WO2015179999A1

WO2015179999A1 - Ratio-type fluorescent probe substrate of cytochrome oxidase cyp1a and use thereof

Info

Publication number: WO2015179999A1
Application number: PCT/CN2014/000958
Authority: WO
Inventors: 杨凌; 戴子茹; 崔京南; 葛广波; 冯磊; 宁静
Original assignee: 中国科学院大连化学物理研究所
Priority date: 2014-05-30
Filing date: 2014-10-31
Publication date: 2015-12-03
Also published as: CN105219374A; CN105219374B

Abstract

Disclosed are a ratio-type fluorescent probe substrate of cytochrome oxidase CYP1A and a use thereof, which belong to the technical field of biomedicine. The specific probe substrate has a structure of a hydroxy-naphthalimide-alkanoic acid, which can be used for determining the enzyme activity of the CYP1A in a biosystem. The flow process for determining the enzyme activity of CYP1A is as follows: selecting the demethylation of hydroxy-naphthalimide-alkanoic acids as the probe reaction; and the enzyme activity of the CYP1A in a variety of biological samples were determined by quantitative detection of the amount of produced demethylation metabolic product in unit time. The present invention can be used for the quantitative evaluation of the enzyme activity of CYP1A in the biological samples from various individuals, various individual sources, and for the quantitative determination of the enzyme activity of CYP1A in animal tissue cell culture media and the cell preparations from different sources, so as to achieve the evaluation for the ability of the drug disposition of the important drug metabolic enzyme CYP1A. Furthermore, the probe reaction can also be used for rapid screening of the inhibitor of CYP1A in vitro and evaluating the inhibiting ability thereof.

Description

Ratio-type fluorescent probe substrate for cytochrome oxidase CYP1A and application thereof

Technical field

The invention belongs to the technical field of biomedicine, and particularly relates to a ratio type fluorescent probe reaction of cytochrome oxidase CYP1A and application thereof.

Background technique

The cytochrome P450 (P450) superfamily is the most important drug-metabolizing enzyme in the body. The main clearance of about 60% of drugs (including most clinical drugs and pesticides) is mediated by CYP. . The cytochrome P450 enzyme system is a superfamily of proteins and is a large class of heme-containing proteins. The complex formed with CO in the reduced state has a maximum absorption peak at 450 nm. Because of its catalytic phase I reaction is a key step in the metabolism of compounds in the body, because this step is usually the rate-limiting step of drug removal from the body, which can affect the kinetics of compound half-life, clearance, etc., and P450 enzyme activity often Changes in genetic factors, age, disease state, or other drug interactions. The effect of the drug on the body's P450 enzyme can cause clinically significant drug interactions.

CYP1A is an important phase I metabolic enzyme, mainly including two subtypes: CYP1A1 and CYP1A2, of which CYP1A1 is mainly expressed in human lung, while CYP1A2 is mainly expressed in human liver and accounts for 13% of total CYP in human liver. . CYP1A is also involved in a variety of drugs such as theophylline, caffeine, antipyrine, etc., as well as the metabolism of environmental toxins and endogenous substrates, and is activated in a variety of procarcinogens to become genotoxic intermediates or final carcinogens. The process plays an important role, for example, to a certain extent, activation of caffeine induces the occurrence of cirrhosis (MOLASPECTS MED. 1999.20: 1-137). In addition, the activity of CYP1A is also very different among different ethnic groups. Population studies have found that CYP1A may exhibit unimodal, bimodal or even trimodal distribution in different races (Eur J Clin Pharmacol. 1995.47: 423 -430). Therefore, the study of individual differences in CYP1A enzyme activity is of great significance for clinical personalized safety medication. At present, domestic and foreign pharmaceutical giants need to evaluate the ability of each candidate new drug to inhibit CYP1A in vitro during drug development. Therefore, the development of efficient and sensitive specific CYP1A probe substrates is essential for the efficient screening of CYP1A inhibitors and for the quantitative determination of CYP1A activity in biological systems.

