WO2017049423A1 - Bioluminescence assay kit of human carboxylesterase 1, method for use and application thereof - Google Patents

Abstract

Provided are a bioluminescence assay kit of human carboxylesterase 1, a method for use and an application thereof. The kit comprises an A liquid, a B liquid, a C liquid, and a quality control standard. The A liquid is a 100 mM phosphate buffer solution, with pH = 5.5-10, and preferably a buffer system with pH of 6.5. The B liquid is a 0.1-10 mM solution which contains a human carboxylesterase 1 specific biological probe substrate, wherein a solvent is dimethyl sulfoxide. The C liquid is a luciferin detection reagent. The quality control standard is a phosphate solution with 5 mg/ml of hCE1 and pH of 6.5. The human carboxylesterase 1 specific biological probe substrate is 4,5-dihydro-2(6-hydroxy-2-benzimidazole)4-carboxylic acid thiazole methyl ester or 4,5-dihydro-2(6-amino-2-benzimidazole)4-carboxylic acid thiazole methyl ester. The kit can be used for detection of existence of hCE1 in various biological samples and quantitative assay of enzyme activity thereof, and can also be used for quickly selecting an inhibitor/inducer for the hCE1.

Description

Bioluminescence detection kit for human carboxylesterase 1 and use method and application thereof Technical field

The invention belongs to the field of medical biotechnology, and particularly relates to a bioluminescence detection kit for human carboxylesterase 1 and a method and application thereof.

Background technique

Human carboxylesterase 1 (hCE1) is an important functional protein distributed in the human body. It is highly abundant in the liver of the human population, and its protein expression ranks in the top ten in the liver distribution protein. hCE1 is a membrane-bound protein localized in the endoplasmic reticulum [CANCER RESEARCH 1998; 58: 3627-32 & Drug Metab. Pharmacokinet. 2006; 21: 173-85], which is also secreted outside the cell into the blood circulation. System [Proteomics2009; 9: 3989-99]. The basic metabolic reaction of hCE1 is to catalyze the hydrolysis of smaller alcohol groups and larger amide substrates. Although the protein expression of hCE1 was detected in various tissues, it was much higher in human liver tissue than in other tissues. hCE1 is involved in the metabolic clearance of a variety of exogenous substances, such as the drugs oseltamivir, methylphenidate and so on. At the same time, hCE1 plays a non-negligible role in maintaining normal physiological functions of the human body, participating in the transport and metabolism of cholesterol esters and free fatty acids in the human body, and maintaining lipid metabolism balance [Chemistry & Biology, Vol. 10, 341-349, April, 2003] . In normal healthy humans, hCE1 is distributed in cells of liver tissue in addition to higher content, and a small amount of hCE1 is detected in plasma. More importantly, hCE1 in plasma of hepatocellular carcinoma patients is healthy 2 - 5 times, hCE1 is considered to be a good potential serological marker for early diagnosis of hepatocellular carcinoma [Proteomics 2009, 9, 3989-3999]. In addition, hepatocyte damage In patients with related liver diseases, hCE1 is also released into the blood, causing a change in the concentration of hCE1 in plasma. Therefore, hCE1 in plasma can be used as a marker for acute liver injury. There is a large individual difference in the distribution of hCE1. The expression and function of hCE1 enzyme itself are affected by many factors such as human heredity, age, disease, gender, environment and co-administration. Therefore, the hCE1 enzyme activity assay has broad clinical application prospects and can be used to evaluate liver dysfunction, hepatotoxicity caused by drugs and poisons, early diagnosis of hepatocellular carcinoma, individualized evaluation of liver function, and personality of some prodrugs. Use and so on. In addition, hCE1 is an important protein for maintaining the balance of lipid metabolism in the human body. Its inhibitor can be used as a hypolipidemic drug, and hCE1 has also been approved by the FDA as an important drug target. The rapid characterization method of hCE1 activity can also be used to discover and evaluate novel high-efficiency inhibitors of hCE1 in the early stage of new drug development. Therefore, the development of efficient, ultra-sensitive, highly specific hCE1 activity assays is essential for the detection of hCE1 activity in clinical samples, as well as high-content, high-throughput screening of new drugs.

