Specific fluorescent probe for glucuronyl transferase UGT1A1 and application thereof

A glucuronic acid and fluorescent probe technology is applied in the field of specific fluorescent probe substrates for glucuronyltransferase UGT1A1, which can solve the problems of low detection sensitivity, low single-enzyme selectivity, poor chemical stability, etc. The detection cost is low, the synthesis process is simple and feasible, and the effect of sensitive detection

Active Publication Date: 2015-05-06
CHANGSHU RES INST OF DALIAN UNIV OF TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although bilirubin has high single-enzyme selectivity, it has poor chemical stability and low detection sensitivity
However, the single-enzyme selectivity

Method used

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  • Specific fluorescent probe for glucuronyl transferase UGT1A1 and application thereof
  • Specific fluorescent probe for glucuronyl transferase UGT1A1 and application thereof
  • Specific fluorescent probe for glucuronyl transferase UGT1A1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1. In vitro determination of the selectivity of human recombinant UGT single enzyme

[0029] (1) Prepare 95 μL UGT metabolic reaction system in advance, including Tris-HCl buffer solution (50mM) at pH 7.4, each single enzyme of recombinant human UGT (0.1mg / mL), the final substrate concentration is 100μM, shake at 37°C Pre-incubation for 3 minutes;

[0030] (2) Add 5 μL of UDPGA with a concentration of 40 mM (final concentration 2 mM) to the reaction system to initiate the reaction;

[0031] (3) After 60 minutes, add 100 μL of glacial acetonitrile, shake vigorously, and terminate the reaction;

[0032] (4) Use a high-speed refrigerated centrifuge to centrifuge at 4°C and 20,000×g for 20 minutes at high speed, take the supernatant, and perform fluorescence detection (Ex=460nm, Em=612nm); selectivity of recombinant human UGT1A1 enzyme The highest is about 8 times that of other single enzymes ( figure 1 ).

Embodiment 2

[0033] Example 2. Inhibition experiment of UGT1A1 in human liver microsomes in vitro

[0034] (1) Prepare 190 μL human liver microsomes and UGT1A1 metabolic reaction system in advance, including Tris-HCl buffer (50mM) at pH 7.4, human liver microsomes (0.25mg / mL), UGT1A1 (0.1mg / mL), substrate The final concentration is 10 μM, add 10 μM niflumic acid, 10 μM nilotinib and 500 μM fluconazole respectively, and shake for 3 minutes at 37°C;

[0035] (2) Add 10 μL of UDPGA with a concentration of 40 mM to the reaction system to initiate the reaction;

[0036] (3) After 30 minutes, add 200 μL of glacial acetonitrile, shake vigorously, and terminate the reaction;

[0037](4) Use a high-speed refrigerated centrifuge under the condition of 4°C and 20,000×g, after high-speed centrifugation for 20 minutes, take the supernatant, and perform fluorescence detection (Ex=460nm, Em=612nm); Suppress results such as figure 2 .

Embodiment 3

[0038] Example 3 The linear incubation time in the recombinant single enzyme UGT1A1

[0039] (1) Prepare 95 μL UGT metabolic reaction system in advance, including Tris-HCl buffer solution (50 mM) at pH 7.4, recombinant human UGT1A1 single enzyme (0.1 mg / mL), and the final substrate concentration is 10 μM. Incubate for 3 minutes;

[0040] (2) Add 5 μL of UDPGA with a final concentration of 40 mM (final concentration 2 mM) to the reaction system to initiate the reaction;

[0041] (3) Fluorescence scanning detection (Ex=460nm, Em=610nm) is carried out every 15 minutes; Calculate the linear reaction time of recombinant human UGT1A1 enzyme (see image 3 ).

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Abstract

The invention discloses a specific fluorescent probe for glucuronyl transferase UGT1A1 and an application thereof. The substrate of the specific probe is (E)-2-(4-(4-hydroxystyryl)-3- cyano-5,5-dimethyl furan-2(5H)-ylene) malononitrile, which is named TCF for short and can be used for determining the enzymatic activity of UGT1A1 in a biosystem. A flow for determining the enzymatic activity of UGT1A1 is as follows: selecting a glucuronyl transferase acidification reaction of TCF acids as a probe reaction, and determining the activity of the UGT1A1 enzyme in various biological samples, cells, carriers and overall organs by quantitatively detecting the generation amount of glucuronyl transferase acidification metabolites in a unit time. The specific fluorescent probe disclosed by the invention can be used for quantitative evaluation for the activity of the UGT1A1 enzyme in different species and different individual sources of biological samples and for quantitative determination for the activity of the UGT1A1 enzyme in animal tissue cell culture fluids and cell prepared products from different sources, so as to realize evaluation for the medicine disposition capacity of the important medicine metabolizing enzyme UGT1A1. In addition, the probe reaction can also be used for rapidly screening a UGT1A1 inhibitor in vitro and evaluating the inhibition capacity of the inhibitor.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a specific fluorescent probe substrate of glucuronosyltransferase UGT1A1 and an application thereof. Background technique [0002] The glucuronosyltransferase (Uridine diphosphate-glucuronosyltransferase, UGT) superfamily is the most important phase II drug-metabolizing enzyme in the body. Glucuronic acid (GA) is combined, thereby increasing the hydrophilicity of the substrate, so that it can be more effectively excreted from the body in urine or bile. Usually the glucuronide conjugation reaction mediated by UGT enzyme is an important detoxification process of the body. Many endogenous compounds, mutagens, drugs, and their metabolites are substrates of UGTs, such as endogenous substances such as bilirubin and estradiol, and exogenous substances SN-38 and nitrous The detoxification of amine compounds is achieved through the glucuronidation pathway. [0003] Huma...

Claims

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Application Information

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IPC IPC(8): C09K11/06C07D307/68C12Q1/48C12Q1/04
Inventor 崔京南冯磊
Owner CHANGSHU RES INST OF DALIAN UNIV OF TECH CO LTD
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