WO2016049998A1

WO2016049998A1 - Fluorescent probe substrate for detecting activity of human intestine carboxylesterase, and uses thereof

Info

Publication number: WO2016049998A1
Application number: PCT/CN2015/000326
Authority: WO
Inventors: 杨凌; 崔京南; 冯磊; 葛广波; 刘兆明; 宁静
Original assignee: 中国科学院大连化学物理研究所
Priority date: 2014-09-29
Filing date: 2015-05-13
Publication date: 2016-04-07
Also published as: CN105441062B; CN105441062A

Abstract

A fluorescent probe substrate for detecting the activity of human intestine carboxylesterase and uses thereof, which relate to the technical field of pharmaceuticals. The specific probe substrate is an aromatic ester (TCFE for short) of (E)-2-(4-(4-hydroxybenzene vinyl)-3-cyan-5,5-dimethylfuran-2(5H)-subunit)malononitrile (TCF for short). The substrate can be used for quick quantitative detection of the activity of the human intestine carboxylesterase and for quick screening and estimation of an inhibitor of the human intestine carboxylesterase. The process of the quick quantitative detection of the activity of the human intestine carboxylesterase comprises: selecting a TCFE hydrolysis reaction as a probe reaction, adding a proper substrate into a buffer system comprising hCE2, and determining the actual activity of hCE2 enzyme in each in-vitro organism sample by quantitatively detecting the production of a hydrolysis metabolism product TCF in a unit time in a linear reaction interval. The probe has good selectivity, the hydrolysis product TCF has a long fluorescence emission wavelength (612 nm), and the probe can reduce background fluorescence of the organism sample and increase the sensitivity, and has good practicability and application prospect.

Description

Fluorescent probe substrate for detecting intestinal carboxylesterase activity in human and application thereof

Technical field

The invention belongs to the technical field of medicine, and particularly relates to a fluorescent probe substrate for detecting the intestinal carboxylesterase activity of human and its application.

Background technique

Carboxylesterase (CE) is an important phase I metabolic enzyme in the body, which can catalyze ester bond cleavage of various endogenous and exogenous ester compounds, and release more polar alcohol after hydrolysis. Molecular and carboxylic acid molecular fragments, which in turn are further catalyzed by other metabolic enzymes such as cytochrome P450 (CYP450) or uridine diphosphate glucuronyltransferase (UGTs), allowing drug molecules to be excreted more efficiently. Drug molecules containing ester bonds in various structures, such as irinotecan hydrochloride (CPT-11), oseltamivir (Duffy), clopidogrel, benyl ester, etc., are all activated by carboxylesterase catalysis. Or metabolic elimination.

Carboxylesterases that mediate drug metabolism in humans are mainly divided into two subtypes: carboxylesterase 1 (hCE1) and carboxylesterase 2 (hCE2). The two carboxylesterases have different tissue distribution specificity and substrate selectivity, and hCE1 is mainly distributed in human liver, while hCE2 is mainly in small intestine, and only a small amount of hCE1 is distributed. Therefore, human carboxylesterase 2 is also known as human intestine carboxylesterase. In recent years, it has been found that the expression level of hCE2 is up-regulated in various tumor cells, and the purpose of targeting and selectively killing tumor cells by hCE2-specific hydrolysis of anti-tumor prodrugs can be designed by using this feature. In addition, most oral prodrugs need to pass through the intestines before they can be absorbed into the blood circulation, so the intestines The metabolism of ester drugs by the carboxylesterase 2 enzyme is considered to be an important component of its first-pass metabolism. The difference in carboxylesterase 2 activity has a significant impact on the bioavailability of prodrugs. In addition, some intravenously administered prodrugs, such as irinotecan hydrochloride, are secreted into the intestine via bile excretion, which in turn is catalyzed by hCE2 in the intestine to release the toxic metabolite SN-38, which is considered to be The main cause of delayed side effects of irinotecan delayed diarrhea. Thus, a high-throughput technique based on fluorescent probes was established for high-throughput screening of potent inhibitors of hCE2 and its application to slowing delayed diarrhea caused by irinotecan and real-time quantitative detection of hCE2 function to predict potential The drug-drug interaction is important.

The present invention provides a class of (E)-2-(4-(4-hydroxyphenylethyl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene)malononitrile The use of an ester derivative as a substrate for the hCE2 fluorescent probe, which is hydrolyzed by hCE2, produces a hydrolyzate having good fluorescence properties. The enzymatic reaction has the characteristics of high selectivity, easy detection of metabolites, rapid and efficient evaluation of enzyme activity and inhibition activity.

