ± standard deviation for experimental data

All experimental data are shown to be processed using SPSS 12.0 software.

3) RNA pull-down and protein mass spectrometry technology for detecting the binding effect of dsRNA-184-U and ATP5A

RNA 3-terminal desthiobiotinylation labeling and paramagnetic RNA protein binding kits were purchased from Pierce. 50pmol of RNA was mixed with 1nmol of biotin-labeled cytidine diphosphate, 40U T4RNA ligase, 40U RNase inhibitor and 15% polyethylene glycol in RNA ligase reaction buffer at 16 ℃ overnight. Unlabeled RNA and RNA ligase were removed by ethanol precipitation. The 3-desthiotyped single-stranded RNA anneals to a non-labeled complementary single strand. Then, 50pmol of the single-stranded or double-stranded RNA complex was gently stirred with 50. mu.l of streptavidin magnetic beads for 30 minutes. After the RNA-magnetic bead complex is generated, the RNA-magnetic bead complex is mixed with cell lysate for 60 minutes at 4 ℃, then the cell lysate is put into a magnetic bead collecting tube to collect combined magnetic beads, finally, 50 microliters of eluent is used for eluting the RNA protein conjugate, and the eluted protein complex is sent to Prot Tech company of Suzhou for protein mass spectrometry to obtain a protein sequence.

4) Western Blot (immunoblotting test) technology for verifying ATP5A expression in mitochondrial membrane and cytoplasm

RNA (dsRNA-184-U) drug (final concentration 30nM) and

cells were treated with RNAimax (mock control), and after 48 hours, cell membrane and cytoplasmic proteins were extracted, respectively, and Western Blot was performed using ATP5A antibody.

5) ATP synthase Activity assay

Normal culture lens epithelial cells were seeded in 6-well plates and transfected with dsRNA-184-U into the crystal cells at a final concentration of 30 nM. At the same time

RNAiMAX was directly mixed with cell culture medium and added to lens epithelial cells as a control. After further incubation for 24 and 48 hours, ATP synthase activity was measured. The experiments were performed in three groups simultaneously and the average was calculated.

4. The experimental results are as follows:

1) the results of the ATP drop expression experiments are shown in the following table.

ATP values at different time points of dsRNA-184-U treated cells Table 1

^**P in comparison with the control group<0.01；

As can be seen from Table 1, dsRNA-184-U was able to reduce cellular ATP expression very significantly at 72 hours (P <0.01) compared to the control group;

2) the protein extract pulled down by dsRNA-184-U is found to contain ATP5A protein peptide segment by RNA-pull down and protein mass spectrum technology; the sequences of these ATP5A protein peptides are shown in the following table:

3) RNA drugs induce cytosolic release of the alpha subunit of F1F0-ATP synthase in the mitochondrial membrane

Wb (western blot) results show (see fig. 2): RNA drug-treated cytosol had ATP5A expression, while control lip-treated cytosol had no ATP5A expression; it can be seen that RNA drug treatment promotes cytosolic release of the alpha subunit of F1F0-ATP synthase.

4) dsRNA-184-U drugs decrease cellular F1F 0-ATPase activity

F1F0-ATP synthetase Activity assay it was found that dsRNA-184-U treated cells reduced F1F0-ATP synthetase activity (p <0.05), see FIG. 3.

Sequence listing

<110> Zhejiang university

<120> RNA inhibitor of F1F0-ATP synthetase alpha subunit and application thereof

<160>2

<210>1

<211> 22

<212>RNA

<213> Artificial sequence

<220>

<222>（1）…（22）

<400>1

　　UGGAU GGAGA ACUGA UAAGG GU 22

<110> Zhejiang university

<160>2

<210>2

<211> 22

<212>RNA

<213> Artificial sequence

<220>

<222>（1）…（22）

<400>2

　　UGGAU GGAGA ACUGA UAAGG GU 22

Claims

1. Use of an RNA for the preparation of an RNA inhibitor of F1F0-ATP synthase that binds to the alpha subunit, characterized in that: the sequence of the RNA is as follows:

5’-UGGAUGGAGAACUGAUAAGGGU-3’；

the complementary sequence of the RNA is: 5'-ACCCUUAUCAGUUCUCCAUCCA-3', respectively;

the RNA inhibitor is applied to diseases with abnormally high expression of ATP, or applied to vascular proliferative diseases and tumor treatment.

2. Use according to claim 1, characterized in that: the preparation method of the RNA inhibitor comprises the following steps:

1) synthesizing single chains:

obtaining an RNA inhibitor sequence 5'-UGGAUGGAGAACUGAUAAGGGU-3' and a complementary sequence 5'-ACCCUUAUCAGUUCUCCAUCCA-3' by an ABI394RNA automatic synthesizer, and respectively purifying and separating;

2) and (3) double strand synthesis:

adding the same amount of DEPC water into each single-stranded RNA sample with the same mole number, then adding the mixture into an annealing buffer solution in a ratio of 1 to 1, denaturing at 90 ℃ for 1 minute, and naturally cooling to room temperature to obtain a product, namely the required double-stranded RNA;

the step 1) of synthesizing the single strand comprises the following steps:

a. removing a protecting group;

b. activating;

c. performing nucleophilic reaction;

d. removing the protecting group;

e. purifying and separating;

f. and (5) centrifugally drying and freezing.