WO2015014092A1 - Specific fluorescent probe substrate of human carboxylesterase subtype and use thereof - Google Patents

Abstract

A specific fluorescent probe substrate of a human carboxylesterase subtype and a use thereof. The specific fluorescent probe substrate is an ester derivative of 2-(2'-carboxyl-3'-methoxyphenyl) benzothiazole (HMBT) compound 2'-carboxyl. The specific fluorescent probe substrate can be used for detecting whether CES1 exists in different biological samples and can be used for quantitative measurement of the activity of the CES1. A process of enzymatic activity measurement is also follows: selecting an ester derivative of an HMBT hydrolysis reaction as a probe reaction, and detecting the activity of CES1 in a biological sample, a cell, a body and a whole organ by detecting the production of a hydrolytic metabolism product in quantitative measurement unit time in a linear reaction interval. The present invention can be used for quantitative evaluation of the enzyme activity of CES1 from different biological samples, and can be used to detect trace CES1 existing in a patient suffering a liver disease (especially an early liver cancer). In addition, by means of the probe reaction, an inhibitor of CES1 can be rapid screened in vitro.

Description

 Specific fluorescent probe substrate for human carboxylesterase subtype and its application

 The invention belongs to the technical field of medicine, and particularly relates to a specific fluorescent probe substrate of human carboxylesterase 1 (CES1) and application thereof.

Background technique

 Carboxylesterase (CES) is an important one-phase hydrolysis and metabolism enzyme in the body, which catalyzes ester bond cleavage of ester compounds and produces two products with polar groups (hydroxy or carboxyl groups). It is further catalyzed by other metabolic enzymes such as UGTs in the body, making it easier to excrete. Carboxylesterase is not only involved in the metabolism of endogenous substances such as fatty acids, but also a variety of exogenous substances, such as irinotecan (CPT-11), oseltamivir (Duffy) and other ester structure prodrugs and structures. Major metabolic enzymes of other compounds containing ester bonds [J Pharmacol Exp Ther. 2006 Dec; 319(3): 1477-84. & J Pharmacol Exp Then 2006 Dec; 319(3): 1467-76.L Many precursors Drugs require activation or metabolic elimination via a carboxylactone-catalyzed pathway.

Human body Λ mediated drug metabolism There are two main families before the carboxylic acid chymase enzymes: CES1 and CES2, of which CES1 can continue to be divided into two kinds of CESlb and CESlc [Mamm & name, 2010 Oct; 21 (9- 10) 427-41, J _e CES1 and CES2 have different group distribution specificity and substrate selectivity. The liver mainly expresses CES1 and expresses a small amount of CES2. In the small intestine, CES2 subtype is dominant, only a small amount of distribution. Llife Sci. 2007, 81(11): 924-932 ]o In recent years, some scholars have found that the level of CES1 in plasma of patients with liver cancer is significantly increased. iPr tee ics. 2009 Αι ;9 ): 39 ^99, blood CES1 in the slurry is expected to become a potential marker for the diagnosis of liver cancer.

 The present invention provides a class of 2-(2,-hydroxy-3,-methoxyphenyl)benzothiazole (HMBT) compounds 2, a hydroxyl ester ester street organism and its as a substrate for the €ES1 enzyme probe The application, which is hydrolyzed by the carboxylesterase CES1, produces a hydrolyzed product having a different fluorescence emission spectrum than the prototype. The crane-promoting reaction has the characteristics of high selectivity, easy detection of metabolites, rapid activity and inhibition of activity evaluation.

Summary of the invention

 It is an object of the present invention to provide a specific fluorescent probe substrate for human carboxylesterase 1 (CES1) and its use, which have significant differences in fluorescence emission wavelengths before the substrate prototype and the hydrolyzate, and the product is easier to detect. The probe reaction allows quantitative assessment of CES1 cake distribution and function in a variety of biological systems.

The present invention provides a specific fluorescent probe substrate of human carboxylesterase 1, which can be specifically hydrolyzed by €ESi to a corresponding hydrolyzate and exhibits an emission spectrum that is not compatible with the substrate; The substrate is a 2'-hydroxyl ester derivative of 2-(2'-hydroxy-3'-methoxyphenyl)benzothiazole (oxime) compound, and its structural formula is as shown in formula (1), wherein , R is -H or -CH ₃ , -CF ₃ , -C ₂ H ₅ , -C _{3 3⁄4,} etc., or one of -OCH ₃ , -OC ₂ H ₅ , or -Br, -Cl -F One of the alternatives to the base. The R group can be in the ortho, coordination or para position of the benzene ring.