Summary of the invention

It is an object of the present invention to provide a fluorescent probe substrate which is capable of quantitatively detecting the activity of a human intestinal carboxylesterase and an application thereof, the hydrolysis product of which has good fluorescence properties. The probe reaction can be used to quantitatively evaluate the distribution and function of hCE2 in various biological systems.

A specific fluorescent probe substrate for human intestinal carboxylesterase (hCE2), the substrate is (E)-2-(4-(4-hydroxyphenylethyl)-3-cyano-5 , 5-dimethylfuran-2(5H)-ylidene)malononitrile (abbreviated as TCF) aromatic ester (abbreviated as TCFE), its structural formula is:

Wherein R is phenyl, p-methylphenyl, p-ethylphenyl, p-propylphenyl, p-hexylpropyl, p-nitrophenyl, p-chlorophenyl, 1-nyl, 2-furyl Any one of a 2-thienyl group, a (4-phenyl)phenyl group or a 4-ethoxyphenyl substituent.

A method for the bioassay of a specific fluorescent probe substrate of human intestinal carboxylesterase (hCE2), which is specifically catalyzed by human intestinal carboxylesterase and produces a hydrolyzate TCF, the product has fluorescence emission properties, and the product can be rapidly and ultra-sensitively detected by a fluorescence detector; the TCF fluorescence detection condition is: excitation wavelength 560 nm, fluorescence emission spectrum detection at 590-675 nm; the fluorescent probe substrate can be It is used for rapid quantitative detection of carboxylesterase activity in human intestinal tract, as well as rapid screening and evaluation of human intestinal carboxylesterase inhibitors.

The rapid quantitative detection method of human intestinal carboxylesterase activity is as follows: TCFE hydrolysis reaction is a specific probe reaction of hCE2, hCE2 specifically catalyzes TCFE and produces hydrolysate TCF, which has good fluorescence properties and can be quantitatively detected. The true activity of hCE2 was determined by the change in fluorescence intensity per unit time.

The rapid screening and evaluation method of human intestinal carboxylesterase inhibitors is as follows: TCFE hydrolysis reaction is used as a specific probe reaction of hCE2, and the amount of TCF produced per unit time in the presence and absence of inhibitors is quantitatively compared. The residual activity of hCE2 in the biological system, thereby achieving rapid screening of hCE2 inhibitors and quantitative evaluation of inhibitory ability.

The specific steps for rapid quantitative detection of human intestinal carboxylesterase activity are as follows:

(1) using a common buffer, the reaction temperature is between 20 ° C and 60 ° C; the incubation system pH is between 5.5 and 10.5;

(2) (E)-2-(4-(4-Hydroxyphenylethyl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene)malononitrile ester Derivatives as specific probe substrates; substrate concentration selection 1/10 ~ 10K _m

(3) The reaction time is 5 to 120 minutes, and the reaction is terminated while ensuring that the hydrolysis product reaches the limit of quantitation and the substrate conversion rate is between 0.1% and 20%;

(4) Quantitative determination The amount of hydrolyzed product produced per unit time is substituted into the standard curve and converted to obtain the activity and amount of hCE2.

The specific steps for screening and evaluating human intestinal carboxylesterase inhibitors are as follows:

(2) (E)-2-(4-(4-Hydroxyphenylethyl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene)malononitrile ester The derivative is used as a specific probe substrate; the substrate concentration is selected from 1/10 to 10 K _m , preferably K _m ;

(3) adding different concentrations of inhibitor and setting a blank control group, incubating for 5 to 120 minutes, and terminating the reaction after the hydrolysis product reaches the limit of quantitation and the substrate conversion rate is between 0.1% and 20%;

(4) The fluorescence value of the hydrolyzate per unit time was quantitatively determined and the percentage of the decrease in the fluorescence value added to the inhibitor group was calculated as the evaluation standard for the inhibitory activity.

The field of application of the fluorescent probe substrate for detecting the activity of human intestinal carboxylesterase (hCE2) according to the present invention includes, but is not limited to, recombinantly expressed hCE2 enzyme, containing hCE2 Quantitative determination of hCE2 activity in biological samples such as cell and tissue preparations.

The application of the fluorescent probe substrate for detecting the activity of human intestinal carboxylesterase (hCE2) 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%. between.

