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UGT1A9/1A8 specific probe substrate and use thereof

A technology for probe substrates and compounds, applied in the field of medicine, can solve problems such as lack of specific probe substrates, and achieve the effects of easy availability, high safety, and high LD50 value.

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

AI Technical Summary

Problems solved by technology

[0004] Multiple subtypes in UGT1A have high homology in amino acid sequence, so the catalyzed substrates have a wide range of crossover, and currently there is a lack of specific probe substrates for each subtype of UGT

Method used

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  • UGT1A9/1A8 specific probe substrate and use thereof
  • UGT1A9/1A8 specific probe substrate and use thereof
  • UGT1A9/1A8 specific probe substrate and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Shikonin is used for the quantitative determination of the enzyme activity of UGT1A9 in the recombinant single enzyme

[0029] (1) Prepare 180 μl UGT metabolic reaction system in advance, including Tris-HCl buffer (5mM) at pH 7.4, 5mM MgCl 2 , recombinant human UGT1A9 (0.015mg / ml), the final concentration of shikonin is 35μM, pre-incubated at 37°C for 5 minutes;

[0030] (2) Add 20 μl of 40 mM UDPGA (final concentration: 4 mM) to the reaction system to initiate the reaction; (3) After 20 minutes, add 100 μl of ice-cold methanol and shake vigorously to terminate the reaction;

[0031] (4) Use a high-speed refrigerated centrifuge at 4 °C, 20,000×g, after high-speed centrifugation for 20 minutes, take the supernatant for UFLC-UV detection;

[0032](5) Quantitative detection of shikonin and its glucuronidation products at 517nm. The calculated maximum catalytic rate of recombinant human UGT1A9 enzyme was 150.3 pmol / mim / mg.

Embodiment 2

[0033] Example 2: Shikonin is used to quantitatively determine the enzyme activity of UGT1A9 in human liver microsomes

[0034] (1) The reaction system is the same as above. The UGT1A9 in the system was replaced with human liver microsomes, the protein concentration of the microsomes was 0.025 mg / ml, and the reaction time was 20 minutes.

[0035] (2) After high-speed centrifugation at 4°C and 20,000×g for 20 minutes, take the supernatant for UFLC-UV detection;

[0036] (3) Quantitative detection of shikonin and its glucuronidation products at 517nm. The maximum catalytic rate of UGT1A9 in human liver microsomes was measured to be 92.6 pmol / min / mg.

Embodiment 3

[0037] Embodiment 3: detect the enzymatic activity in recombinant human UGT1A8 single enzyme

[0038] The reaction conditions and operation process are the same as those in Example 1, except that UGT1A8 is used instead of UGT1A9 for shikonin metabolism incubation. The same method can be used to measure the activity of different batches of recombinant human UGT1A8. The maximum reaction rate of recombinant human UGT1A8 determined in this experiment was 9.67pmol / min / mg.

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PUM

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Abstract

The invention discloses naphthoquinone compound alkannin, which can be used for quantitatively testing the enzyme activity of glucuronic acid transferase UGT1A9 and UGT1A8 as UGT1A9 and UGT1A8 specific probe substrate. The use is implemented by the following step of: selecting alkannin as a specific probe substrate, carrying out the UGT catalytic reaction of a specific substrate by using a UGT in-vitro incubation system, quantitatively testing the enzyme activity of UGT1A9 and UGT1A8 in biological samples and cells by quantitatively testing the lost amount of the substrate or the generated amount of the product. The substrate can be used for quantitative evaluation of the enzyme activity of UGT1A9 / 1A8 in different species, individuals and biological samples, calibration of propofol anesthetic metabolizing capacity of certain tissues, and calibration and estimation of the elimination rate of compounds which are mainly metabolized by UGT1A9 in specific organisms based on in-vitro metabolism dynamic data.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a UGT1A9 / 1A8 specific probe substrate and application thereof. Background technique [0002] UGT (uridine diphosphate-glucuronosyltransferase, Uridine diphosphate-glucuronosyltransferase) enzyme family is an important metabolic enzyme system in the human body, which is responsible for clearing a variety of endogenous compounds such as bilirubin, serotonin, thyroxine, etc. A large number of exogenous compounds such as irinotecan, raloxifene, acetaminophen and other drugs, as well as food and environmental toxicants such as diethylstilbestrol and bisphenol A (Drug Metabolism Disposition. 2004, 32: 1201-1208). The 1 and 2 families of UGT enzymes are the two main families of UGT enzymes, and the UGT1 family has nine subtypes: 1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9 and 1A10. The nine isoforms of UGT1A have obvious tissue specificity in human tissue distribution. Among t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07C50/32C12Q1/48
Inventor 杨凌何桂元葛广波宁静朱亮亮夏杨柳
Owner ZHANGJIAGANG IND TECH RES INST CO LTD DALIAN INST OF CHEM PHYSICS CHINESE ACADEMY OF SCI
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