阅读说明：本技术 一种检测细胞色素p450 2c9的近红外型荧光探针及其应用 (Near-infrared fluorescent probe for detecting cytochrome P4502C 9 and application thereof ) 是由 马骁驰 冯磊 于 2020-06-08 设计创作，主要内容包括：一种检测细胞色素P450 2C9的近红外型荧光探针及其应用,其属于生物医药技术领域。该特异性探针底物可用于测定生物体系中CYP2C9的酶活性。CYP2C9酶活性测定的流程如下：选择1,3-二氯-7-烷氧基-9,9-二甲基-2(9H)-吖啶酮O-脱烷基反应为探针反应,通过定量检测单位时间内底物1,3-二氯-7-烷氧基-9,9-二甲基-2(9H)-吖啶酮的消除量或其脱烷基代谢产物的生成量来测定各类生物样品中CYP2C9酶的活性。本发明可用于不同来源、不同种属、不同个体的生物样本中CYP2C9酶活的定量评估以及生物体中CYP2C9的成像,以期实现CYP2C9处置药物能力的评估。还可用于CYP2C9活性调节剂的体外快速筛选,评估药物及候选化合物等由于抑制或激活CYP2C9催化活性而导致药物药物相互作用的潜在性。(A near-infrared fluorescent probe for detecting cytochrome P4502C 9 and application thereof belong to the technical field of biological medicine. The specific probe substrate can be used for measuring the enzymatic activity of CYP2C9 in a biological system. The protocol for measuring CYP2C9 enzyme activity is as follows: selecting 1, 3-dichloro-7-alkoxy-9, 9-dimethyl-2 (9H) -acridone O The dealkylation reaction is a probe reaction, and the activity of the CYP2C9 enzyme in various biological samples is measured by quantitatively detecting the elimination amount of the substrate 1, 3-dichloro-7-alkoxy-9, 9-dimethyl-2 (9H) -acridone or the generation amount of dealkylation metabolites thereof in unit time. The invention can be used for CYP2C9 enzyme in biological samples of different sources, different species and different individualsQuantitative assessment of viability and imaging of CYP2C9 in organisms in an attempt to achieve an assessment of CYP2C9 ability to treat drugs. The method can also be used for in vitro rapid screening of CYP2C9 activity regulators, and evaluating the potential of drug-drug interaction caused by inhibiting or activating CYP2C9 catalytic activity of drugs, candidate compounds and the like.)

1. The application of the near-infrared fluorescent probe for detecting cytochrome P4502C 9 is characterized in that: the probe substrate can be specifically catalyzed by CYP2C9 to generate a corresponding dealkylation product, and the substrate has a 1, 3-dichloro-7-alkoxy-9, 9-dimethyl-2 (9H) -acridone structure, and the structural general formula is as follows:

Wherein R is methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, cyclobutyl, isobutyl, n-pentyl, cyclopentyl, phenyl or benzyl;

the probe is applied to the enzymatic reaction after being mixed with a biological sample by taking the probe as a substrate, and the activity of cytochrome P4502C 9 is quantitatively determined by quantitatively detecting the substrate elimination rate in unit time or the generation rate of dealkylation products or the change of a fluorescence imaging signal.

2. The use of the near-infrared fluorescent probe for detecting cytochrome P4502C 9 as claimed in claim 1, wherein: the specific measurement method and conditions for the quantitative detection are as follows:

A. 1, 3-dichloro-7-alkoxy-9, 9-dimethyl-2 (9H) -acridone is taken as a probe substrate in the system; the concentration of the substrate is selected from 1/10-10K_m；

B. In a buffer containing NADPH, the reaction temperature is between 20 ℃ and 60 ℃; the pH value of the incubation system is between 5.5 and 10.5;

C. the reaction time is 5-120 minutes, so that the dealkylation products corresponding to the substrates reach the quantitative limit, and the reaction is terminated when the substrate conversion rate does not exceed 20%;

D. measuring the decrease in the amount of the substrate or the amount of the dealkylated product produced per unit time as an index for evaluating the activity of cytochrome P4502C 9;

The fluorescent signals of the probe substrate and the dealkylation product thereof need to be excited by adopting the wavelength of 600nm, and the emission wavelength is 630-690 nm.