Since each subtype of the CYP1A subfamily has a similar amino acid sequence, its substrates usually overlap each other, so each subtype of enzyme has a few specific substrates. At present, three fluorescent probe substrates have been reported for CYP1A, namely 3-cyano-7-ethoxycoumarin, ethoxy resorufin and fluorescein-ME-EGE. This Some known fluorescent substrates are off-on type probes, the single enzyme selectivity is not high and is susceptible to interference by biological matrices, and the quantitative error is large. The blue shift/red shift of the emission spectrum of the ratio probe can be used for ratio detection, and the prototype of the probe molecule can be used as internal calibration to reduce the illumination intensity, probe concentration, sample non-uniformity, instrument parameters, etc. Impact. Therefore, the development of highly selective CYP1A ratio-type fluorescent probe reactions and their associated high-throughput detection methods have important practical value.

Summary of the invention

The object of the present invention is to provide a ratiometric fluorescent probe substrate of cytochrome oxidase CYP1A and an application thereof, wherein the fluorescence emission wavelength of the ratio-type fluorescent probe substrate and the demethylated product are significantly different, and the product is The fluorescence quantum yield is higher and easier to detect. The probe reaction can be used to quantitatively evaluate the distribution and function of CYP1A in various biological systems.

The present invention provides a ratiometric fluorescent probe substrate for cytochrome oxidase CYP1A, which is specifically catalyzed by CYP1A2 to produce a corresponding O-demethylation product having 1,8- The naphthalimide structure has the following structural formula:

Wherein R is any one of -COOH, benzoic acid, and -SO ₃ H, and n is 2 to 10.

The present invention also provides a ratio of a cytochrome oxidase CYP1A fluorescent probe substrate, using the specific substrate of the CYP1A sub-enzyme, mixed with a biological sample containing CYP1A, followed by enzymatic reaction, through quantitative detection unit Quantitative determination of the activity of CYP1A in different biological systems during the time of substrate elimination rate or its demethylation product formation rate. The specific determination methods and conditions are as follows:

A. System 1,8-naphthalimide type compounds as a ratio probe substrate; substrate concentration to select 1/10 ~ 10K _m; single-point concentration is preferably measured K _m of substrate;

B. In PBS buffer, the reaction temperature is between 20 ° C and 60 ° C, preferably 37 ° C is the optimal reaction time; the incubation system pH is between 5.5 and 10.5, preferably pH 7.4 is the optimal reaction pH;

C. The reaction time is 5 to 120 minutes, and the reaction is terminated when the corresponding O-demethylation product of the above substrate reaches the limit of quantification and the substrate conversion rate does not exceed 20%;

D. The amount of substrate reduction or the amount of O-demethylated product produced per unit time was determined as an evaluation index of CYP1A activity.

The ratiometric fluorescent probe substrate application of the cytochrome oxidase CYP1A is further characterized in that the biological system is recombinantly expressed CYP1A single enzyme, human or animal tissue preparation liquid, various mammalian tissue cells and preparation thereof Any one of them.

The fluorescent signal of the probe substrate and its demethylated product should be detected by different detection wavelengths. The fluorescence detection conditions of the demethylated product and the substrate are: excitation wavelength 450, 372 nm, and maximum emission wavelength are 564, respectively. , 452nm.

The probe substrate can also be used for rapid screening of CYP1A inhibitors and quantitative evaluation of inhibitory capacity.

The probe substrate can also be used as a probe substrate for the in vivo and whole CYP1A of experimental animals to assess individual and species differences in the metabolic enzyme CYP1A.

The use of the ratiometric fluorescent probe reaction of the cytochrome oxidase CYP1A provided by the present invention, the probe substrate and the O-demethylation product thereof have fluorescence properties, and the two have different optical properties, and the fluorescence can be used. The detector simultaneously detects the rapid and sensitive detection of the substrate and the product; the O-demethylation product and the substrate fluorescence detection conditions are: excitation wavelength 372,450 nm, maximum emission wavelength is 450, 564 nm as shown in FIG. 4 .

The specific probe substrate is a ratiometric fluorescent probe, which is not easily interfered by biological system matrix and impurities during the detection process of CYP1A activity, and can be used for various recombinant CYP1A, human and animal tissue preparation liquids, and CYP1A in various tissue cells. Quantitative determination of enzyme activity; also as a probe substrate for CYP1A in vivo and in vivo, to assess individual and species differences in the metabolic enzyme CYP1A. The probe substrate and the fluorescence detection method of O-demethylated metabolites can also be used for rapid screening of CYP1A inhibitors and quantitative evaluation of inhibitory ability.