The invention provides a fluorescent probe substrate for specifically detecting the activity of human intestinal carboxylesterase (hCE2), the hydrolysis product of the probe molecule has good fluorescence property, and the product and the substrate can be realized by using a fluorescence detector. Rapid and sensitive detection; fluorescence detection conditions are: excitation wavelength 560nm, fluorescence emission spectrum detection at 590 ~ 675nm. In addition, the specific probe substrate and the corresponding hCE2 activity detection process are not interfered by the matrix and impurities of the biological system, and can be used for quantitative determination of hCE2 enzyme activity in various biological systems.

The specific probe reaction can be used for quantitative determination of hCE2 enzyme activity in various biological systems such as recombinant carboxylesterase, cell or tissue preparation liquid, and can also be used for rapid screening of hCE2 enzyme inhibitors and quantitative evaluation of inhibitory ability.

The hCE2 single enzyme or liver microsome incubation system was used to investigate (E)-2-(4-) through correlation analysis, specific inhibition experiments, recombinant single enzyme metabolic reactions, and enzyme reaction kinetics. (4-Hydroxystyrene)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene) Malononitrile derivatives can be specifically metabolized by carboxylesterase hCE2 The corresponding hydrolyzate is produced (as shown in Figures 5-8).

The specific probe substrate of hCE2 of the present invention is used to detect the in vitro activity of hCE2 enzyme with the following outstanding advantages:

(1) High specificity: (E)-2-(4-(4-hydroxyphenylethyl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene) Nitrile ester derivatives can be metabolized to a single generation by hCE2 Xie products, other human esterases or proteins with hydrolytic activity are not involved in the hydrolysis of their compounds.

(2) High sensitivity: product (E)-2-(4-(4-hydroxyphenylethyl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene) Nitrile has good fluorescence properties and has a long fluorescence emission wavelength (maximum emission wavelength at 612 nm), which can reduce the background fluorescence of biological samples and enhance the sensitivity of detection. It has a good application prospect in biological samples.

(3) High accuracy: By plotting the standard curve of the fluorescence intensity and enzyme concentration of the metabolite, as well as the intra-day difference and the inter-day difference, it is found that the fluorescence intensity of the product TCF has a good linear relationship with the enzyme concentration of hCE2, for different biological samples. The quantitative detection error of hCE2 activity is less than 5%.

(4) High-throughput: 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) The advantages.

DRAWINGS

Figure 1. (E)-2-(4-(4-Hydroxyphenylethyl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene)malononitrile derivative Structural formula of matter;

Figure 2. (E)-2-(4-(4-Benzoyloxyphenylethyl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene) ¹ H-NMR spectrum of nitrile;

Figure 3. (E)-2-(4-(4-Benzoyloxyphenylethyl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene) ¹³ C-NMR spectrum of nitrile;

Figure 4. (E)-2-(4-(4-Benzoyloxyphenylethyl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene) High resolution mass spectrum of nitrile;

Figure 5. (E)-2-(4-(4-Benzoyloxyphenylethyl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene) Nitrile human recombinant single enzyme screening test results;

Figure 6. (E)-2-(4-(4-Benzoyloxyphenylethyl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene) Fluorescence spectrum of nitrile with increasing hCE2 protein concentration;

Figure 7. (E)-2-(4-(4-Benzoyloxyphenylethyl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene) Linear fit of the fluorescence intensity of the nitrile hydrolysate to the incubation time;

Figure 8. (E)-2-(4-(4-Benzoyloxyphenylethyl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene) a rice kinetics fit curve for nitrile catalyzed hydrolysis by hCE2;

Figure 9. (E)-2-(4-(4-Benzoyloxyphenylethyl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene) a synthetic route map of nitrile;

Figure 10. (E)-2-(4-(4-Benzoyloxyphenylethyl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene) Fitting map of residual activity curves of nitrile inhibition by loperamide in human liver microsomes, human intestinal microsomes and hCE2;

Figure 11. Screening of various inhibitors in vitro.

detailed description

The invention is further illustrated by the following examples, which are not intended to limit the invention.