式〈i ) 当 R为 -H时，该底物为 2-(2，-轻基 -3 甲氧苯基)苯并噻唑 (ffldBT) 2'-羟基苯甲酰酯。

Formula <i) When R is -H, the substrate is 2-(2,-lightyl-3-methoxyphenyl)benzothiazole (ffldBT) 2'-hydroxybenzoyl ester.

 The present invention also provides the use of a specific fluorescent probe substrate of human carboxylesterase 1, which is a specific substrate of CES1, which undergoes a hydrolysis reaction, and is determined by quantitatively detecting the amount of hydrolysis produced in a unit. Biological samples such as enzymes or cell preparation fluids and activities of CES1 in cells; specific determination methods are:

- 2-(2'-hydroxy-3'-methoxyphenyl)benzothiazepine (HMBT) compound 2,-hydroxyl ester derivative as a specific probe substrate; substrate concentration select 1 / 10~10 K _m; Choi single point measurement of substrate concentration is preferably K _m.

 ——In a common buffer such as PBS or Tris-HCl, the reaction temperature is 2 () ° C to 60 ° C, preferably 31 TC is the optimal reaction to the servo; the incubation system pH is hand-held 5. ΪΘ 伺, preferred Η 7.4 is the optimal reaction ρ Η value;

——The reaction time is 5~120 minutes, ensuring the corresponding hydrolyzate of the above substrate To the limit of quantitation and the substrate conversion rate does not exceed 20% to terminate the reaction;

 - The amount of hydrolyzate produced per unit time was measured as an evaluation index of carboxylesterase CES1 activity.

 The application of the specific fluorescent probe substrate of human carboxylesterase 1 provided by the present invention, the substrate elimination rate or the production rate of hydrolysis and production should be between D.1% and 2%.

 The application of the specific fluorescent probe substrate of the human carboxylesterase 1 provided by the invention, the probe substrate and the hydrolyzed product thereof have the fluorescent property, and the fluorescence detector can be used for rapid and sensitive detection of the product and the substrate; The detection conditions are as follows: The excitation wavelength is 304 nm, and the fluorescence emission spectrum is detected at 450 to 560 mn.

 The application of the specific fluorescent probe substrate of human carboxylesterase 1 provided by the invention, the specific probe substrate and the corresponding detection process of the activity of Si are not interfered by the matrix and the cockroach of the biological system, and can be used for Quantitative determination of CES1 enzyme activity in various biological systems.

 The present hair tree provides the application of a specific fluorescent probe substrate of human carboxylesterase 1, and the specific probe substrate and its hydrolysis reaction can also be used for rapid screening and tanning ability of human carboxylesterase CES1 inhibitor. Quantitative evaluation of rhyme.

 The present invention provides the use of the specific fluorescent probe substrate for quantitative determination of CES1 enzyme activity in recombinant carboxylesterase, human and animal tissue preparation solutions, and various tissue cells, And an inhibitor that rapidly screens the CES1 enzyme using the probe reaction.

 Recombinant carboxylesterase €-ESi single enzyme, liver microsome incubation system was examined, through correlation analysis, specific inhibition experiments, recombinant single enzyme metabolic reactions, and enzyme reaction kinetics. 2-(2'-hydroxy-3'-methoxyphenyl)

The compound 2, a hydroxyl ester derivative can be specifically metabolized by the carboxylesterase CES1 (eg 3⁄4 5-3⁄4 «shown), produces a hydrolysate that breaks at the €-2' ester bond. The metabolic netting system such as freshly extracted hepatocytes, primary cultured hepatocytes, liver sections, and hepatic perfusion of various mammals was examined and found to have very good specificity.

 As a fluorescent probe substrate for the highly specific carboxylesterase CES1, this compound can be used to detect 3⁄4«W€ES1, especially for bacteria, insect silver cells, mammalian cells and yeast clones. The carboxylesterase CES1 recombinase pre-enzyme activity assay produced by the expression system, and the activity calibration of CES1 in the preparation of tissue microsomes, 9 and other preparations of various tissues and tissues of the suckling animal.

 The in vitro activity of the specific ester probe substrate enzyme esterase CES1 enzyme of the present invention is the following advantages:

 (1) High specificity: The ester derivative of 2'-hydroxyl of 2-<2'-hydroxy-3'-methoxyphenyl)benzothiazole (H BT) compound can be highly specific by carboxylesterase CES1 Metabolized into a metabolic calving, that is, €-2, the ester bond breaks the hydrolysate.

