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Method for screening glutamine transporter inhibitor, inhibitor screened by adopting method and application of inhibitor

A technique for glutamine and transporters, which is applied in the field of screening inhibitors of glutamine transporters, and can solve problems such as high cost, low throughput, and poor specificity

Active Publication Date: 2022-03-01
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this screening method has the disadvantages of poor specificity, low throughput, and high cost. Therefore, it is of great significance to establish a reasonable inhibitor screening system.

Method used

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  • Method for screening glutamine transporter inhibitor, inhibitor screened by adopting method and application of inhibitor
  • Method for screening glutamine transporter inhibitor, inhibitor screened by adopting method and application of inhibitor
  • Method for screening glutamine transporter inhibitor, inhibitor screened by adopting method and application of inhibitor

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] The construction of embodiment 1 plasmid

[0060] The hSLC1A5 gene sequence (NM_005628.3) was obtained from a gene bank, and the hSLC1A5 target gene was obtained by gene synthesis using synthetic biology methods, wherein the nucleotide sequence of the hSLC1A5 target gene was shown in SEQ ID No.1. The above-mentioned genes were cloned into the vector pcDNA5-TO (Hygromycin / Ampicillin) through 5'NotI and 3'XbaI (such as figure 1 shown), prepare the mini-scale recombinant plasmid DNA and the puncture bacteria containing the recombinant plasmid, and construct the pcDNA5-TO-hSLC1A5 plasmid for subsequent experiments.

Embodiment 2

[0061] Example 2 Stable transfection cell line construction and verification

[0062] (1) Source and culture of cells:

[0063] The transfected cells were selected as human embryonic kidney cells (HEK293 cells), and the HEK293 cells were purchased from ATCC (American type culture collection). The cells were grown in a 100mm cell culture dish and passaged every three days at a ratio of 1:5. The medium was DMEM containing 10% FBS, and cultured in an incubator containing 5% carbon dioxide, 95% humidity, and 37°C.

[0064] (2) Antibiotic concentration screening:

[0065] HEK293 cells were seeded in 6-well plates, and 0, 4, 8, 16, and 32 μL of 50 mg / mL hygromycin B prepared in PBS were added to make the final concentrations 0, 100, 200, 400, 800, and 1000 μg / mL. For cell state, select the final screening concentration as 200 μg / mL and maintain the concentration as 100 μg / mL.

[0066] (3) Plasmid transfection:

[0067] HEK293 cells in the logarithmic growth phase were taken, spr...

Embodiment 3

[0084] Example 3 Condition Optimization of SLC1A5 Inhibitor Screening Experimental System

[0085] (1) LC-MS / MS method establishment for deuterated glutamine (L-glutamine-2,3,3,4,4-D5):

[0086] The transport function of HEK293-hSLC1A5-positive monoclonal cells was tested using the SLC1A5 transporter substrate deuterated glutamine (L-glutamine-2,3,3,4,4-D5) to evaluate the function of the monoclonal cell transporter. Firstly, a LC-MS / MS method capable of effectively determining the content of L-glutamine-2,3,3,4,4-D5 was developed, the chromatogram is as follows Figure 5 as shown, Figure 5 In , the standard concentration of deuterated glutamine is 10 μM. The conditions for LC-MS / MS analysis of the concentration of deuterated glutamine are as follows: the chromatographic column is VenusilHLP C185 μm (4×150mm); mobile phase A phase: water mixed with 10mM ammonium acetate containing 0.1% formic acid, B phase: acetonitrile, sample injection run time is 5min, elution mode is ...

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Abstract

The invention relates to a method for screening a glutamine transporter inhibitor, a novel glutamine transporter inhibitor mianserin is screened on the basis of a human-derived glutamine transporter inhibitor screening system, and a novel application field is expanded for mianserin. The invention discloses a novel application of mianserin. The compound contributes to the development of medicines for preventing and treating various tumors clinically.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for screening glutamine transporter inhibitors, the inhibitors screened by the method and applications thereof. Background technique [0002] To adapt to the biosynthetic and redox demands of proliferation and metastasis, tumor cells readjust cellular metabolism to meet energy (glucose), amino acids, fatty acids, and other nutrients. Research has shown that interfering with or blocking certain metabolic pathways, such as glutamine metabolism, can effectively reverse tumor cell resistance to anticancer therapies. Glutamine, the most abundant non-essential neutral amino acid in plasma, can be obtained from dietary breakdown or produced in muscle, liver, adipose tissue, lung, and brain, and is a key intermediate in many metabolic processes, including energy formation, oxidation Reductive homeostasis, macromolecular synthesis and signal transmission, etc. During rapid ...

Claims

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Application Information

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IPC IPC(8): C12Q1/02G01N33/68A61K31/55A61K31/513A61P35/00
CPCG01N33/5011G01N33/68A61K31/55A61K31/513A61P35/00A61K2300/00
Inventor 张洪建周志芸段泽琳
Owner SUZHOU UNIV
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