3. The use of the near-infrared fluorescent probe for detecting cytochrome P4502C 9 as claimed in claim 2, wherein: the concentration of the substrate in the single-point measurement is K_m(ii) a In NADPH-containing buffer, the reaction temperature was 37 ℃; the pH of the incubation system was 7.4.

4. The use of the near-infrared fluorescent probe for detecting cytochrome P4502C 9 as claimed in claim 2, wherein: the biological system is a recombinant expression enzyme, a human or animal tissue preparation sample, various mammalian cells and a preparation thereof.

5. The use of the near-infrared fluorescent probe for detecting cytochrome P4502C 9 as claimed in claim 1, wherein: the probe substrate is used for rapid screening of cytochrome P4502C 9 inhibitor or activator and quantitative evaluation of regulation capacity.

6. The use of the near-infrared fluorescent probe for detecting cytochrome P4502C 9 as claimed in claim 1, wherein: the probe substrate is used as an experimental animal carrier and a probe substrate of the whole cytochrome P4502C 9, and individual and species differences of the metabolic enzyme cytochrome P4502C 9 are evaluated.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a near-infrared fluorescent probe for detecting cytochrome P4502C 9(CYP2C9) and application thereof.

Background

Cytochrome P450 enzymes are a class of heme-thiolated protein superfamily that are widely distributed in nature. In human body, it can mediate the biotransformation of endogenous substances such as fatty acid, steroid hormone and cholesterol, and also play an important role in the metabolic clearance or biological activation of exogenous substances such as drugs, carcinogens and environmental pollutants.

CYP2C9 is a member of cytochrome P450 superfamily, and is mainly distributed in important human metabolic organs such as liver and intestinal tract. It is counted that CYP2C9 is involved in the metabolism of more than 20% of clinical commonly used drugs (more than 100 drugs), such as phenytoin, warfarin, tolbutamide, glipizide, diclofenac, losartan, etc., which are all substrates of CYP2C9 (Curr Med chem.2011,18: 667-. Due to the wide range of CYP2C9 substrates, it is common in the clinic for co-drugs to induce drug-drug interactions by modulating CYP2C9 activity. Notably, CYP2C9 mediates the metabolic clearance of drugs with narrow therapeutic windows such as phenytoin, warfarin, tolbutamide, etc. (Drug Metab Dispos.1996,24: 1401-2231403; blood.2018,132: 2230-2239; Drug Metab Dispos.2000,28: 354-359). Thus, inhibition of CYP2C9 metabolism by co-administered drugs is highly likely to result in increased drug exposure levels and the induction of serious drug side effects. Thus, the catalytic ability of CYP2C9 is closely related to the safe use of drugs. The real-time activity detection of CYP2C9 in biological samples and organisms is helpful for accurately characterizing the activity change level of CYP2C9, and quantitatively and qualitatively evaluating the CYP2C 9-mediated drug metabolism condition and the drug interaction potential.

The fluorescent molecular imaging technology has the characteristics of low interference degree of a biological system, real-time imaging, matching with high-flux detection equipment and the like, and has increasingly prominent application in-vitro detection of biological samples and in-vivo detection of biological targets. Therefore, the development of the high-selectivity near-infrared fluorescent probe of CYP2C9 and the high-throughput detection method matched with the same have important practical value.

Disclosure of Invention

The invention aims to provide a near-infrared fluorescent probe for detecting cytochrome P4502C 9(CYP2C9) and application thereof, wherein a substrate of the near-infrared fluorescent probe has no fluorescence, and can emit fluorescence in a near-infrared region after being excited by dealkylation products. The probe reaction can be used for quantitatively evaluating the expression and function level of CYP2C9 in various biological systems or organisms.

The invention provides a near-infrared fluorescent probe for detecting CYP2C9, which can be specifically catalyzed by CYP2C9 to generate a corresponding dealkylation product 1, 3-dichloro-7-hydroxy-9, 9-dimethyl-2 (9H) -acridone, wherein the structural general formula of the probe is shown as formula (1), and the structural general formula of the probe is shown as follows:

wherein R is methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, cyclobutyl, isobutyl, n-pentyl, cyclopentyl, phenyl or benzyl.