Summary of the invention

It is an object of the present invention to provide a bioluminescence detection kit for human carboxylesterase 1 and a method and use thereof. The kit uses 4,5-dihydro-2(6-hydroxy-2-benzimidazole) 4-carboxylic acid thiazole methyl ester or 4,5-dihydro-2(6-amino-2-benzimidazole) 4- The carboxylic acid thiazolyl methyl ester serves as a human carboxylesterase 1 probe substrate (Fig. 1), and the probe is hydrolyzed by hCE1 to form a substrate for firefly luciferase. The activity of the enzyme is expressed based on the amount of product produced per unit time. The enzymatic reaction has the characteristics of high selectivity, easy detection of metabolites, rapid and efficient evaluation of enzyme activity and inhibition activity.

A bioluminescence detection kit for human carboxylesterase 1, the kit comprises four reagents: liquid A, liquid B, liquid C and quality control standard;

The liquid A is: 100 mM phosphate buffer, pH=5.5-10,

The solution B is a solution of 0.1 to 10 mM of a specific bioprobe substrate containing human carboxylesterase 1, and the solvent is dimethyl sulfoxide.

The C solution is a luciferase detection reagent containing 0.5 mM ATP, 10 mM Mg ²⁺ and 50 g/ml luciferase

The quality control standard is 5 mg/ml of an aqueous solution of hCE1;

The specific bioprobe substrate of the human carboxylesterase 1 is 4,5-dihydro-2(6-hydroxy-2-benzimidazole) 4-carboxylic acid thiazole methyl ester or 4,5-dihydro- 2(6-Amino-2-benzimidazole) 4-carboxylic acid thiazole methyl ester, the structural formula of which is:

Wherein R is -OH or -NH ₂ ;

In a buffer system of pH 6.5, the methyl ester bond of the above compound can be specifically hydrolyzed by hCE1 to the corresponding hydrolyzate 4,5-dihydro-2(6-hydroxy-2-benzimidazole) 4-carboxylic acid thiazolecarboxylic acid or 4 , 5-dihydro-2(6-amino-2-benzimidazole) 4-carboxylic acid thiazolecarboxylic acid.

The liquid A preferably has a pH of 6.5.

A method for using a bioluminescence detection kit for human carboxylesterase 1 is specifically carried out according to the following steps:

(1) Pre-incubation: Mix the sample to be tested with solution A, incubate for 3 to 5 minutes at 20 to 60 degrees; the total volume of the mixture is 20 μL, and the concentration of the sample to be tested is: 0 to 0.5 mg/ml.

(2) initial reaction: add B solution, mix, incubate at 20 to 60 degrees for 2 to 120 minutes; The volume of solution B was 30 μL and the final concentration was 3 μM.

(3) Detection: Add liquid C, mix, incubate at 37 ° C for 20-30 min, and measure the sample with a microplate reader or a chemiluminescence analyzer. The amount of the C solution added is 50 μL.

The test sample is recombinantly expressed hCE1, mammalian cell/tissue preparation liquid containing hCE1, plasma or other common biological samples.

A bioluminescence detection kit for human carboxylesterase 1 is an annoying application for rapid quantitative detection of human carboxylesterase 1 activity, as well as rapid screening and evaluation of the enzyme inhibitor.

The kit is used for rapid quantitative detection of human carboxylesterase 1 activity: probe substrate 4,5-dihydro-2(6-hydroxy-2-benzimidazole) 4-carboxylic acid thiazolyl methyl ester Or 4,5-dihydro-2(6-amino-2-benzimidazole) 4-carboxylic acid thiazole methyl ester can be specifically hydrolyzed by human carboxylesterase 1 to a substrate of firefly luciferase; according to the kit The method is used to characterize the amount of hydrolyzed product by quantitatively detecting the bioluminescence intensity of the target in the sample per unit time, and then determining the activity of human carboxylesterase 1 in different samples.

The rapid screening and evaluation method for the human carboxylesterase 1 inhibitor is as follows: the hydrolysis reaction of the specific bioprobe substrate of human carboxylesterase 1 is used as a specific probe reaction of hCE1, according to the reagent The method of using the cartridge is to quantitatively compare the residual activity of hCE1 in the biological system by quantitatively comparing the presence of the inhibitor with the amount of hydrolyzed product per unit time in the missing state, thereby realizing the rapid screening of the hCE1 inhibitor and the quantitative evaluation of the inhibitory ability.

