CN113491771A

CN113491771A - Application of small molecular compound in preparation of GLUT5 inhibitor for uptake and transportation of fructose

Info

Publication number: CN113491771A
Application number: CN202010192247.3A
Authority: CN
Inventors: 张洪建; 周丹丹; 倪瑶
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2020-03-18
Filing date: 2020-03-18
Publication date: 2021-10-12

Abstract

The invention relates to application of a small molecular compound in preparation of an inhibitor for GLUT5 to uptake and transport fructose and application of the small molecular compound in preparation of a medicine for resisting growth and proliferation of tumor cells.

Description

Application of small molecular compound in preparation of GLUT5 inhibitor for uptake and transportation of fructose

Technical Field

The invention relates to the field of GLUT5 transporter inhibitors and tumor treatment drugs, in particular to application of a small molecular compound in preparation of an inhibitor for GLUT5 to take up and transport fructose.

Background

The metabolism of normal cells, usually with glucose as the basic substance, enters the tricarboxylic acid cycle, generates ATP to provide energy, forms basic units of biological macromolecules such as bases, amino acids, lipids and the like, maintains the redox balance, and realizes and completes physiological functions. The metabolism of tumor cells, i.e., the production of pyruvate by sugars, mainly proceeds to glycolysis for energy production, and even in the presence of sufficient oxygen, this is a short, low-energy-production pathway through glycolysis, which is known as the Warburg effect in oncology. The process of sugar entry into tumor cells (basal unit supply) is important because of the large energy consumption and sugar consumption required by tumor cells to continually divide and proliferate due to uncontrolled growth cycles. Since the cell membrane is a non-polar fat membrane (nonpolarator lipid bilayer) and the sugar molecule is a polar molecule, the process of sugar entry into tumor cells, except for a small amount of passive diffusion, is primarily dependent on uptake and transport of specific sugar transporters.

Sugar transporters belong to the SLC2 gene family and can be classified into three categories according to their physiological function differences: (1) transport of 6-membered ring glucose in blood cells, adipose tissue and skeletal muscle, such as SLC2a1(GLUT 1); (2) transport of 5-membered ring fructose, if the sugar-specific transporter SLC2a5(GLUT 5); (3) bifunctional GLUT7 and GLUT11, which are capable of transporting both 6-membered ring sugars, such as glucose, and 5-membered ring sugars, such as fructose.

The cell cycle of the tumor cells is out of control, and a large amount of energy is consumed for the continuous division and proliferation; this metabolic reprogramming is revealed as the wolberg effect: the tumor cells usually adopt glycolysis pathway with lower productivity, and tend to consume more glucose, resulting in glucose deficiency in the process of tumor growth. In this case, the tumor cells will develop a fructose replacement mechanism, i.e., make up for the glucose deficiency with fructose to provide an energy source. Research proves that the use of fructose can cause the proliferation of tumor cells to be accelerated, the cloning formation to be increased, the infiltration to be stronger, and the cell characteristics of various tumor deteriorations to be shown. Therefore, in tumor cells with insufficient glucose supply, the fructose uptake is blocked or reduced, and the effect of inhibiting the growth and proliferation of tumor cells can be achieved. Based on this hypothesis, the possibility of treating cancer by modulating GLUT5 transport function was explored, using the fructose specific transporter GLUT5 as a potential novel target.

GLUT5 is a transporter for specifically transporting fructose, a plurality of GLUT5 inhibitors for absorbing and transporting fructose have been developed at present, and the development of novel GLUT5 inhibitors based on small molecular compounds is significant, so that more medicaments and methods can be provided for treating tumors.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide an application of a small molecular compound in preparing an inhibitor for GLUT5 to uptake and transport fructose, and discloses a plurality of small molecular compounds capable of inhibiting GLUT5 to uptake and transport fructose and an application of the small molecular compounds in preparing a medicine for resisting tumor cell growth and proliferation.