The 1, 3-dichloro-7-alkoxy-9, 9-dimethyl-2 (9H) -acridone compound has the characteristics of high selectivity of metabolic enzyme (mainly activated by CYP2C9 metabolism), easiness in detection of metabolic products, high sensitivity and the like.

The invention also provides a near-infrared fluorescent probe for detecting CYP2C9, which adopts the compound of the formula (1) as a specific substrate of CYP2C9 enzyme to carry out enzymatic reaction, and quantitatively determines the CYP2C9 activity in different biological systems (including recombinant expression enzyme, biological samples such as human or animal tissue preparations, various cells and preparations and the like) by quantitatively detecting the substrate elimination rate in unit time or the generation rate of dealkylation products thereof or the change of fluorescence imaging signals; the specific determination method comprises the following steps:

the system takes 1, 3-dichloro-7-alkoxy-9, 9-dimethyl-2 (9H) -acridone compounds as near-infrared probe substrates; the concentration of the substrate is selected from 1/10-10K_m(ii) a The substrate concentration at the time of single-point measurement is preferably K_m。

-in a buffer containing NADPH, the reaction temperature is between 20 ℃ and 60 ℃, preferably 37 ℃ for an optimal reaction time; the pH value of the incubation system is between 5.5 and 10.5, and the preferable pH value of 7.4 is the reaction pH value;

the reaction time is 5-120 minutes, so that the dealkylation products corresponding to the substrates reach the quantitative limit, and the reaction is terminated when the substrate conversion rate does not exceed 20%;

-measuring the amount of production of the dealkylated product per unit time (amount of decrease in the substrate) or the change in the fluorescence imaging signal as an index for evaluating the CYP2C9 activity.

The CYP2C9 near-infrared fluorescent probe and the application thereof have the advantages that the probe substrate does not have fluorescence, the dealkylation product has near-infrared fluorescence property, and a fluorescence detector can be adopted to realize the quick and sensitive detection of the product; the dealkylation product fluorescence detection conditions are respectively as follows: the excitation wavelength is 600nm, and the emission wavelength is 630-690 nm.

The specific probe substrate is a near-infrared fluorescent probe, is not easily interfered by a biological system matrix and impurities in the CYP2C9 activity detection process, and can be used for quantitative determination of the CYP2C9 enzyme activity in various recombinant CYP2C9 enzymes, human and animal cell and tissue preparations; meanwhile, the probe can also be used as a probe substrate of the whole CYP2C9 of an in-vivo animal and an animal, and the individual and species difference of the metabolic enzyme CYP2C9 is evaluated. The fluorescence detection method of the probe substrate and the dealkylation metabolite can also be used for rapid screening of CYP2C9 activity regulator (inhibitor or activator) and quantitative evaluation of regulating ability.

Experiments such as correlation analysis, recombinant single enzyme metabolic reaction, specificity inhibition and the like prove that the 1, 3-dichloro-7-alkoxy-9, 9-dimethyl-2 (9H) -acridone compound can be specifically converted by CYP2C9 to generate the dealkylation product 1, 3-dichloro-7-hydroxy-9, 9-dimethyl-2 (9H) -acridone.

As a near-infrared fluorescent probe substrate with high CYP2C9 specificity, the compound can be used for detecting the activity calibration of CYP2C9 in various preparations such as tissue slices, cells, microsomes, S9 and the like of mammal sources in various clone expression systems.

The near-infrared fluorescent probe reaction for detecting the CYP2C9 enzyme activity of the CYP2C9 has the following outstanding advantages:

(1) high specificity: 1, 3-dichloro-7-alkoxy-9, 9-dimethyl-2 (9H) -acridones can be metabolized by CYP2C9 with high specificity to a metabolite, namely dealkylation product 1, 3-dichloro-7-hydroxy-9, 9-dimethyl-2 (9H) -acridones.

(2) The method is cheap and easy to obtain: the 1, 3-dichloro-7-alkoxy-9, 9-dimethyl-2 (9H) -acridone compounds can be obtained by chemical synthesis, the synthesis process is simple and feasible, and the detection cost of the fluorescence method is low.