Recombinant cytochrome oxidase CYP1A single enzyme, liver microsome incubation system was investigated, and the correlation analysis (as shown in Figure 6), recombinant single enzyme metabolic reaction (as shown in Figure 7), specific inhibition experiments (as shown in Figure 8), as well as evidence of several aspects of enzyme reaction kinetics, demonstrate that 1,8-naphthylimide compounds can be specifically metabolized by the cytochrome oxidase CYP1A (as shown in Figure 9), generating O - Demethylation of the oxidation product. Furthermore, a variety of mammalian freshly extracted hepatocytes, primary cultured hepatocytes, liver sections, liver perfusion and other metabolic evaluation systems were examined and found to have very good specificity.

As a fluorescent probe substrate for the highly specific cytochrome oxidase CYP1A single enzyme, this compound can be used to detect the activity of CYP1A, especially for the production of bacterial, insect cells, mammalian cells and yeast cloning expression systems. The enzyme activity assay of CYP1A, as well as the activity calibration of CYP1A in preparations of microsomes, S-9 and the like from various mammalian tissues and organs.

The ratio of the cytochrome oxidase CYP1A single enzyme in vitro activity using the cytochrome oxidase CYP1A single enzyme of the present invention has the following outstanding advantages:

(1) High specificity; 1,8-naphthylimide compounds can be highly specifically metabolized by the cytochrome oxidase CYP1A single enzyme into a metabolite, that is, an O-demethylated product.

(2) Cheap and easy to obtain: 1,8-naphthylimide compounds can be obtained by chemical synthesis, the synthesis process is simple and easy, and the detection cost of the fluorescent method is low.

(3) High sensitivity: Compounds with a 1,8-naphthalimide core structure have good fluorescence emission characteristics (450-700 nm), and the substrate and its O-demethylation metabolites are different. The fluorescence emission spectrum characteristics can be better differentiated and detected, and the lower limit of detection of CYP1A single enzyme can be quantitatively determined by the establishment of the ratio type standard curve to be 0.2 nM/ml.

DRAWINGS

Figure 1.1, the structural formula of the 8-naphthylimide compound;

Figure 2. ¹ H-NMR spectrum of N-(3-carboxypropyl)-4-methoxy-1,8-naphthalimide;

Figure 3. ¹³ C-NMR spectrum of N-(3-carboxypropyl)-4-methoxy-1,8-naphthalimide;

Figure 4. Ultraviolet absorption spectrum of N-(3-carboxypropyl)-4-methoxy-1,8-naphthalimide and its O-demethylated metabolites (maximum at 372 nm and 450 nm, respectively) absorb);

Figure 5.14 is a metabolic diagram of HLM versus N-(3-carboxypropyl)-4-methoxy-1,8-naphthalimide;

Figure 6. N-(3-carboxypropyl)-4-methoxy-1,8-naphthalimide and its O-demethylation rate and the rate of O-deethylation of phenacetin Correlation analysis experiment;

Figure 7. Results of human CYP recombinant single enzyme screening test of N-(3-carboxypropyl)-4-methoxy-1,8-naphthalimide;

Figure 8. Results of chemical inhibition experiments of N-(3-carboxypropyl)-4-methoxy-1,8-naphthalimide in human liver;

Figure 9. CYP1A mediates the metabolic pathway of N-(3-carboxypropyl)-4-methoxy-1,8-naphthalimide;

Figure 10. Scheme for the synthesis of N-(3-carboxypropyl)-4-methoxy-1,8-naphthalimide.

detailed description

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

The apparatus and model of the invention are: fluorescence emission/excitation spectroscopy is completed by SynergyH1 full-function microplate detector; ¹ H-NMR spectrum and ¹³ C-NMR spectrum are obtained by NMR spectrometer (Avance) II 400MHz) Detection completed.

Example 1

Synthesis route of N-(3-carboxypropyl)-4-methoxy-1,8-naphthalimide

(1) Synthesis of Compound 1

4.2 mmol of 4-aminobutyric acid was added to a solution containing 1 g (3.61 mmol) of 4-bromo-1,8-naphthalene anhydride in 50 ml of ethanol, and after reacting at 70-80 ° C overnight, 200 ml of water was added to precipitate a large amount of solid, which was filtered and vacuumed. Drying gave a beige solid N-(3-carboxypropyl)-4-bromo-1,8-naphthalimide in a yield of 80-90%.