The invention discloses an application of a small molecular compound in preparing an inhibitor for GLUT5 to uptake and transport fructose, wherein the small molecular compound comprises one or more of imatinib, dapagliflozin, quercitrin, quinidine, pazopanib, scutellarin, clopidogrel sulfate, quercetin-3-oxyglucoside, enalapril, diclofenac sodium, atenolol, nifedipine and aconitine.

Further, the administration mode of the small molecule compound is oral or intravenous injection.

Furthermore, GLUT5 absorbed fructose in the transporter at a fructose concentration of 3mM and a compound concentration of 100. mu.M.

Preferably, the small molecule compound comprises one or more of dapagliflozin, quercitrin, scutellarin and quercetin-3-oxyglucoside. These small molecule compounds all have sugar structures. The inhibition effect of the small molecular compound on the uptake and transport of fructose by GLUT5 is related to sugar structure, and is also related to 'aglycon' structure.

On the other hand, the invention discloses the application of small molecular compounds in preparing medicines for resisting the growth and proliferation of tumor cells, wherein the small molecular compounds comprise one or more of imatinib, dapagliflozin, quercitrin, quinidine, pazopanib, scutellarin, clopidogrel sulfate, quercetin-3-oxyglucoside, enalapril, diclofenac sodium, atenolol, nifedipine and aconitine.

Further, the tumor cells include human gastric cancer cell SGC7901, human cervical cancer cell HeLa, human breast cancer cell MCF-7, human pancreatic cancer cell PANC-1 and human colon cancer cell HCT-116.

Preferably, the small molecule compound comprises one or more of dapagliflozin, quercitrin, scutellarin and quercetin-3-oxyglucoside.

According to the invention, a plurality of GLUT5 inhibitors including natural products and molecular small compounds with different and diverse structures are screened out through a GLUT5 inhibitor screening experiment system. The research on tumor cells finds that different compounds have specific inhibition effects on the survival and proliferation of different tumor cells and are influenced by the content of fructose and glucose in a culture environment. For tumor cells which proliferate depending on fructose, under the culture condition of only fructose, the GLUT5 inhibitor can obviously inhibit the proliferation and survival of the tumor cells by inhibiting fructose transport mediated by the inhibitor. In the culture environment, once glucose exists, the inhibition effect of the glucose is weakened or even disappears along with the increase of the glucose concentration. The inhibitor can block or reduce the uptake of fructose by inhibiting the activity of GLUT5 under the condition of glucose deficiency, and can be used for preparing medicaments for inhibiting the growth and proliferation of tumors.

In the invention, the inhibitor of GLUT5 for absorbing and transporting fructose is screened by a human GLUT5 stable transfected cell strain, and the construction method comprises the following steps:

(1) assembling the hGLUT5 target gene with pcDNA3.1/G418(+) Ampicillin expression vector to construct pcDNA3.1/G418(+) -hGLUT5 plasmid; wherein, the nucleotide sequence of the hGLUT5 target gene is shown as SEQ ID No. 1;

(2) transfecting the plasmid constructed in the step (1) to HEK293 cells by adopting a liposome method, after the cells are transfected for 24h, screening positive clones by using G418 antibiotics, and confirming that the cell strain stably expresses GLUT5 gene and protein thereof at the same time by using positive compounds to carry out transport or metabolic function verification, thereby constructing the human GLUT5 stably transfected cell strain.

Further, in the step (1), the method for synthesizing the hGLUT5 target gene comprises the following steps: the SLC2A5 gene sequence (NM-001328619.2) was obtained from a gene bank and the gene was synthesized using synthetic biology methods. Compared with the traditional gene cloning technology, the synthetic biology has the characteristics of rapidness, accuracy, high efficiency and the like, and has more advantages in constructing a gene expression system.

Further, in the step (1), the endonucleases used in constructing the plasmid are a5 '-end KpnI and a 3' -end XhoI restriction endonuclease.

In the step (2), compared with a physical transfection method, the method is time-consuming and labor-consuming, and low in efficiency, an artificial liposome method in a chemical transfection method has high transfection efficiency, cell lines which are difficult to transfect by other methods can be transfected, and DNA plasmids, small interfering RNAs (microRNAs, shRNA and the like) and even proteins can be transfected by the liposome method. In addition, the liposome transfection method is simultaneously suitable for transient expression and stable expression, wherein the former has the characteristics of rapidness and flexibility, and the latter has the advantages of stability, strong reproducibility and the like.