(3) High sensitivity: the dealkylation product of the 1, 3-dichloro-7-alkoxy-9, 9-dimethyl-2 (9H) -acridone compound has good fluorescence spectrum characteristics, can emit fluorescence in a near infrared region after being excited, and can well reduce background fluorescence interference in a biological system.

Drawings

FIG. 1 shows the general structural formula of 1, 3-dichloro-7-alkoxy-9, 9-dimethyl-2 (9H) -acridone compounds.

FIG. 2 preparation of 1, 3-dichloro-7-methoxy-9, 9-dimethyl-2 (9H) -acridone¹H-NMR spectrum.

FIG. 3 preparation of 1, 3-dichloro-7-methoxy-9, 9-dimethyl-2 (9H) -acridone¹³C-NMR spectrum.

FIG. 4 is a high resolution mass spectrum of 1, 3-dichloro-7-methoxy-9, 9-dimethyl-2 (9H) -acridone.

FIG. 5 results of human CYP recombinant single enzyme screening assay for 1, 3-dichloro-7-methoxy-9, 9-dimethyl-2 (9H) -acridone

FIG. 6 results of human liver microsome activity assay of 1, 3-dichloro-7-methoxy-9, 9-dimethyl-2 (9H) -acridone

FIG. 7 is a standard curve of CYP2C9 protein concentration

FIG. 8 is a diagram of laser confocal imaging of cells.

Figure 9 high throughput screening for modulators of CYP2C9 activity.

FIG. 10 is a synthetic route for 1, 3-dichloro-7-methoxy-9, 9-dimethyl-2 (9H) -acridone.

Detailed Description

The following examples further illustrate the invention but are not intended to limit the invention thereto.

Example 1, 3-dichloro-7-methoxy-9, 9-dimethyl-2 (9H) -acridone

61.6mg (0.2 mmol)) The 1, 3-dichloro-7-hydroxy-9, 9-dimethyl-2 (9H) -acridone was dissolved in 30mL of acetonitrile, 69.5mg (0.5mmol) of potassium carbonate and 31. mu.L (0.5mmol) of methyl iodide were added, the mixture was refluxed for 8 hours and then cooled to room temperature, filtered, the acetonitrile was distilled off under reduced pressure, the remaining solid was dissolved in dichloromethane, and the solution was separated by thin layer chromatography on silica gel using a developing solvent of dichloromethane: n-hexane 1:1 (vol/vol), 15.6mg of an orange solid was obtained as 1, 3-dichloro-7-methoxy-9, 9-dimethyl-2 (9H) -acridone. The data are represented by a nuclear magnetic resonance hydrogen spectrum, a nuclear magnetic resonance carbon spectrum and a high-resolution mass spectrum, and are as follows: ¹H NMR(600MHz,CDCl₃)δ7.63(s,1H),7.61(d,J＝8.7Hz,1H),7.00(d,J＝2.7Hz,1H),6.92(dd,J＝8.7,2.7Hz,1H),3.92(s,3H),1.88(s,6H).¹³C NMR(150MHz,CDCl₃) Delta 173.22,163.06,147.47,140.66,139.44,136.62,135.86,134.47,134.14,113.13,112.94,55.80,39.15,26.82 HRMS (ESI positive) theoretical value [ M + H]⁺322.0396, found 322.0395.

Note: process for preparing 1, 3-dichloro-7-methoxy-9, 9-dimethyl-2 (9H) -acridone¹H-NMR spectrum,¹³The C-NMR spectrum and the high-resolution mass spectrum are shown in figures 2, 3 and 4.