 (2) Cheap and easy to obtain: 2-(2'-hydroxy-3'-methoxyphenyl)benzothiazole (HMBT) compound 2'-hydroxyl ester derivatives and their hydrolysis products can be obtained by chemical synthesis The synthesis process is simple and easy.

(3) High sensitivity: Compounds with HMBT core structure have good fluorescence emission characteristics (430~650 nm), and the substrate and its hydrolyzed metabolites have different fluorescence emission spectra, which can distinguish well. The detection can be quantitatively determined by drawing a standard curve by a ratio method. Figure 1. 2-(2'-hydroxy-3'-methoxyphenyl)benzothiazole (HMBT) compound 2, -hydroxyl Record the structural formula of the cool derivative;

Figure 2. ¹ H-M spectrum of 2-(2'-hydroxy-3'-methoxyphenyl)benzothiazole (HMBT) 2,-hydroxybenzoyl ester;

Figure 3 2-(2'-hydroxy-3'-methoxyphenyl)benzothiazole (HMBT) 2'-hydroxybenzoyl ester fishing ¹³ spectrum;

 Figure 4 High resolution mass spectrum of 2-(2'-hydroxy-3'-methoxyphenyl)benzothiazole (HMBT) 2,-hydroxybenzoyl ester;

 Figure 5 Human recombinant single enzyme screening test of 2-(2'-hydroxy-3'-methoxyphenyl)benzothiazole (HMBT) 2'-hydroxybenzoyl ester;

 Figure 6 Results of human recombinant single enzyme screening test of 2-(2,-hydroxy-3'-methoxyphenyl)benzothiazole (HMBT) 2,-hydroxy-p-methylbenzoyl ester;

 Figure 7 2-(2,-hydroxy-3'-methoxyphenyl)benzothiazole (HMBT) 2,-hydroxy-p-methoxybenzoyl ester human recombinant single-rejection screening test results;

 Figure 8 Results of human recombinant single enzyme screening test of 2-(2'-hydroxy-3,-methoxyphenyl)benzothiazole (HMBT) 2,-hydroxy-p-bromobenzoate;

 Figure 9. Fluorescence emission intensity of 2'-hydroxybenzoyl esterified substituted 2-(2'-hydroxy-3'-methoxyphenyl)benzothiazole (HMBT) debenzoyl metabolite <at 304 nm Ff excitation) increases as the concentration of the two subtypes of the carboxylesterases CESlb and CESlc increases;

 The amount of W-debenzoyl hydrolyzed metabolites is linearly matched with the light-to-finance change.

Figure 11 ² ,-hydroxyl-methylformamidine-substituted 2-(2,-hydroxy-3,-methoxyphenyl) thiazole (HMBT) is a metabolic pathway catalyzed by CES1; m 12 2-(2,-Hydroxy-3,-methoxyphenyl ketone ί*3⁄4Η ΒΤ) 2,-Hydroxybenzoyl thios.

detailed description

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

Example 1. 2-(2,-Hydroxy-3,-methoxyphenyl)benzothiazole (HMBT) 2,-hydroxybenzoyl ester Female synthesis

(1) To a 10 mL solution containing 0.5 mmol of 2-(2'-hydroxy-3'-methoxyphenyl)benzothiazine and 0.625 mmol of triethylamine, slowly add .6 mmol. Benzoyl chloride (dissolved in 5 mL of tetrahydrofuran), controlled at 0 ° C _;

 (2) After mixing i h, heat the reaction solution to room temperature and react overnight (see Figure 12);

(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 (i: 3 v/v)

 <4) Characterization of compound structures by high resolution mass spectrometry and nuclear magnetic resonance (see Figure 2-m)

4).

Implementation of the reverse 2. Recombination in the selectivity of human single enzyme

) /Α清 白蛋白 （500 g/ml) /血浆（1%) /丁酰胆碱酯酶（25U/L) /磷酸缓冲 液， 于 37°C条件下震荡预孵 10分钟； (1) Prepare a 99 μΐ metabolic reaction system, including ρΗ 7.4 in PBS buffer (!O toM). Recombinantly expressed human CESlb (5)

/ Α albumin (500 g / ml) / plasma (1%) / butyrylcholinesterase (25U / L) / phosphate buffer, pre-incubation for 10 minutes at 37 ° C;