In the step (2), the HEK293 cell is used as a transfected cell, the HEK293 cell is a human embryonic kidney cell, and the biochemical characteristic examination finds that most of metabolic enzymes, uptake transporters and efflux transporters are lacking in the HEK293 cell. Therefore, the HEK293 cell has the advantages of short culture period, low background, easy culture and the like, and the stably transfected HEK293 cell strain is an effective means for drug metabolism and transport research.

Further, in step (2), cells in the logarithmic growth phase are taken for cell transfection.

Further, in step (2), the concentration of the plasmid at the time of transfection of the plasmid was 0.001. mu.g/. mu.L.

Further, in step (2), the concentration of the G418 antibiotic was 800. mu.g/mL. At the above concentrations, cells without the corresponding resistance died 60% in about 2 days, whereas cells with G418 resistance did not die in principle. Too high a concentration will kill resistant cells and the morphology of cells cultured for a long period of time will be subject to change.

Further, in the step (2), when the cell fusion degree exceeds 25% in the screening of the G418 antibiotics, the cells are immediately passaged, the culture solution containing the G418 antibiotics is replaced every 3 to 4 days for culture, the steps are repeated until the cells are passaged to 3 to 4 generations, and positive monoclonals are selected and continuously cultured until the cells grow into cell strains.

Further, in step (2), the medium used for plasmid transfection is serum-free DEME medium.

Further, in step (2), plasmid transfection was performed using PolyJet transfection reagent.

Further, the amount of transfection reagent used was 8. mu.L per 100. mu.L of medium.

Further, in step (2), the expression and function of mRNA and protein are verified to select a positive clone cell strain which stably expresses hGLUT5 gene and has activity.

The method for screening GLUT5 uptake transport fructose by using the human GLUT5 stable transfected cell strain comprises the following steps:

(S1) culturing the human GLUT5 stable transfected cell strain and Mock cells in a high-sugar culture medium, and when the cells grow to 75-85%, plating the cells in a porous plate coated with polylysine;

(S2) replacing the high-sugar culture solution with sugar-free DMEM, and pre-incubating the solution of the compound to be tested prepared from the sugar-free DMEM to remove the influence of glucose in the culture environment; adding substrate fructose and a compound to be tested into the pre-incubation environment for incubation, removing the incubation liquid and cleaning the pore plate after the incubation is ended so as to remove the influence of the fructose in the culture environment; wherein the same compound to be tested is added before and after the pre-incubation;

(S3) detecting the content and protein concentration of fructose in cells of each hole, normalizing the content of fructose in cells of each hole by using the protein concentration, calculating the uptake and transport rate of the cells to fructose, and screening an inhibitor of the uptake and transport of fructose by GLUT5 if the compound to be detected has an inhibiting effect on the uptake and transport of fructose by GLUT 5.

Further, in the step (S1), the cells were cultured in 100mm cell culture dishes, passaged every four days, in a medium containing 10% fetal bovine serum, and placed in an incubator maintained at 37 ℃ with 5% carbon dioxide and 95% humidity.

Further, in the step (S1), the density of the cells in plating is 2X 10^ 5/well, and the cell culture solution in plating is high-sugar DMEM containing 10% fetal bovine serum.

Further, in step (S1), HEK293-Mock cells and HEK293-GLUT5 cells in 100mm cell culture dishes were plated in polylysine-coated 24-well plates. The specific operation steps are that firstly, 180 mu L of 100 mu g/mL polylysine solution is added into a 24-well plate to soak the bottom surface, the bottom surface is absorbed by a pipette after being placed for 5 minutes, and the bottom surface is washed for three times by ultrapure water.

Further, the next day of cell plating, the step (S2) is performed.