Example 1

Synthesis of (E)-2-(4-(4-benzoyloxystyrene)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene) method

(1) containing 0.5 mmol of (E)-2-(4-(4-hydroxystyrene)-3-cyano-5,5-dimethylfuran-2(5H)-subunit to 10 mL N,N two of malononitrile and 0.625 mmol of triethylamine In the methylformamide solution, 0.6 mmol of p-benzoyl chloride (dissolved in 5 mL of N, N-dimethylformamide) was slowly added dropwise, and the temperature was controlled at 0 ° C;

(2) After stirring for 1 hour in an ice bath, the solution was naturally warmed to room temperature and stirred overnight;

(3) The reaction solution was subjected to a solvent under reduced pressure, and the residue was purified by silica gel chromatography eluting with ethyl acetate-hexane (1:3, v/v) to give 30.6 mg of orange solid powder. The route is shown in Figure 9). ¹ H NMR (400 MHz, DMSO) δ 8.16 (d, J = 7.9 Hz, 2H), 8.05 (d, J = 8.5 Hz, 2H), 7.97 (d, J = 16.5 Hz, 1H), 7.78 (t, J = 7.2 Hz, 1H), 7.63 (t, J = 7.7 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 16.5 Hz, 1H), 1.82 (s, 6H) ¹³ C NMR (100 MHz, DMSO) δ 177.54, 175.57, 164.74, 153.91, 146.65, 134.73, 132.73, 131.34, 130.36, 129.50, 129.10, 123.36, 116.06, 113.09, 112.26, 111.26, 100.14, 99.95, 55.08, 25.56 .HRMS (ESI positive) calcd for [ M + Na] + 430.1162, found430.1147. (1 H NMR and ¹³ C NMR spectra are shown in Figures 2 and 3, HRMS spectrum shown in Figure 4).

Example 2

Selectivity in recombinantly expressed human single enzyme

(1) Prepare 99 μL of metabolic reaction system in advance, including PBS buffer (10 mM) at pH 7.4, recombinantly expressed human hCE1 (10 μg/mL) / human hCE2 (10 μg/mL) / serum albumin (10 μg/mL) / acetylcholine Esterase (10μg/mL) / butyrylcholinesterase (1.5U / L) / paraoxonase 1 (10μg / mL) / paraoxonase 2 (10μg / mL) / phosphate buffer, at 37 ° C Pre-incubation for 10 minutes under shock;

(2) Add 1 μL of (E)-2-(4-(4-Benzene) with a final concentration of 20 μM to the reaction system. Starting reaction of acyloxyphenethyl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidenemalononitrile;

(3) After 30 minutes, 100 μL of ice acetonitrile was added, and after violent shaking, the reaction was terminated;

(4) Fluorescence detection (E _x = 560 nm, E _m = 612 nm) was carried out; the fluorescence intensity in each system was calculated (see Fig. 5).

Example 3

Protein concentration of hCE2 catalyzed linear reaction in recombinant single enzyme

(1) Preparing 99 μL of hCE2 metabolic reaction system, including PBS buffer (10 mM) at pH 7.4, recombinant human hCE2 (0-15 μg/mL), and pre-incubating for 10 minutes at 37 °C;

(2) 1 μL of (E)-2-(4-(4-benzoyloxystyrene)-3-cyano-5,5-dimethylfuran at a final concentration of 20 μM was added to the reaction system. -2(5H)-subunit) malononitrile starting reaction;

(4) Perform fluorescence detection (E _x = 560 nm, E _m = 612 nm); calculate the fluorescence intensity in each system (see Figure 6); obtain a standard curve as follows: Y = 101.9 * X - 97.05, where Y represents the fluorescence intensity of the product X represents the concentration of hCE2 in μg/mL and the X range is 0-15 μg/mL.

Example 4

Linear incubation time in recombinant single enzyme hCE2

(1) Prepare 99 μL of hCE2 metabolic reaction system in advance, including PBS buffer (10 mM) at pH 7.4, recombinant human hCE2 (5, 10, 15, 20 μg/mL) at 37 °C. Under shock for 10 minutes;

(3) Fluorescence scanning detection was performed every 5 minutes (E _x = 560 nm, E _m = 612 nm); the linear reaction time of the recombinant human hCE2 enzyme was calculated (see Fig. 7).

Example 5

Quantitative determination of hCE2 in recombinant single enzyme by in vitro pH=7.4

(1) Preparing 99 μL of hCE2 metabolic reaction system, including PBS buffer (10 mM) at pH 7.4, recombinant human hCE2 (2 μg/mL), and pre-incubation for 10 minutes at 37 °C;

(4) Fluorescence detection (E _x = 560 nm, E _m = 612 nm); the maximum catalytic rate of the recombinant human hCE2 enzyme was calculated to be 5.84 ± 0.23 nmol/min/mg (see Fig. 8).