(2) Add 1 μΐ of 2-(2'-hydroxy-3'- at a final concentration of 25 μΜ to the reaction system. Methoxyphenyl)benzothiazepine (HMBT) 2'-hydroxyphenyl acyl ester / p-methylbenzoyl ester / p-methoxybenzoyl ester / p-bromobenzoyl ester initial reaction;

 (3) After 30 minutes, add ΙΟΘμΙ ice acetonitrile, and after violent shaking, terminate the reaction;

(4) Perform fluorescence detection (Ex=304 nm, Em=490 nm); Calculate the fluorescence intensity in each system (see Huai 5-Fig. 8);

Example 3. Linear protein concentration of CESlb or CESlc in recombinant single enzyme

 (1) Prepare the μΐ CES1 metabolic reaction system, including PBS buffer (10 mM) of ρΗ 7.4, recombinant human CESlb or lc (0-100 ug/ml), and shake for 1⁄2 minutes at 37 °C;

 (2) adding to the reaction system 1 a final concentration of 25 μΜ of 2-(? hydroxyl-3'-methoxyphenyl)benzothiazole (ΗΜΒΤ) 2'-hydroxybenzoyl ester to initiate the reaction;

 (3) After 30 minutes, add 100 μl of ice acetonitrile, and vortex, stop the reaction; 4 >3⁄4 lines of light detection < Ex=304 nm, Em=490 nm); calculate recombinant human €ES 1% or lc enzyme Linear protein concentration (see Figure 9).

Example 4. Recombinant Single Enzyme Linear Incubation in CESlb or CESle

 (1) Pre-prepare 99 μΐ CES1 metabolic reaction system, including ρΗ 7.4 in PBS buffer (i0 mM), recombinant human CESib or ie (5/i0/i5/20ug/mi), shake pre-incubation at 37 °C 10 minutes;

 (2) adding 1 μΐ of a final concentration of 25 μΜ rhyme 2<2'-hydroxy-3'-methoxyphenyl)benzothiazole (ΗΜΒΤ) 2,-hydroxybenzoyl ester to the reaction system;

(3) Subfluorescence scanning was performed every 5 minutes for cadmium (Ex=304 nm, Em=490 nm); the linear reaction time of recombinant human CESlb or lc enzyme was calculated (see Figure 10). Example 5. Quantitative determination of the activity of CESifc or CESie in recombinant single enzyme in vitro

 (1) Prepare a 99 μΐ CES1 metabolic reaction system, including ρΗ 7.4 PBS buffer < 10 mM), recombinant human CESlb or ie (2 ug mi), and incubate for 10 minutes at 37 °C;

 (2) adding 2 μm of 2-(2'-hydroxy-3'-methoxyphenyl)benzothiazole (ΗΜΒΤ) 2'-hydroxybenzoyl ester with a final concentration of 25 μM to the reaction system;

 (3) After 30 minutes, add Η)β μ water acetonitrile, and after violent shaking, terminate the reaction;

(4) Fluorescence detection (Ex=304 nm, Em=490 nm); calculation of the maximum catalytic rate of recombinant human CESlb or ie enzyme is 140i ± 33ϋ nmoi/min/mg and 245i ± 135

Example 6. Quantitative determination of the activity of CES1 in human intestinal microtubules in vitro

 (1) Prepare a 99 μΐ human intestinal microsomal metabolic system, including Tris-HCi buffer (5 mM) of ρΗ 7.4, human intestinal microsomes (20 iig/mi), and shake pre-incubation at 37 °C. Minute

 (2) adding a 2-μ2--hydroxy-3,-methoxyphenyl)benzothiazole (ΗΜΒΤ) 2'-hydroxybenzoyl ester of i μΐ at a final concentration of 25 μΜ to the reaction system;

 (3) After 30 minutes, add Η)θ μί ice acetonitrile, and after violent shaking, terminate the reaction;

(4) Fluorescence detection (Ex=304 nm, Em=490 nm); The maximum catalytic rate of the probe compound in the human small intestine was 442 ± 43 Ling Ol/m1⁄2 mg.

Example 7. Quantitative determination of CES1 activity in human lung adenocarcinoma A549 cells

(i) Human lung adenocarcinoma A549 cell line was cultured on coverslips using DMEM medium (containing 10% calf serum) and 100 ug/ml double antibody, culture environment It is a 5% carbon dioxide incubator at 37 °C.

 (2) Prior to use, adherent cells were washed 3 times with serum-free DMEM medium and added to a final concentration of IO uM of 2-(2'-hydroxy-3'-methoxyphenyl)benzothiazole (HMBT). 2'-hydroxybenzoyl ester, incubated at 37 ° C for 30 minutes.