Further, in step (S2), in order to remove the interference of glucose in the culture environment, the high-sugar culture solution needs to be aspirated and washed with Phosphate Buffered Saline (PBS) and dried by aspiration. Additional 450 μ L of sugarless DMEM, 25 μ L of 2mM test compound in sugarless DMEM was preincubated.

Further, in step (S2), the concentration of sugar-free DMEM is 450 μ L; in the pre-incubation process, the final concentration of the compound to be detected is 100 mu M; the final concentration of substrate fructose was 3mM upon incubation.

Further, in step (S2), the pre-incubation time was 1 hour.

Further, in step (S2), 25. mu.L of a 60mM fructose solution prepared with sugar-free DMEM was added to each well on the basis of the pre-incubation to give a final fructose concentration of 3mM as the start of the incubation.

Further, in step (S2), after adding the substrate fructose and the test compound, incubation was performed at 37 ℃ for 5 minutes.

Further, in step (S2), when the incubation was terminated, the incubation solution was poured off, washed three times with pre-ice Phosphate Buffer Solution (PBS) and blotted dry.

Further, in the step (S3), the method of detecting the fructose content and the protein concentration in the cells per well includes the steps of:

taking 100 mu L of cell disruption solution per hole, adding 200 mu L of acetonitrile containing 2 mu g/mL of internal standard betamethasone, precipitating protein after 2min of vortex, centrifuging at 13000rpm for 10min at 4 ℃, transferring 100 mu L of supernatant to a sample injection bottle, sucking 10 mu L of supernatant for LC-MS/MS analysis to detect the content of fructose in cells of each hole;

in addition, 25. mu.L of cell disruption solution was taken per well, and the BCA kit by TaKara was used to detect the protein concentration in the cells of each well.

Further, the preparation method of the cell disruption solution comprises the following steps: 250. mu.L of ultrapure water was added to each well, and the cells were disrupted by an ultrasonic disrupter.

Further, the chromatographic column is Syrergi 4 μm Hydro-RP during LC-MS/MS analysis

(75X 4.6 mm); mobile phase a phase: 5mM ammonium acetate with 0.1% formic acid in water and acetonitrile in phase B; the sample injection running time is 4min, and the elution mode is isocratic elution (the mobile phase ratio is 1:1, v/v). The detected ion information of fructose is 179.1 → 89.1. The ion source is an ESI source, the scanning mode is a multi-reaction detection (MRM) mode, and the scanning time is 100 ms.

By the scheme, the invention at least has the following advantages:

the invention discloses a plurality of small molecule compound inhibitors for absorbing and transporting fructose by GLUT5, wherein the small molecules can be used as anti-tumor growth and proliferation medicaments based on the inhibition effect of the small molecules on absorbing and transporting fructose by GLUT5, and are applied to treatment of a plurality of tumors.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.

Drawings

FIG. 1 shows the comparison of the mRNA levels of hGLUT5 in HEK293 cells and CHO cells after the same transfection with plasmids of the invention;

FIG. 2 is a schematic diagram of HEK293 cells transfected by plasmids with hGLUT5 target genes according to the invention;

FIG. 3 shows the result of verifying the mRNA expression level of SLC2A5 gene in the hGLUT5 monoclonal cells of the present invention;

FIG. 4 is a typical chromatogram of fructose (3. mu.M) according to the present invention;

FIG. 5 shows the result of the GLUT 5-mediated fructose uptake assay in monoclonal cell lines;

FIG. 6 shows the results of the protein level verification of the HEK293-hGLUT5 monoclonal cell strain (17 #);

FIG. 7 shows the effect of SiRNA transfection on SLC2A5 gene level in HEK293-hGLUT5 monoclonal cell line (17 #);

FIG. 8 shows the fructose uptake of HEK293-hGLUT5 monoclonal cell line (17#) at different incubation times;

FIG. 9 shows the fructose uptake of HEK293-hGLUT5 monoclonal cell line (17#) by preincubation with the inhibitor Apigenin of the present invention for various periods of time;

FIG. 10 shows the screening results of GLUT5 uptake transport fructose inhibitor using HEK293-hGLUT5 monoclonal cell strain and GLUT5 inhibitor screening test system successfully constructed in the present invention.