Example 6

Quantitative determination of hCE2 in recombinant single enzyme by in vitro pH=7.8

(4) Fluorescence detection (E _x = 560 nm, E _m = 612 nm); the maximum catalytic rate of the recombinant human hCE2 enzyme was calculated to be 5.32 ± 0.21 nmol/min/mg.

Example 7

Quantitative determination of hCE2 in human intestinal microsomes by pH=7.4

(1) Preparing 99 μL of human intestinal microsomal microsome metabolic reaction system, including Tris-HCl buffer (50 mM) at pH 7.4 and human intestinal microsome (20 ug/ml), and pre-incubating for 10 minutes at 37 °C;

(4) Fluorescence detection (E _x = 560 nm, E _m = 612 nm) was carried out; the maximum catalytic rate of the probe compound in the human small intestine was calculated to be 12.08 ± 0.15 nmol/min/mg (see Fig. 8).

Example 8

Quantitative determination of hCE2 in human intestinal microsomes by pH=7.8

(1) Prepare 99 μL of human intestinal microsome metabolic reaction system in advance, including Tris-HCl buffer (50 mM) at pH 7.4, human intestinal microsome (20 ug/ml), at 37 ° C Under the shock, pre-incubate for 10 minutes;

(4) Fluorescence detection (E _x = 560 nm, E _m = 612 nm) was carried out; the maximum catalytic rate of the probe compound in the human small intestine was calculated to be 11.98 ± 0.16 nmol/min/mg.

Example 9

Quantitative determination of hCE2 in human liver microsomes by pH=7.4

(1) Preparing 99 μL of human liver microsome metabolic reaction system, including Tris-HCl buffer (50 mM) at pH 7.4 and human liver microsomes (20 μg/mL), and pre-incubating for 10 minutes at 37 °C;

(4) Fluorescence detection (E _x = 560 nm, E _m = 612 nm); the maximum catalytic rate of the probe compound in the human small intestine was calculated to be 14.17 ± 0.29 nmol/min/mg (see Fig. 8).

Example 10

Quantitative determination of hCE2 in human liver microsomes by pH=7.8

(1) Prepare 99 μL of human liver microsome metabolic reaction system in advance, including pH 7.4 Tris-HCl buffer (50 mM), human liver microsomes (20 μg/mL), pre-incubated for 10 minutes at 37 ° C;

(4) Fluorescence detection (E _x = 560 nm, E _m = 612 nm); the maximum catalytic rate of the probe compound in the human small intestine was calculated to be 14.08 ± 0.32 nmol/min/mg.

Example 11

Inhibition experiment of loperamide

(1) hCE2 recombinant expression of single enzyme (10 μg / mL), human liver microsomes (10 μg / mL), human intestinal microsomes (10 μg / mL) each 196 μL, shaking at 37 ° C conditions pre-incubation for 10 minutes;

(2) adding 2 μL of the positive inhibitor of carboxylesterase hCE2, loperamide (0-100 μM), to the reaction system, and continuing to incubate for 10 minutes;

(3) Add 2 μL of (E)-2-(4-(4-benzoyloxystyrene)-3-cyano-5,5-dimethylfuran-2 at a final concentration of 20 μM ( 5H)-subunit) malononitrile starting reaction;

(4) After 40 minutes, 200 μL of ice acetonitrile was added, and after violent shaking, the reaction was terminated;

(5) Fluorescence detection (E _x = 560 nm, E _m = 612 nm); fluorescence intensity was calculated, and the inhibition intensity of hCE2 was calculated from the fluorescence intensity at 612 nm of each group and the fluorescence intensity ratio of the DMSO group (see Fig. 10).

Example 12

Rapid screening of inhibitors of hCE2 in vitro

(1) hCE2 recombinantly expressed 196 μl of single enzyme (10 μg/mL), and pre-incubated for 10 minutes at 37 °C;

(2) adding 2 μl of Chinese medicine 95% ethanol extract to the reaction system, and continuing to incubate for 10 minutes;

(3) adding 2 μl of a TCFB initial reaction with a final concentration of 10 μM;

(5) Perform fluorescence detection (E _x =560 nm, E _m =612 nm); calculate the fluorescence intensity, and calculate the residual activity of hCE2 in the reaction system according to the ratio of the fluorescence intensity at 612 nm of the traditional Chinese medicine extract group to the fluorescence intensity of the DMSO group. The inhibitory intensity of Chinese herbal extracts on CES2 (see Figure 11).