The kit detection principle utilizes human carboxylesterase 1 to catalyze the hydrolysis of ester bonds, specifically identifying and catalyzing the methyl ester in the specific bioprobe substrate structure of human carboxylesterase 1, at physiological pH conditions. Subsequent hydrolysis produces a substrate for firefly luciferase. Use the probe The reaction allows quantitative assessment of the distribution and function of hCE1 in a variety of biological systems. The detection principle is shown in Figure 8.

The application field of the kit for specifically detecting human carboxylesterase (hCE1) of the present invention includes, but is not limited to, quantification of hCE1 activity in recombinantly expressed hCE1 enzyme, hCE1-containing cells and tissue preparations, plasma and other biological samples. Determination.

The use of the kit for detecting human carboxylesterase 1 (hCE1) provided by the present invention requires attention that the substrate elimination rate or the production rate of the hydrolyzate should be between 0.1% and 20%.

The kit for detecting human carboxylesterase 1 (hCE1) provided by the invention, the product 4,5-dihydro-2(6-hydroxy-2-benzimidazole) 4-carboxylic acid thiazolecarboxylic acid or 4,5-di Hydrogen-2(6-amino-2-benzimidazole) 4-carboxylic acid thiazolecarboxylic acid is a substrate for firefly luciferase, and a chemiluminescent detector can be used to achieve rapid and sensitive detection of the product. In addition, the specific probe substrate and the corresponding hCE1 activity detection process are not interfered by the matrix and impurities of the biological system, and can be used for quantitative determination of hCE1 enzyme activity in various biological systems.

The kit of the invention can be used for quantitative determination of hCE1 enzyme activity in various biological systems such as recombinant carboxylesterase, cell or tissue preparation liquid, and can also be used for rapid screening of inhibitors of hCE1 enzyme and quantitative evaluation of inhibitory ability.

The human carboxylesterase 1 detection kit and the method of using the same according to the present invention have the following outstanding advantages:

(1) High specificity: 4,5-dihydro-2(6-hydroxy-2-benzimidazole) 4-carboxylic acid thiazole methyl ester or 4,5-dihydro-2(6-amino-2-benzazole The 4-carboxylic acid thiazole methyl ester can be metabolized by hCE1 to a metabolite with high specificity, that is, a hydrolysis product of methyl ester bond cleavage;

(2) cheap and easy to obtain: substrate 4,5-dihydro-2(6-hydroxy-2-benzimidazole) 4-carboxylic acid thiazole Methyl ester or 4,5-dihydro-2(6-amino-2-benzimidazole) 4-carboxylic acid thiazole methyl ester and its hydrolysis product can be obtained by chemical synthesis, and the synthesis process is simple and easy;

(3) High sensitivity: The hydrolysate is a substrate for luciferase, and the luciferase detection reagent can be used for high sensitivity detection, and the minimum limit of quantification is 0.1 nM.

(4) High-throughput detection: Rapid detection of 96,386-well plates can be achieved by using this type of fluorescent probe substrate, which can achieve high-throughput detection of 20,000 samples per day, with a single test cost (<0.5 yuan) Advantage.

DRAWINGS

Figure 1. Structural formula of the hCE1 bioluminescent probe substrate;

Figure 2.4, results of human recombinant single enzyme screening test of 5-dihydro-2(6-hydroxy-2-benzimidazole) 4-carboxylic acid thiazolyl methyl ester;

Figure 3. Linear reaction concentration of hCE1 hydrolyzed 4,5-dihydro-2(6-hydroxy-2-benzimidazole) 4-carboxylic acid thiazole methyl ester

Figure 4. Active chemical inhibition experiment of hCE1

Figure 5.12 shows the difference in hydrolysis rate of HLM to 4,5-dihydro-2(6-hydroxy-2-benzimidazole) 4-carboxylic acid thiazole methyl ester;

Figure 6.6 Differences in the rate of hydrolysis of tumor cell S9 to 4,5-dihydro-2(6-hydroxy-2-benzimidazole) 4-carboxylic acid thiazolyl methyl ester;

Figure 7.8 shows the difference in hydrolysis rate of 4,5-dihydro-2(6-hydroxy-2-benzimidazole) 4-carboxylic acid thiazolyl methyl ester in healthy human plasma;

Figure 8.4, Metabolic detection of 5-dihydro-2(6-hydroxy-2-benzimidazole) 4-carboxylic acid thiazole methyl ester intention.

detailed description

The invention is further illustrated by the following examples, which are not intended to limit the invention. The structural formula of the hCE1 bioluminescent probe substrate is shown in Figure 1. The detection principle of the hCE1 kit is shown in Figure 8. The specific application of the kit is as follows.