FIG. 11 is a graph showing the effect of Scutellarin, an inhibitor of GLUT5, screened by the present invention, on the proliferation of various tumor cells.

Detailed Description

The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

EXAMPLE 1 construction of plasmid

Obtaining an SLC2A5 gene sequence (NM-001328619.2) from a gene bank, and obtaining an hGLUT5 target gene by gene synthesis by using a synthetic biology method, wherein the nucleotide sequence of the hGLUT5 target gene is shown as SEQ ID No. 1. The genes are respectively connected with pcDNA3.1/G418(+) Ampicillin expression vectors, and after transformation and amplification, purified plasmid pcDNA3.1/G418(+) -hGLUT5 recombinant plasmids are extracted. And carrying out electrophoretic identification on the recombinant plasmid to obtain a corresponding target band, carrying out double enzyme digestion identification on the recombinant plasmid by taking the 5 'end KpnI and the 3' end XhoI as restriction enzyme digestion sites to obtain a corresponding carrier band and a corresponding target gene band, wherein the result shows that the plasmid is successfully constructed and can be used for subsequent experiments.

EXAMPLE 2 transfection of plasmids

(1) Source and culture of cells:

for the selection of host cells, the levels of hGLUT5 mRNA in HEK293 cells and CHO cells transfected with plasmids carrying the hGLUT5 target gene were compared. The results are shown in FIG. 1.

The transfected cells were finally selected as HEK293 cells, and human embryonic kidney cells (HEK293) were purchased from ATCC (American type culture collection). Cells were grown in 100mm cell culture dishes every three days at a rate of 1: passage 5 was once performed in DMEM (100U/mL penicillin, 100. mu.g/mL streptomycin) containing 10% FBS, and cultured in an incubator containing 5% carbon dioxide, 95% humidity, 37 ℃.

(2) And (3) screening antibiotic concentration:

HEK293 cells in the logarithmic growth phase are taken for carrying out antibiotic (G418) concentration screening, the antibiotic concentration is different from 100 mu G/mL to 1600 mu G/mL, the cells are cultured for 2-3 weeks, and the lowest antibiotic concentration at which all the cells are killed is selected as the antibiotic concentration adopted when constructing the stable transfer cell strain. The results show that the screening concentration G418 of the HEK293 cell is 800 mug/mL.

(3) Plasmid transfection:

a) HEK293 cells in the logarithmic growth phase are taken and paved on a 6-well plate according to 10 ten thousand per well of HEK293 cells, and the transfection experiment is carried out after 24 hours.

b) HEK293 cells were transfected with plasmid pcDNA3.1 empty vector, pcDNA3.1/G418(+) -hGLUT5 according to the Polyjet transfection reagent instructions. And (3) screening out HEK293-hGLUT5 positive monoclonals by using G418 antibiotics, and confirming that the cell strain stably expresses GLUT5 genes and proteins thereof at the same time through metabolic function verification.

The plasmid transfection comprises the following specific steps:

mu.g of the plasmid was added to 100. mu.l of serum-free DMEM, then 100. mu.l of serum-free DMEM containing 8. mu.l of lipofectin Polyjet was added, mixed well, left for 15min and dropped into 6-well plates of the corresponding cell line for transfection.

c) 24hr after cell transfection, DMEM cell culture medium containing a certain concentration of selective antibiotic (G418 at 800. mu.g/mL) was replaced for selection. Once the cell fusion degree exceeds 25%, the cells are immediately passaged, the culture solution containing antibiotics with specific concentration is replaced every 3-4 days, and the circulation is repeated for 2-3 weeks until the cells are passaged to 3-4 generations, monoclones (48 cells) are selected to a 24-well plate to expand the cells, and a monocloned cell strain with G418 resistance and good growth state is selected for mRNA expression verification, functional expression verification and seed preservation. And selecting the cell strain with higher expression level for carrying out a function verification experiment continuously for four weeks, and selecting the cell strain with highest mRNA expression level and most stable function according to the result, namely constructing the human GLUT5 stable transfection cell strain which is used as a stable over-expression cell model.