Claims

A fluorescent probe substrate for detecting intestinal carboxylesterase activity in humans, characterized in that the substrate is (E)-2-(4-(4-hydroxyphenylethyl)-3-cyano- An aromatic ester of 5,5-dimethylfuran-2(5H)-ylidenemalononitrile (abbreviated as TCF) (abbreviated as TCFE), which has the structural formula:

Wherein R is phenyl, p-methylphenyl, p-ethylphenyl, p-propylphenyl, p-hexylpropyl, p-nitrophenyl, p-chlorophenyl, 1-nyl, 2-furyl Any one of a 2-thienyl group, a (4-phenyl)phenyl group or a 4-ethoxyphenyl substituent.
Use of a fluorescent probe substrate for detecting intestinal intestinal carboxylesterase activity according to claim 1, wherein said fluorescent probe substrate is specifically catalyzed by human intestinal carboxylesterase And the hydrolysis product TCF is generated, the product has fluorescence emission property, and the product can be quickly and ultra-sensitively detected by a fluorescence detector; the TCF fluorescence detection condition is: excitation wavelength 560 nm, fluorescence emission spectrum detection at 590-675 nm; The needle substrate can be used for rapid quantitative detection of carboxylesterase activity in human intestinal tract, as well as rapid screening and evaluation of human intestinal carboxylesterase inhibitors.
The use of a fluorescent probe substrate for detecting human intestinal carboxylesterase activity according to claim 2, characterized in that the rapid quantitative detection method of human intestinal carboxylesterase activity is: using TCFE hydrolysis reaction as hCE2 Specific probe reaction, hCE2 specifically catalyzes TCFE and produces hydrolysate TCF, which has good fluorescence properties and can be passed The true activity of hCE2 was determined by quantitatively detecting changes in fluorescence intensity per unit time.
The use of a fluorescent probe substrate for detecting human intestinal carboxylesterase activity according to claim 2, characterized in that the rapid screening and evaluation method of human intestinal carboxylesterase inhibitor is: hydrolysis with TCFE The reaction is a specific probe reaction of hCE2, and the residual activity of hCE2 in the biological system is evaluated by quantitatively comparing the amount of hydrolysate TCF produced per unit time in the presence and absence of the inhibitor, thereby realizing rapid screening and inhibition of hCE2 inhibitor. Quantitative evaluation.
The use of a fluorescent probe substrate for detecting human intestinal carboxylesterase activity according to claim 3, characterized in that said method for rapid quantitative detection of human intestinal carboxylesterase activity is characterized in that The specific steps for rapid quantitative detection of human intestinal carboxylesterase activity are as follows:

(1) using a common buffer, the reaction temperature is between 20 and 60 ° C; the incubation system pH is between 5.5 and 10.5;

(2) (E)-2-(4-(4-Hydroxyphenylethyl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene)malononitrile ester The derivative is used as a specific probe substrate; the substrate concentration is selected from 1/10 to 10 K m ;

(3) The reaction time is 5 to 120 minutes, and the reaction is terminated while ensuring that the hydrolysis product reaches the limit of quantitation and the substrate conversion rate is between 0.1 and 20%;

(4) Quantitative determination The amount of hydrolyzed product produced per unit time is substituted into the standard curve and converted to obtain the activity and amount of hCE2.
Use of a fluorescent probe substrate for detecting human intestinal carboxylesterase activity according to claim 4, characterized in that the human intestinal carboxylesterase inhibitor is screened and evaluated The application of the method for screening and evaluating human intestinal carboxylesterase inhibitors is as follows:

(1) using a common buffer, the reaction temperature is between 20 and 60 ° C; the incubation system pH is between 5.5 and 10.5;

(2) (E)-2-(4-(4-Hydroxyphenylethyl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene)malononitrile ester The derivative is used as a specific probe substrate; the substrate concentration is selected from 1/10 to 10 K m , preferably K m ;

(3) adding different concentrations of inhibitor and setting a blank control group, incubating for 5 to 120 minutes, and terminating the reaction after the hydrolysis product reaches the limit of quantitation and the substrate conversion rate is between 0.1 and 20%;

(4) The fluorescence value of the hydrolyzate per unit time was quantitatively determined and the percentage of the decrease in the fluorescence value added to the inhibitor group was calculated as the evaluation standard for the inhibitory activity.
The use of a fluorescent probe substrate for detecting a human intestinal carboxylesterase activity according to any one of claims 2 to 6, characterized in that the field of application includes, but is not limited to, recombinantly expressed hCE2 enzyme, containing Quantitative determination of hCE2 in biological samples such as cells and tissue preparations of hCE2.