(2) Synthesis of Compound 2

800 mg of Compound 1 and 2.54 g of potassium carbonate were placed in a 100 ml single-mouth bottle, 30 ml of methanol was added, and the reaction was carried out at 60-70 ° C overnight, followed by cooling, the pH was adjusted to be acidic with 1 M hydrochloric acid, a large amount of a yellow solid was precipitated, filtered, and washed with a large amount of water. Drying under vacuum gave N-(3-carboxypropyl)-4-methoxy-1,8-naphthalimide as a yellow solid, yield 80-90%.

The structures of

compounds

1, 2 are shown in Figure 10. Ultraviolet absorption spectra of N-(3-carboxypropyl)-4-methoxy-1,8-naphthalimide and its O-demethylated metabolites As shown in Fig. 4, there is maximum absorption at 372 nm and 450 nm, respectively; the nuclear magnetic resonance spectrum analysis of the prepared product is shown in Fig. 2 and Fig. 3, as follows:

¹ H NMR (400 MHz, DMSO) δ = 12.05 (s, 1H), 8.49 (ddd, J = 8.4, 7.8, 1.1, 2H), 8.43 (d, J = 8.3, 1H), 7.80 (dd, J = 8.3) , 7.4, 1H), 7.31 (d, J = 8.4, 1H), 4.13 (s, 3H), 4.06 (t, J = 7.0, 2H), 2.30 (t, J = 7.4, 2H), 1.88 (p, J = 7.2, 2H). ¹³ C NMR (100 MHz, DMSO) 23.59, 31.82, 39.33, 56.99, 116.36, 114.39, 122.01, 122.86, 126.45, 131.15, 133.39, 160.48, 163.27, 163.93, 174.45.HRMS[M+H ] ⁺ 313.0950, found 314.1025.

Example 2

Synthesis of N-(carboxypentyl)-4-methoxy-1,8-naphthalimide

(1) Synthesis of the compound N-(3-carboxypentyl)-4-bromo-1,8-naphthalimide

4.2 mmol of 6-aminocaproic acid was added to a solution containing 1 g (3.61 mmol) of 4-bromo-1,8-naphthalene anhydride in 50 ml of ethanol, and after reacting at 70-80 ° C overnight, 200 ml of water was added to precipitate a large amount of solid, which was filtered and vacuumed. Drying gave a beige solid N-(3-carboxypentyl)-4-bromo-1,8-naphthalimide in a yield of 80-90%.

(2) Synthesis of the compound N-(3-carboxypentyl)-4-methoxy-1,8-naphthalimide

800 mg of the compound N-(3-carboxypentyl)-4-bromo-1,8-naphthalimide and 2.54 g of potassium carbonate were placed in a 100 ml single-mouth bottle, added with 30 ml of methanol, and reacted at 60-70 ° C overnight, followed by cooling. The pH was adjusted to be acidic with 1 M hydrochloric acid, a large amount of a yellow solid was precipitated, filtered, washed with a large amount of water and dried in vacuo to give a yellow solid N-(3-carboxypentyl)-4-methoxy-1,8-naphthalimide. The yield is 80-90%.

The nuclear magnetic resonance spectrum of the prepared product is as follows:

^{1 H NMR (400MHz, DMSO)} δ11.99 (s, 1H), 8.55-8.38 (m, 3H), 7.83-7.74 (m, 1H), 7.30 (d, J = 8.4Hz, 1H), 4.12 (s , 3H), 4.05-3.94 (m, 2H), 2.22 (t, J = 7.3 Hz, 2H), 1.67-1.48 (m, 4H), 1.40-1.25 (m, 2H). ¹³ C NMR (100 MHz, DMSO ) δ 174.87, 163.63, 162.96, 160.34, 133.18, 130.97, 128.47, 128.17, 126.25, 122.72, 121.84, 114.27, 106.17, 56.80, 33.96, 27.70, 26.54, 24.68. HRMS [M+H] ⁺ 341.1236, found342. 1335.