 3) After that, rinse 3 times with PBS buffer. The cells were observed under a laser confocal fluoroscopy, and the distribution and intensity of CES1 in the cells were shown by the position and intensity of the fluorescence distribution.

Example 8. Quantitative determination of CES1 in human blood

 (1) Add CESlc recombinant expression single enzyme (100 g/mL) 20 μΐ to 3SO μΐ of PBS diluted 1 ϋ% ϊΕ human blood dish, and incubate for 10 minutes at 37 °C;

(2) adding 1 μΐ of 2<2′-hydroxy-3′-methoxyphenyl)benzothiazole (ΗΜΒΤ) 2′-hydroxybenzoyl ester to a reaction solution at a final concentration of 25 μM;

 (3) After 3 minutes, add 400 μί of ice acetonitrile, violently shake, and finally react;

(4) Fluorescence detection (Ex=304 ran, Em=490 ran); Calculate the signal-to-noise ratio S/N>W, and the limit of quantification of CESi in plasma is 5 tg/mL.

Example 9. Rapid screening in vitro CES1 inhibitor

 (1) CESife CESle recombinantly expressed single (5 pg mL) ίΜ≠, pre-incubated for 10 minutes under 37t conditions;

 (2) Add 2 μΐ of Chinese medicine 95% ethanol extract to the reaction system and continue to incubate for 1⁄2 minutes;

<3) adding a final concentration of 2 pi of 1 ύ μΜ of 2-(2,-hydroxy-3,-methoxyphenyl) 3⁄4 and thiazole (oxime) 2,-hydroxybenzoyl ester to initiate the reaction; (3) After 30 minutes, add 200 μl of ice acetonitrile, and after violent shaking, the final it reaction;

(4) Fluorescence detection (Ex=304 nm, Em=490 nm); Calculating the fluorescence intensity, the inhibition intensity of CES1 was calculated according to the ratio of the fluorescence intensity at 490 mi of the Chinese herbal extract group to the fluorescence intensity of the group.

Claims

claims 1. A specific fluorescent probe substrate for human carboxylesterase 1, characterized in that: the ester bond of the substrate can be specifically hydrolyzed by CES1 to the corresponding hydrolysis product and displays an emission spectrum different from that of the substrate; The substrate is an ester derivative of the 2'-hydroxyl group of 2-(2'-hydroxy-3'-methoxyphenyl)benzothiazole (HMBT) compound, and its general structural formula is as shown in formula (1) , where R is one of -H, -C¾, -CF ₃ , -C ₂ H ₅ , -C ₃ He, -OCH ₃ , -OC ₂ , -Br, -Cl, and -F substituents.

Formula <U 2. Application of the specific fluorescent probe substrate of human carboxylesterase 1 according to claim 1, characterized in that: as a specific substrate of CESi, the substrate undergoes a hydrolysis reaction and quantitatively detects hydrolysis per unit time. The amount of product produced was used to determine the activity of CES 1 in different enzyme sources. 3. According to the specific fluorescent probe substrate of human carboxylesterase 1 according to claim 2 The application is characterized in that: the enzyme source is a recombinantly expressed CESi single enzyme, a human or animal tissue preparation, or various tissue and cell biological systems. 4. Application of human carboxylesterase 1 specific fluorescent probe substrate according to claim 2, characterized in that: the hydrolysis reaction system is phosphoric acid or Tris-HCl buffer, and the probe substrate is concentrated acid The range is between /^~^^; the incubation system is between ^^~^^; the reaction temperature is between 20~60°C. 5. Application of the specific fluorescent probe substrate of human carboxylesterase 1 according to claim 2, characterized in that: the generation rate of the hydrolyzate should be between 0.1% and 20%. 6. Application of the specific fluorescent probe substrate of human carboxylesterase 1 according to claim 2, characterized in that: the probe substrate and its hydrolyzate have fluorescent properties, and a fluorescence detector can be used to realize the product and Rapid and sensitive detection of substrates; fluorescence detection conditions are: excitation wavelength 304 nm, detection of fluorescence emission spectrum at 450~560 nm. 7. Application of the specific fluorescent probe substrate of human carboxylesterase 1 according to claim 2, characterized in that: the specific probe substrate and its hydrolysis reaction can also be used to inhibit human carboxylesterase CES1 Rapid screening of agents and quantitative evaluation of their production capabilities.