Example 3 human recombinant CYP Single enzyme incubation experiments

(1) Preparing a CYP metabolic reaction incubation solution in advance, wherein the CYP metabolic reaction incubation solution comprises a phosphate buffer solution (100mM) with pH 7.4 and human recombinant CYP monoenzyme, and the concentration of 1, 3-dichloro-7-methoxy-9, 9-dimethyl-2 (9H) -acridone is 10 mu M, and shaking the solution for pre-incubation for 3 minutes at 37 ℃;

(2) to the reaction system was added 20. mu.L of NADP at a concentration of 10mM⁺Carrying out solution initial reaction;

(3) after reacting for 30 minutes, adding 100 mu L of glacial acetonitrile, and terminating the reaction after violent shaking;

(4) after centrifuging the sample at 4 ℃ at 20,000 Xg for 20 minutes at high speed, the supernatant was collected and subjected to fluorescence detection (E)_x＝600nm，E_m＝658nm)。

Example 4 quantitative evaluation of CYP2C9 Activity in human liver microsomes from different sources

(1) Selecting multiple individual Human Liver Microsomes (HLM), adding into reaction system for in vitro incubationAnd (5) carrying out experiments. The incubation system included phosphate buffer (100mM) pH 7.4, human liver microsomes, glucose-6-phosphate (10mM), glucose-6-phosphate dehydrogenase (1unit/mL), MgCl ₂(4mM), 1, 3-dichloro-7-methoxy-9, 9-dimethyl-2 (9H) -acridone (10. mu.M), and pre-incubating for 3 minutes at 37 ℃ with shaking;

(2) to the reaction system was added 20. mu.L of NADP at a concentration of 10mM⁺Starting reaction;

(3) after 30 minutes, adding 100 mu L of glacial acetonitrile, and terminating the reaction after violent shaking;

(4) after centrifugation at 20,000 Xg for 20 minutes at 4 ℃ the supernatant was collected and subjected to fluorescence detection (E)_x＝600nm，E_m658nm), the rate of the HLM catalyzed the dealkylation reaction of 1, 3-dichloro-7-methoxy-9, 9-dimethyl-2 (9H) -acridone was determined (fig. 6).

Example 5 CYP2C9 protein concentration Standard Curve assay

The incubation system included phosphate buffer (100mM) pH 7.4, human liver microsomes, glucose-6-phosphate (10mM), glucose-6-phosphate dehydrogenase (1unit/mL), MgCl₂(4mM), 1, 3-dichloro-7-methoxy-9, 9-dimethyl-2 (9H) -acridone (10. mu.M), and 1.25 to 12.5pmol/mL of the CYP2C9 monoose. After incubation at 37 ℃ for 30min, fluorescence detection was performed by a microplate reader (Ex ═ 600nm, Em ═ 658nm) to obtain a standard curve of fluorescence intensity of the dealkylated product versus concentration of CYP2C9 protein (fig. 7).

Example 6 confocal imaging of cell laser

LoVo and HepG2 cells were cultured in RPMI-1640 medium at 1X 10 ⁵The cells were seeded in six well plates at 37 ℃ with 5% CO₂Incubate under conditions overnight. After discarding the suspension cells, the probe substrate 1, 3-dichloro-7-methoxy-9, 9-dimethyl-2 (9H) -acridone was added and incubated at 37 ℃ for 1 hour. The residual probe was subsequently washed with phosphoric acid buffer solution and photographed by means of a laser confocal microscope (E)_x＝633nm，E_m650 and 690 nm). (FIG. 8)

Example 7 high throughput screening study of modulators of CYP2C9 Activity

(1) Using human liver microsomes, recombinant monoenzymes, etcThe enzyme source was subjected to in vitro incubation experiments. The incubation system included phosphate buffer pH 7.4 (100mM), glucose-6-phosphate (10mM), glucose-6-phosphate dehydrogenase (1unit/mL), MgCl₂(4mM), 1, 3-dichloro-7-methoxy-9, 9-dimethyl-2 (9H) -acridone (10. mu.M). Fully and uniformly mixing the pre-screened compound with an incubation system, adding the mixture into a 96-well plate, and oscillating the mixture at the temperature of 37 ℃ for pre-incubation for 3 minutes;

(2) to the reaction system was added 20. mu.L of NADP at a concentration of 10mM⁺Starting reaction;

(3) after 30 minutes, 100. mu.L of glacial acetonitrile is added to terminate the reaction;

(4) fluorescence detection by means of a multispectral laser imager (E)_x＝635nm，E_m670nm), the fluorescence intensity of each well sample was measured (fig. 9).