Example 3

Synthesis of N-(4-carboxyphenyl)-4-methoxy-1,8-naphthalimide

(1) Synthesis of the compound N-(4-carboxyphenyl)-4-bromo-1,8-naphthalimide

4.2 mmol of 4-aminobenzoic acid was added to a solution of 1 g (3.61 mmol) of 4-bromo-1,8-naphthalene anhydride in 50 ml of acetic acid, and reacted at 100-110 ° C overnight, filtered while hot, and the filter cake was washed with acetic acid, vacuum Drying gave a beige solid N-(4-carboxyphenyl)-4-bromo-1,8-naphthalimide in a yield of 30-40%.

(2) Synthesis of the compound N-(4-carboxyphenyl)-4-methoxy-1,8-naphthalimide

800 mg of the compound N-(4-carboxyphenyl)-4-bromo-1,8-naphthalimide and 2.54 g of potassium carbonate were placed in a 100 ml single-mouth bottle, and 30 ml of methanol was added thereto, and the reaction was carried out at 60-70 ° C overnight. After cooling, the pH was adjusted to be acidic with 1M hydrochloric acid, and a large amount of yellow solid was precipitated, filtered, washed with a large amount of water and dried in vacuo to give a yellow solid N-(4-carboxyphenyl)-4-methoxy-1,8-naphthyl Amine, yield 60-70%.

The nuclear magnetic resonance spectrum of the prepared product is as follows:

^{1 H NMR (400MHz, DMSO)} δ13.05 (s, 1H), 8.62 (d, J = 7.9Hz, 1H), 8.51 (dd, J = 12.4,7.5Hz, 2H), 8.12-8.03 (m, 2H ), 7.87 (t, J = 7.9 Hz, 1H), 7.52 (d, J = 8.3 Hz, 2H), 7.38 (d, J = 8.4 Hz, 1H), 4.16 (s, 3H).

Example 4.

Determination of the selectivity of human recombinant CYP single enzyme in vitro

(1) Prepare 90 μl of CYP metabolic reaction system in advance, including PBS buffer (100 mM) at pH 7.4, recombinant human CYP single enzyme (0.75 nm/ml), N-(3-carboxypropyl)-4-methoxy -1,8-Naphthalimide final concentration of 10 μM, pre-incubation for 3 minutes at 37 ° C;

(2) adding 10 μl of 10 mM NADP ⁺ starting reaction to the reaction system;

(3) After 40 minutes, 50 μl of ice acetonitrile was added, and after violent shaking, the reaction was terminated;

(4) After high-speed centrifugation at 20,000 × g for 20 minutes at 4 ° C in a high-speed refrigerated centrifuge, the supernatant was taken for fluorescence detection (Ex = 372 nm, Em = 450 nm); recombinant human CYP1A (1A1 and 1A2) The selectivity of the enzyme is about 10 times higher than that of other single enzymes (Fig. 7).

Example 5.

Quantitative assessment of CYP1A2 activity in liver microsomes from different individuals

(1) 14 human liver microsomes (HLM) were diluted to 2.5 mg/ml to prepare CYP1A metabolic reaction system, including pH 7.4 PBS buffer (100 mM), human liver microsomes (0.25 mg/ml), NADP ⁺ 10 mM, glucose 6-phosphate phosphate 100 mM, glucose-6-phosphate dehydrogenase 1 unit/ml, MgCl ₂ 40 mM, N-(3-carboxypropyl)-4-methoxy-1,8-naphthalimide final concentration For 10 μM, pre-incubate for 3 minutes at 37 °C;

(2) adding 10 μl of 10 mM NADP ⁺ starting reaction to the reaction system;

(3) After 30 minutes, 10 μl of ice acetonitrile was added, and after violent shaking, the reaction was terminated;

(4) After high-speed centrifugation at 20,000 × g for 20 minutes at 4 ° C in a high-speed refrigerated centrifuge, the supernatant was taken for fluorescence detection (Ex = 372 nm, Em = 450 nm), and the obtained fluorescence intensity was substituted into a standard curve. The metabolic rate of 14 human liver microsomes (HLM) versus N-(3-carboxypropyl)-4-methoxy-1,8-naphthalimide was obtained (Fig. 5).

Example 6.