(3) Construction and verification of stably transfected cell strains: in the steps b) and c), pcDNA3.1/G418(+) -hGLUT5 is firstly transfected into HEK293 cells, a plurality of groups of parallel experiments are carried out, the numbers of the experiments are respectively 1-48, and a plurality of HEK293-hGLUT5 positive monoclonals are screened out. The positive monoclonals obtained in parallel experiments were subjected to mRNA identification (fluorescent quantitative PCR) and protein expression (Western blot analysis). Total RNA from selected cell clones was extracted using RNAioso Plus and the concentration and purity of the RNA was determined using a Q5000 UV-Vis spectrophotometer (Quawell). RNA was reverse transcribed into cDNA according to PrimerScriptTM (Takara) kit method, GAPDH was selected as an internal reference gene, and hGLUT5 was quantified using iQTM SYBR Green Supermix (Bio-Rad) reagent and CFX 96Real-Time fluorescent quantitative PCR instrument (Bio-Rad). The PCR reaction conditions are as follows: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 2min, circulation for 35 times, and extension at 72 deg.C for 10 min. The expression of GLUT5 at the mRNA level was examined. The results are shown in FIG. 3, where Mock refers to wild-type cells of HEK293 and the numbers in the graph represent the numbers of HEK293-hGLUT5 positive monoclonals. The primers used therein are shown in table 1.

TABLE 1 primers for GAPDH, GLUT5 and SiRNA used in the present invention

(4) Verification of stably transformed cell lines

Transport function of HEK293-hGLUT5 positive monoclonals the function of the transporters of each monoclonal cell was evaluated using the GLUT5 transporter substrate fructose (fructose). First, an LC-MS/MS method capable of effectively measuring the Fructose content was developed, and a chromatogram is shown in FIG. 4, in which the standard concentration of Fructose is 3. mu.M in FIG. 4. The conditions for analyzing the fructose concentration by LC-MS/MS are as follows: the chromatographic column is Syrergi 4 μm Hydro-RP

(75X 4.6 mm); mobile phase a phase: 5mM ammonium acetate with 0.1% formic acid in water and acetonitrile in phase B; the sample injection running time is 4min, and the elution mode is isocratic elution (the mobile phase ratio is 1:1, v/v). Detection of fructose ion information is179.1 → 89.1. The ion source is an ESI source, the scanning mode is a multi-reaction detection (MRM) mode, and the scanning time is 100 ms.

Secondly, the positive clone cells after screening are processed according to the proportion of 2.0 multiplied by 10⁵Individual cell/cm²The density of (2) was inoculated on a 24-well (100. mu.g/mL polylysine PDL-coated) plate, and the experimental requirements were met after 1 day of culture. The transmembrane transport experiment was performed by incubating fructose at 3mM as a substrate in a water bath at 37 ℃ for 24hr, and the uptake rate of fructose in each positive monoclonal cell line was examined, and the results are shown in FIG. 5.

The method comprises the following steps of:

250. mu.L of ultrapure water was added to each well, and the cells were disrupted by an ultrasonic disrupter. Taking 100 mu L of cell disruption solution per well, adding 200 mu L of acetonitrile containing 2 mu g/mL of betamethasone as an internal standard, precipitating protein after 2min of vortex, centrifuging at 13000rpm for 10min at 4 ℃, transferring 100 mu L of supernatant to a sample injection bottle, sucking 10 mu L of supernatant for LC-MS/MS analysis to detect the content of fructose in cells of each well. The screened positive clone cell HEK293-hGLUT5(17# cell strain in figure 3) is inoculated on a 6-well plate and grows to 75% -85% per well, thus meeting the experimental requirements. Removing culture solution from each hole, washing with PBS for three times, extracting total cell protein, quantifying and normalizing, performing deglycosylation treatment, and performing Western blotting experiment. The results are shown in FIG. 6.

Wherein, the total cell protein quantification step is as follows:

after disrupting the cells, 25. mu.L of cell disruption solution was additionally taken per well, and the BCA kit by TaKara was used to detect the protein concentration within the cells of each well.

