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Specificity probe substrate of glucuronic acid transferase UGT1A3 and application of specificity probe substrate

A technology of glucuronic acid and probe substrate, applied in the field of medicine, can solve problems such as limited application, and achieve the effects of easy acquisition, good ultraviolet absorption characteristics and simple source

Active Publication Date: 2013-01-30
ZHANGJIAGANG IND TECH RES INST CO LTD DALIAN INST OF CHEM PHYSICS CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

UGT2B4 and UGT2B7 are significantly more expressed in the liver than UGT1A3, which severely limits their application in human liver microsomes

Method used

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  • Specificity probe substrate of glucuronic acid transferase UGT1A3 and application of specificity probe substrate
  • Specificity probe substrate of glucuronic acid transferase UGT1A3 and application of specificity probe substrate
  • Specificity probe substrate of glucuronic acid transferase UGT1A3 and application of specificity probe substrate

Examples

Experimental program
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Effect test

Embodiment 1

[0029] 3-Table-16-Deacetylated cinobufagin used in the determination of UGT1A3 enzyme activity in 12 cases of individual human liver microsomes

[0030] Twelve commercialized human liver microsomal samples from different individuals were purchased, and the enzyme activity of UGT1A3 in human liver samples was determined by using 3-epi-16-deacetyl cinobufagin. The specific operation procedure is as follows:

[0031] (1) In 200 μl of in vitro metabolic reaction system, 25 μg / ml alamethicin and 5 mM MgCl 2 , the concentration of human liver microsomes is 0.2 mg / ml, the final concentration of 3-epi-16-deacetyl cinobufagin is 30 μM, and pre-incubated at 37°C for 3 minutes;

[0032] (2) Add 10 μl UDPGA (final concentration 40 mM) to the reaction system to initiate the reaction;

[0033] (3) After 10 minutes, add 200 μl of methanol and shake vigorously to terminate the reaction;

[0034] (4) Use a high-speed refrigerated centrifuge to centrifuge the above system for 10 minutes under...

Embodiment 2

[0037] 3-keto-16-deacetyl cinobufagin used to detect enzyme activity of human recombinant UGT1A3 system

[0038] Using 3-keto-16-deacetyl cinobufagin to detect the catalytic activity of human recombinant single enzyme between different batches, the specific steps are as follows:

[0039] (1) In 200 μl of in vitro metabolic reaction system, 25 μg / ml alamethicin and 5 mM MgCl 2 , the concentration of recombinant UGT1A3 was 0.1 mg / ml, the final concentration of 3-keto-16-deacetyl cinobufagin was 30 μM, and pre-incubated at 38°C for 10 minutes;

[0040] (2) Add 10 μl UDPGA (final concentration 40 mM) to the reaction system to initiate the reaction;

[0041] (3) After 10 minutes, add 200 μl of methanol and shake vigorously to terminate the reaction;

[0042] (4) Use a high-speed refrigerated centrifuge to centrifuge the above system for 10 minutes under the condition of 20,000×g, and then take the supernatant for UFLC-UV detection and analysis;

[0043] Quantitatively detect the g...

Embodiment 3

[0045] 3-Acetyl-16-deacetyl Cinobufagin used in the determination of the enzyme activity of UGT1A3 in human hepatocytes

[0046] (1) Dilute the cells with hepatocyte incubation solution, place in a 6-well culture plate, 4ml per well, put in a metal bath shaker, 80r / min, and incubate at 40°C for 120 minutes with continuous shaking;

[0047] (2) Add 3-acetyl-16-deacetyl cinobufagin to the culture plate with a final concentration of 50 μM;

[0048] (3) After 30 minutes, draw 200 μl of the incubation solution and place it in a -80°C ultra-low temperature refrigerator to stop the reaction;

[0049] (4) Add 200 μl of methanol to the sample to precipitate protein, and use a high-speed refrigerated centrifuge to centrifuge the above system at 20,000×g for 10 minutes at high speed, then take the supernatant for UFLC-ESI-MS detection and analysis.

[0050] UFLC-ESI-MS was used to detect 3-acetyl-16-deacetyl cinobufagin and its metabolites, and SIM mode was used to detect the 3-acetyl-1...

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Abstract

The invention provides a specificity probe substrate of glucuronic acid transferase UGT1A3 and application of the specificity probe substrate. Particular operation flows of enzyme activity measurement comprises the following steps of: selecting a bufanolide compound having 16 bits of hydroxyl to serve as the high specificity probe substrate, a UGT external incubation system is used for developing a UGT catalytic reaction of the specificity substrate, and UGT1A3 enzyme activity in each biological sample and cells is measured through product production quantity or substrate removing quantity in quantitative detection unit time. The specificity probe substrate can be used for quantitative evaluation of the UGT1A3 enzyme activity in the biological samples with different species and different individual resources and quantitative determination of the UGT1A3 enzyme activity in animal tissue cell culture fluid and cell preparation from different sources so as to achieve evaluation of capability of important drug metabolic enzyme UGT1A3 for handling drugs.

Description

technical field [0001] The invention belongs to the technical field of medicine, and specifically relates to a specific probe substrate of a class of glucuronosyltransferase UGT1A3 and an application thereof. Background technique [0002] The glucuronosyltransferase (Uridine diphosphate-glucuronosyltransferase, UGT) superfamily includes important phase II metabolic enzymes in the body, catalyzing the combination of compounds and cofactor uridine diphosphate glucuronic acid (UDPGA), thereby increasing the substrate Hydrophilic, allowing it to be excreted more effectively in urine or bile, an important detoxification process for the body. Currently, glucuronosyltransferases in the human body can be divided into four families according to the gene sequence: UGT1, UGT2, UGT3 and UGT8 (Drug Metab Rev 2010, 42:45-54). Among them, UGT1A3 is the third protein in the UGT1A subfamily, which is mainly distributed in human liver, gastrointestinal and other tissues. It has a wide spect...

Claims

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Application Information

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IPC IPC(8): C07J71/00C12Q1/48
Inventor 杨凌葛广波宁静朱亮亮夏杨柳何桂元
Owner ZHANGJIAGANG IND TECH RES INST CO LTD DALIAN INST OF CHEM PHYSICS CHINESE ACADEMY OF SCI
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