Determination of the lower limit of detection of CYP1A in vitro

The assay was performed on a microplate reader using a 96-well plate, N-(3-carboxypropyl)-4-methoxy-1,8-naphthylenimine 10 μM, NADP ⁺ 10 mM, glucose 6-phosphate glucose 100 mM, glucose -6-phosphate dehydrogenase 1 unit/ml, MgCl ₂ 40 mM, CYP1A2 single enzyme 0.1 nM/ml ~ 2 nM/ml, pH 7.4 PBS buffer 50 mM, total volume 100 μL, incubate at 37 ° C for 1 h and pass the microplate reader For the analysis, the mean of each group was compared with the control group without CYP1A. The results showed that CYP1A at 0.2 nM/ml was statistically significant (P<0.05), so the lower limit of detection for CYP1A was determined to be 0.2 nM/ml.

Example 7.

CYP1A time standard curve determination

The assay was performed on a microplate reader using a 96-well plate, N-(3-carboxypropyl)-4-methoxy-1,8-naphthalimide 10 μM, NADP ⁺ 10 mM, glucose 6-phosphate glucose 100 mM, glucose -6-phosphate dehydrogenase 1 unit/ml, MgCl ₂ 40 mM, CYP1A2 single enzyme 0.1 nM/ml ~ 2 nM/ml, pH 7.4 PBS buffer 50 mM, total volume 100 μL, incubation at 37 ° C for 60 min, every 5 minutes According to the microplate reader analysis, the ratio of the fluorescence intensity of the product to the fluorescence intensity of the substrate and the incubation time were made to a standard curve. The R ² >0.99 of each standard curve indicates that the linear range of the standard curve is broad, and the content of CYP1A can be accurately quantified.

Claims

A ratiometric fluorescent probe substrate for cytochrome oxidase CYP1A, characterized in that the probe substrate is specifically catalyzed by CYP1A to form a corresponding demethylated product having 1,8-naphthoyl The imine structure has the following structural formula:

Wherein R is any one of -COOH, benzoic acid, and -SO 3 H, and n is 2 to 10.
Use of a ratiometric fluorescent probe substrate for cytochrome oxidase CYP1A according to claim 1, characterized in that the specific substrate of the CYP1A sub-enzyme is mixed with a biological sample containing CYP1A for enzymatic The reaction quantitatively measures the substrate elimination rate per unit time or the rate of formation of the demethylated product to quantitatively determine the activity of CYP1A in different biological systems. The specific measurement methods and conditions are as follows:

A. System 1,8-naphthalimide type compounds as a ratio probe substrate; substrate concentration to select 1/10 ~ 10K m; single-point concentration is preferably measured K m of substrate;

B. In PBS buffer, the reaction temperature is between 20 and 60 ° C, preferably 37 ° C is the optimal reaction time; the incubation system pH is between 5.5 and 10.5, preferably pH 7.4 is the optimal reaction pH;

C. The reaction time is 5 to 120 minutes, and the reaction is terminated when the corresponding O-demethylation product of the above substrate reaches the limit of quantification and the substrate conversion rate does not exceed 20%;

D. The amount of substrate reduction or the amount of O-demethylated product produced per unit time was determined as an evaluation index of CYP1A activity.
The ratiometric fluorescent probe substrate for cytochrome oxidase CYP1A according to claim 2, wherein said biological system is recombinantly expressed CYP1A single enzyme, human or animal tissue preparation solution, various mammalian tissues Any of the cells and their preparations.
The use of a ratiometric fluorescent probe substrate for cytochrome oxidase CYP1A according to claim 2, characterized in that the fluorescent signal of the probe substrate and its demethylated product is detected by different detection wavelengths. The fluorescence detection conditions of the demethylated product and the substrate were as follows: the excitation wavelength was 450, 372 nm, and the maximum emission wavelength was 564, 452 nm, respectively.
Use of a ratiometric fluorescent probe substrate for cytochrome oxidase CYP1A according to claim 1, characterized in that the probe substrate can also be used for rapid screening of CYP1A inhibitors and quantitative evaluation of inhibitory ability.
The use of a ratiometric fluorescent probe substrate for cytochrome oxidase CYP1A according to claim 1, wherein the probe substrate is also used as a probe substrate for CYP1A in vivo and in whole animals. Individual and species differences in the metabolic enzyme CYP1A were assessed.