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Method for screening uric acid-lowering small molecule compounds

A small molecule compound and uric acid-lowering technology, applied in the field of pharmacy, to achieve the effects of stable system, low cost, and improved transfection efficiency

Active Publication Date: 2019-11-22
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, this method is not suitable for high-throughput screening

Method used

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  • Method for screening uric acid-lowering small molecule compounds
  • Method for screening uric acid-lowering small molecule compounds
  • Method for screening uric acid-lowering small molecule compounds

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preparation example Construction

[0039] Preparation of Kanamycin: Dissolve 1g of Kanamycin in a sufficient amount of ddH 2 In O, the final volume was adjusted to 20mL, sterilized by filtration, divided into small portions and stored at -20°C, and diluted 1000 times when used.

[0040] Competent cell preparation: Whole-type gold Trans1-T1 competent (product number: CD501-02), thawed on ice before use and ready for use.

[0041] Preparation of liquid medium: Weigh 5g of peptone, 5g of sodium chloride, and 2.5g of yeast extract into 500mL washed bottles, add 500mL of ultrapure water, stir evenly, and sterilize at 121°C for 20min. After sterilization, cool down to 37 °C, add 500 μL of kanamycin (100 μL / 100 mL) and shake well, store at 4 °C for later use.

[0042] Preparation of solid medium: Weigh 3g of peptone, 3g of sodium chloride, 1.5g of yeast extract, and 4.5g of agar into 500mL washed bottles, add 300mL of ultrapure water, and sterilize at 121°C for 20min. After sterilization, place the solid medium at r...

Embodiment 1

[0043] Construction and Identification of Embodiment 1 GLUT9 Recombinant Plasmid

[0044] 1) Construction of pcDNA3.1(-)-GLUT9 recombinant plasmid

[0045] The present invention selects pcDNA3.1(-) as the expression vector; the active fragment of the target gene sequence is the full length of GLUT9 (Gene ID: 117591). Under the action of T4 ligase, clone the target gene fragment into the pcDNA3.1(-) carrier, transform the plasmid into Escherichia coli and culture it at 37°C for 16 hours, pick a single clone, and place it in the prepared liquid culture medium Cultivate and shake the bacteria for 16h. After shaking the bacteria, the plasmid was extracted, followed by double enzyme digestion and identification, and the sample was selected and sent for sequencing for final confirmation.

[0046] result: Figure 1 ~ Figure 3 It is the plasmid map of pcDNA3.1(-)-GLUT9 recombinant plasmid. It is known that the length of pcDNA3.1(-) is about 5.4kb, while that of GLUT9 is about 1.6k...

Embodiment 2

[0058] Example 2 Patch clamp recording current

[0059] 1. Whole Cell Patch Clamp Recording

[0060] (1) Prepare intracellular fluid.

[0061] The formula of the external liquid is: 140mM / L NaCl, 5mM / L KCl, 1mM / L MgCl 2 , 2mM / L CaCl 2 , 10mM / LHEPES, and 10mM / L D-glucose, adjust the pH to about 7.4 with NaOH, filter through a 0.22μm microporous membrane, and store at 4°C for later use.

[0062] The internal liquid formula is: 140mM / 100mL KCl, 1mM / 100mL MgCl 2 , 5mM / 100mL EGTA, and 10mM / 100mL HEPES, adjust the pH to about 7.4 with KOH, filter through a 0.22μm microporous membrane, and store at 4°C for later use.

[0063] (2) Prepare (medicine-containing) uric acid solution: dissolve 8.4 mg of uric acid powder with 0.1 M NaOH to obtain 25 mM uric acid mother solution; add 800 μL of uric acid mother solution to 19.2 mL of external solution and mix well to obtain 20 mL of 1 mM uric acid external solution. It should be prepared and used now. The drug-containing uric acid means...

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Abstract

The invention discloses a method for screening uric acid-lowering small molecule compounds for promoting uric acid excretion based on a patch clamp technology. The method comprises the following stepsof: constructing recombinant plasmids for expressing a GLUT9 gene; instantaneously transfecting the recombinant plasmids to an HEK293T cell; after 18-24 hours, recording the change of a current whenthe GLUT9 cells are expressed to transport uric acid by utilizing a patch clamp technology; and verifying the reliability of the model through positive drugs, namely benzbromarone and probenecid, andnegative drugs, namely RDEA3170 and allopurinol. The method is simple, convenient and rapid to operate; the result is stable, reliable and good in reproducibility; compared with an existing method fordetecting isotope uptake by using xenopus oocytes, the method has the advantages that the consumed time is shorter, the cost is lower, and high-throughput screening of the GLUT9 inhibitor can be achieved.

Description

technical field [0001] The invention relates to the field of pharmacy, in particular to a method for screening uric acid-lowering small molecule compounds. Background technique [0002] Uric acid is the final product of purine metabolism in the body. Hyperuricemia (Hyperuricemia, HUA) refers to the measurement of blood uric acid level on an empty stomach twice on different days under normal purine diet. If it is higher than 420 μmol / L for men and 360 μmol / L for women, it is diagnosed as high. uric acidemia. Hyperuricemia can cause gout, hypertension, cardiovascular disease, diabetes and kidney disease. At the same time, hyperuricemia is also related to excessive uric acid production or insufficient uric acid excretion. More than 80% of patients with hyperuricemia are caused by decreased uric acid excretion. [0003] Uric acid in the body is mainly excreted with urine through the kidneys, and this process depends on the synergistic effect of various uric acid transporters ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/416
CPCG01N27/416
Inventor 吴婷陈演瑜庞建新周平正赵泽安李咏梅李璐
Owner SOUTHERN MEDICAL UNIVERSITY
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