Example 1

In vitro determination of hCE1 selectivity in human recombinant single enzyme

(1) Prepare 20 μl of hCE1 metabolic reaction system in advance, including liquid A and human recombinant single enzyme (5 μg/mL), and incubate for 10 minutes at 37 °C;

(2) adding 30 μl of solution B (final concentration 3 μM) to the reaction system to initiate the reaction;

(3) After 10 minutes, 50 μl of C solution was added, and after incubation at 37 ° C for 20 min, the plate reader was used to detect luminescence.

Recombinant human hCE1 specifically involved in its hydrolysis, and other esterases did not participate in its hydrolysis, confirming that this class of compounds has very good selectivity (Fig. 2).

Example 2

Linear concentration of 4,5-dihydro-2(6-hydroxy-2-benzimidazole) 4-carboxylic acid thiazole methyl ester hydrolyzed by hCE1

(1) Prepare 20 μl of hCE1 metabolic reaction system in advance, including liquid A and human recombinant single enzyme hCE1 (series concentration), pre-incubate for 10 minutes at 37 °C;

(2) adding 30 μl of solution B (final concentration 3 μM) to the reaction system to initiate the reaction;

(3) After 10 minutes, add 50 μl of C solution, incubate at 37 ° C for 20 min, enzyme label The instrument detects luminescence.

The kit detects a minimum of 0.02 ug/ml of hCE1 (Figure 3).

Example 3

Characterization of inhibition ability of hCE1 inhibitor

(1) Preparing the hCE1 metabolic reaction system, including liquid A, human liver microsomes, and different esterase-specific inhibitors (100 μM), pre-incubation for 10 minutes at 37 °C;

(2) adding 30 μl of a B solution having a concentration of 5 μM to the reaction system to initiate a reaction initiation reaction;

(3) After 10 minutes, 50 μl of C solution was added, and after incubation at 37 ° C for 20 min, the plate reader was used to detect luminescence.

The hydrolysis activity of 4,5-dihydro-2(6-hydroxy-2-benzimidazole) 4-carboxylic acid thiazolyl methyl ester in human liver microsomes can be significantly inhibited by the carboxylesterase specific inhibitor BNPP, while others Esterase inhibitors have no significant inhibitory activity on their hydrolysis, confirming that 4,5-dihydro-2(6-hydroxy-2-benzimidazole) 4-carboxylic acid thiazole methyl ester can specifically detect the activity of hCE1 in complex biological samples. (Figure 4).

Example 4

Quantitative assessment of hCE1 activity in liver microsomes from different individuals

(1) 12 individual human liver microsomes (HLM) were selected to prepare hCE1 metabolic reaction system, including liquid A and human liver microsomes, and pre-incubated for 3 minutes at 37 °C;

(2) adding 30 μl of solution B (final concentration 3 μM) to the reaction system to initiate the reaction;

(3) After 10 minutes, 50 μl of C solution was added, and after incubation at 37 ° C for 20 min, the plate reader was used to detect luminescence.

The measured luminescence intensity was substituted into the standard curve to obtain the metabolic rate of 12 human liver microsomes (HLM) to 4,5-dihydro-2(6-hydroxy-2-benzimidazole) 4-carboxylic acid thiazolyl methyl ester ( Figure 5A), this assay showed a good correlation with the hCE1 activity measured by clopidogrel (Fig. 5B).

Example 5

Quantitative assessment of hCE1 activity in tumor cells

(1) Six tumor cell lines were selected, and the hCE1 metabolic reaction system was prepared, including liquid A and cell homogenate (2 μL), and pre-incubated for 3 minutes at 37 °C;

(2) adding 30 μl of solution B (final concentration 3 μM) to the reaction system to initiate the reaction;

(3) After 10 minutes, 50 μl of C solution was added, and after incubation at 37 ° C for 20 min, the plate reader was used to detect luminescence.

The rate of luminescence signal detected was substituted into the standard curve to obtain the metabolic rate of 6 kinds of cells S9 to 4,5-dihydro-2(6-hydroxy-2-benzimidazole) 4-carboxylic acid thiazole methyl ester (Fig. 6).