The screened positive clone cell HEK293-hGLUT5(17#) and Mock cell are normally cultured. 7X 10^ a day before transfection⁴The density of each well was seeded in 24-well plates. Serum-free DMEM is used for diluting SiRNA and Lipofectamin reagents during transfection, and then the fully and uniformly mixed SiRNA/Lipofectamin complex is added into a cell plate. Placing in a cell culture box for 6 hours after transfection, replacing with normal high-sugar DMEM without compound for culturing for 48 hours, extracting total RNA of cells, carrying out Q-PCR quantification after reverse transcription, and inspecting SiRNA interference pairsThe change in GLUT5 mRNA levels is shown in FIG. 7.

Example 3 Condition optimization of GLUT5 inhibitor screening test System

The transport function of HEK293-hGLUT5 positive monoclonal was tested by the rate of GLUT5 transporting substrate fructose (fructose) to evaluate the function of 17# monoclonal cell line transporter. The positive clone cells after screening are 2.0X 10⁵Individual cell/cm²The density of (2) was inoculated on a 24-well (100. mu.g/mL polylysine PDL-coated) plate, and the experimental requirements were met after 1 day of culture. Transmembrane transport experiments were performed by incubating fructose at 3mM as a substrate in a water bath at 37 ℃ for 5min, 10min, 40min and 60min, respectively, and the uptake rate of fructose in each positive monoclonal cell strain was examined, and the results are shown in FIG. 8.

The screened positive clone cell HEK293-hGLUT5(17#) and Mock cell are normally cultured. The effect of known GLUT5 inhibitor Apigenin (Apigenin, 100 μ M) on the rate of fructose transport by GLUT5 was examined at different preincubation times and the results are shown in FIG. 9.

Example 4 application of GLUT5 inhibitor screening test System

The screened positive clone cell HEK293-hGLUT5(17#) and Mock cell are normally cultured. The effect of different known compounds on the fructose transport rate of GLUT5 was examined according to the method of example 3, with a preincubation time of 1 hour, and the results are shown in fig. 10 and table 2. The results show that the known compounds have an inhibitory effect on fructose transport by GLUT5 and can be used as an inhibitor for fructose transport by GLUT 5. The Chinese names and chemical structural formulas of the compounds in the table 2 are shown in the table 3.

TABLE 2 screening results for inhibitors of GLUT5 uptake transport Fructose

TABLE 3 chemical Structure of an inhibitor of GLUT5 uptake transport Fructose

Example 5 Effect of GLUT5 inhibitors on the proliferation of different tumor cells

Human gastric cancer cell SGC7901, human cervical cancer cell HeLa, human breast cancer cell MCF-7, human pancreatic cancer cell PANC-1 and human colon cancer cell HCT-116 were seeded in a 96-well plate, and were co-incubated with 14 GLUT5 inhibitors (100. mu.M) selected in example 4, respectively, in a culture medium with fructose and glucose at different ratios for 72 hours, and it was found that different compounds could inhibit the growth and proliferation of different tumor cells by inhibiting GLUT5, and the results are shown in Table 4.

TABLE 4 comparison of the inhibitory Effect of GLUT5 inhibitor on the survival and proliferation of 5 human tumor cells

Note: in the table, "+ + + + +" indicates strong suppression, "+" indicates weak suppression, "+ + +" indicates medium suppression, and "-" indicates no suppression.

Using the GLUT5 inhibitor Scutellarin as an example, human breast cancer cells MCF-7, human cervical cancer cells HeLa and human colon cancer cells HCT-116 were plated in 96-well plates and incubated with serial concentrations of Scutellarin for 72 hours in DMEM medium containing 6mM Fructose and 25mM Glucose, respectively, and the results are shown in FIG. 11. FIG. 11A shows the results of the effect of Scutellarin on MCF-7 cell proliferation in different media, FIG. 11B shows the results of the effect of Scutellarin on HeLa cells in different media, FIG. 11C shows the results of the effect of Scutellarin on HCT-116 cell proliferation in media containing Fructose, and FIG. 11D shows the results of the effect of Scutellarin on HCT-116 cell proliferation in media containing high concentration Glucose. The result shows that Scutellarin can inhibit the uptake and transport of fructose by different tumor cells.

The results show that the invention successfully constructs the hGLUT5 stable transfected cell strain, and on the basis, the in vitro GLUT5 inhibitor screening method is successfully established, and the screening system of the inhibitor for GLUT5 uptake and transportation fructose based on the hGLUT5 stable transfected cell strain is a rapid, accurate, high-efficiency and strong-reproducibility experimental method, and is an important in vitro research means for screening GLUT5 inhibitors or regulators and evaluating the influence mechanism of drugs on organisms.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Suzhou university

Application of <120> small-molecule compound in preparation of GLUT5 fructose uptake transport inhibitor

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<170> SIPO Sequence Listing 1.0

<210> 1

<211> 747

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<213> (Artificial sequence)

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ggtaccatgg agcaacagga tcagagcatg aaggaaggga ggctgacgct tgtgcttgcc 60

ctggcaaccc tgatagctgc ctttgggtca tccttccagt atgggtacaa cgtggctgct 120

gtcaactccc cagcactgct catgcaacaa ttttacaatg agacttacta tggtaggacc 180

ggtgaattca tggaagactt ccccttgacg ttgctgtggt ctgtaaccgt gtccatgttt 240

ccatttggag ggtttatcgg atccctcctg gtcggcccct tggtgaataa atttggcaga 300

aaaggggcct tgctgttcaa caacatattt tctatcgtgc ctgcgatctt aatgggatgc 360

agcagagtcg ccacatcatt tgagcttatc attatttcca gacttttggt gggaatatgt 420

gcaggtgtat cttccaacgt ggtccccatg tacttagggg agctggcccc taaaaacctg 480

cggggggctc tcggggtggt gccccagctc ttcatcactg ttggcatcct tgtggcccag 540

atctttggtc ttcggaatct ccttgcaaac gtagatggtg agttcaggac atctcgggag 600

cacccccacc ccttcaccac tacccttggc cccctccttg tgttccaaag ccaccaccac 660

aggacaggac tttctgcaga ctggtctctt ctaacaggct ggatgtcctt ggggggccca 720

tcctgtcccg agccaacata gctcgag 747

Claims

1. The application of small molecular compounds in preparing GLUT5 fructose uptake and transport inhibitors, wherein the small molecular compounds comprise one or more of imatinib, dapagliflozin, quercitrin, quinidine, pazopanib, scutellarin, clopidogrel sulfate, quercetin-3-oxyglucoside, enalapril, diclofenac sodium, atenolol, nifedipine and aconitine.

2. Use according to claim 1, characterized in that: the administration mode of the small molecule compound is oral administration or intravenous injection.

3. Use according to claim 1, characterized in that: when GLUT5 ingested the transported fructose, the fructose concentration was 3mM and the compound concentration was 100. mu.M.

4. Use according to claim 1, characterized in that: the small molecule compound comprises one or more of dapagliflozin, quercitrin, scutellarin and quercetin-3-oxyglucoside.

5. The application of small molecular compounds in preparing the medicines for resisting the growth and proliferation of tumor cells, wherein the small molecular compounds comprise one or more of imatinib, dapagliflozin, quercitrin, quinidine, pazopanib, scutellarin, clopidogrel sulfate, quercetin-3-oxyglucoside, enalapril, diclofenac sodium, atenolol, nifedipine and aconitine.

6. Use according to claim 5, characterized in that: the tumor cells comprise one or more of human gastric cancer cells, human cervical cancer cells, human breast cancer cells, human pancreatic cancer cells and human colon cancer cells.

7. Use according to claim 5, characterized in that: the administration mode of the small molecule compound is oral administration or intravenous injection.

8. Use according to claim 5, characterized in that: the small molecule compound comprises one or more of dapagliflozin, quercitrin, scutellarin and quercetin-3-oxyglucoside.