Example 6

Quantitative assessment of hCE1 activity in plasma

(1) The plasma of 8 healthy people was selected to prepare the hCE1 metabolic reaction system, including liquid A and human plasma (2 μL), and pre-incubated for 3 minutes at 37 °C;

(2) adding 30 μl of solution B (final concentration 3 μM) to the reaction system to initiate the reaction;

(3) After 10 minutes, 50 μl of C solution was added, and after incubation at 37 ° C for 20 min, the plate reader was used to detect luminescence.

The intensity of the detected luminescent signal was substituted into the standard curve to obtain the metabolic rate of 8 healthy human plasma to 4,5-dihydro-2(6-hydroxy-2-benzimidazole) 4-carboxylic acid thiazolyl methyl ester (Fig. 7).

Claims (7)

1. A bioluminescent detection kit for human carboxylesterase 1, characterized in that the kit comprises the following four reagents: liquid A, liquid B, liquid C and quality control standard;

   Wherein the A liquid is 100 mM phosphate buffer, pH = 5.5 ~ 10;

   The solution B is a solution of 0.1 to 10 mM of a specific bioprobe substrate containing human carboxylesterase 1, and the solvent is dimethyl sulfoxide;

   The liquid C is a fluorescein detection reagent containing 0.5 mM ATP, 10 mM Mg ²⁺ and 50 g/ml luciferase;

   The quality control standard is a phosphate solution of pH 6.5 of 5 mg/ml of hCE1;

   The specific bioprobe substrate of the human carboxylesterase 1 is 4,5-dihydro-2(6-hydroxy-2-benzimidazole) 4-carboxylic acid thiazole methyl ester or 4,5-dihydro- 2(6-Amino-2-benzimidazole) 4-carboxylic acid thiazole methyl ester, the structural formula of which is:

   

   Wherein R is -OH or -NH ₂ ;

   In a buffer system of pH 6.5, the methyl ester bond of the above compound can be specifically hydrolyzed by hCE1 to the corresponding hydrolyzate 4,5-dihydro-2(6-hydroxy-2-benzimidazole) 4-carboxylic acid thiazolecarboxylic acid or 4 , 5-dihydro-2(6-amino-2-benzimidazole) 4-carboxylic acid thiazolecarboxylic acid.
2. A bioluminescent detection kit for human carboxylesterase 1 according to claim 1, wherein said liquid A preferably has a pH of 6.5.
3. A bioluminescence detection kit for human carboxylesterase 1 according to claim 1 The method of use is characterized in that the detection steps are as follows:

   (1) Pre-incubation: Mix the sample to be tested with solution A, and incubate for 3 to 5 minutes at 20 to 60 degrees;

   (2) initial reaction: add B solution, mix, incubate at 20 to 60 degrees for 2 to 120 minutes;

   (3) Detection: Add liquid C, mix, incubate at 37 ° C for 20-30 min, and measure the sample with a microplate reader or a chemiluminescence analyzer.
4. The method for using the bioluminescent detection kit for human carboxylesterase 1 according to claim 3, wherein the sample to be tested is: recombinantly expressed hCE1, mammalian cell/tissue preparation liquid containing hCE1, plasma or Other common biological samples.
5. The use of the bioluminescent detection kit for human carboxylesterase 1 according to claim 1, characterized in that the kit is used for rapid quantitative detection of human carboxylesterase 1 activity and rapid detection of the enzyme inhibitor Screening and evaluation.
6. The bioluminescent detection kit for human carboxylesterase 1 according to claim 5, characterized in that the kit is used for rapid quantitative detection of human carboxylesterase 1 activity by: using a kit The method is characterized by quantitatively detecting the fluorescence intensity of a target in a sample per unit time to characterize the amount of hydrolyzate produced, and further determining the activity of human carboxylesterase 1 in different samples.
7. The use of the bioluminescent detection kit for human carboxylesterase 1 according to claim 5, characterized in that the kit is used for rapid screening and evaluation of a human carboxylesterase 1 inhibitor by: The method can be used to quantitatively compare the residual activity of hCE1 in the biological system by quantitatively comparing the presence of the inhibitor with the amount of hydrolyzed product per unit time in the missing state, thereby achieving rapid screening of the hCE1 inhibitor and quantitative evaluation of the inhibitory ability.

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