CN112552289B - Near-infrared fluorescent probe substrate of COMT and application thereof - Google Patents

Near-infrared fluorescent probe substrate of COMT and application thereof Download PDF

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CN112552289B
CN112552289B CN202011515633.8A CN202011515633A CN112552289B CN 112552289 B CN112552289 B CN 112552289B CN 202011515633 A CN202011515633 A CN 202011515633A CN 112552289 B CN112552289 B CN 112552289B
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王平
杨凌
王方园
葛广波
魏来
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Wuxi Eryun Technology Co ltd
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Abstract

The invention discloses a near-infrared fluorescent probe substrate of COMT and application thereof, wherein the probe substrate takes 7, 8-dihydroxycoumarin as a mother nucleus, a conjugated double bond is introduced at the C3 position of the probe substrate as a connecting group, and an electron withdrawing group EWG with hydrophilic/hydrophobic properties is connected, wherein the EWG group is DMIC, TCFC, NIFC or BTC. Compared with the prior art, the invention has the following advantages: (1) high specificity and sensitivity: the probe substrate can be specifically metabolized by COMT, and the product has good fluorescence emission characteristics. (2) Easy high-throughput detection: the kit can be used for measuring on various common fluorescent enzyme labeling instruments and clinical large biochemical instruments in laboratories, and can be used for batch detection by utilizing 96 or 386 microporous plates. (3) The anti-interference capability is strong: the methylation product of the probe substrate can emit fluorescence in a near infrared region, has better capability of resisting autofluorescence of biological matrix, and improves the accuracy of enzyme activity method quantification.

Description

Near-infrared fluorescent probe substrate of COMT and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and relates to a functional substrate and application thereof, in particular to a COMT near-infrared fluorescent probe substrate and application thereof.
Background
Catechol-O-methyltransferase (COMT, EC 2.1.1.6) is an important II-binding metabolic enzyme in the human body, and is capable of transferring the methyl group of S-adenosyl-L-methionine (SAM) to the Catechol group of a substrate. Human COMT exists in two major forms, soluble COMT (S-COMT) and membrane-bound COMT (MB-COMT). S-COMT is the predominant form of presence in peripheral tissues, including liver and blood, and plays an important role in the detoxification and metabolism of exogenous catechol compounds and catechol estrogens. MB-COMT is a form that exists mainly in the brain, and its physiological function is to metabolize inactive catecholamines neurotransmitters (such as dopamine, epinephrine and norepinephrine), thereby regulating various behaviors such as mood, sensation and cognitive functions of a human. A great deal of research shows that the high and low COMT activity is closely related to the occurrence and development of diseases such as cognitive dysfunction, schizophrenia, chronic pain, estrogen-induced breast cancer/pancreatic cancer and the like.
COMT has now become an important target for the treatment of parkinson's disease. The use of COMT inhibitors in combination with levodopa is a gold standard and effective strategy for the clinical treatment of parkinson's disease. However, the commercially available COMT inhibitors have the disadvantages of low bioavailability and great hepatotoxicity. Therefore, the COMT probe substrate with high sensitivity and strong specificity is developed, so that the COMT activity under different biological systems and different disease states can be effectively evaluated, a powerful technical support is provided for constructing a high-throughput screening system of the COMT inhibitor, the role of the COMT activity in related human diseases is expected to be further researched, and more efficient COMT related disease treatment drugs are found.
However, the COMT probe substrates currently in common use suffer from the following drawbacks: 1. the detection flux is low: the methylation products which are catalyzed and metabolized into two isomers by COMT need to be separated and enriched by means of HPLC and the like, and the analysis process is complicated and time-consuming; 2. weak anti-interference capability: common substrates are endogenous compounds and are easily interfered by biological matrixes; 3. poor stability: the substrate is easy to be oxidized and degraded in the reaction system, and the accuracy of enzyme activity detection is influenced. In recent years, the detection of target enzyme activity in biological systems by using small-molecule fluorescent probes has been widely applied in the biomedical field. The fluorescent probe molecule has the characteristics of high sensitivity, high specificity, high flux and high space-time resolution, and can realize accurate positioning and real-time dynamic detection of target enzymes on living cells or tissues and living bodies by combining a fluorescent imaging technology. In addition, the near-infrared fluorescent probe also has better tissue penetration capability, less phototoxicity and can effectively avoid the interference of self-absorption and autofluorescence of a biological sample. Therefore, the development of a specific COMT fluorescent probe substrate with the advantage of near infrared and the construction of a high-throughput detection system matched with the substrate have important practical values.
Disclosure of Invention
The technical problem to be solved is as follows: in order to overcome the defects of the prior art, a reaction substrate which can be metabolized by COMT to generate 8-methylated metabolites is obtained, the products have strong fluorescence emission in a near infrared region, and the fluorescence quantum yield of the products is higher by one order of magnitude than that of the substrates, so that the detection is easier, the enzyme activity of COMT is determined by detecting the reaction products, and the accurate quantitative analysis of COMT activity in different systems is realized; in view of this, the invention provides a near-infrared fluorescent probe substrate of COMT and applications thereof.
The technical scheme is as follows: the near-infrared fluorescent probe substrate of COMT takes 7, 8-dihydroxycoumarin as a mother nucleus, a conjugated double bond is introduced at the C3 position of the probe substrate to be used as a connecting group, and the probe substrate is connected with an electron-withdrawing group EWG with hydrophilic/hydrophobic properties, and the structural formula of the probe substrate is as follows:
Figure BDA0002847780440000021
wherein, EWG (electron withdrawing group) is DMIC (1-ethyl-3, 3-dimethyl indole), TCFC (3-cyano-5, 5-dimethyl malononitrile furan), NIFC (4-malononitrile chromone) or BTC (2-benzothiazole). The respective structural formulae are as follows:
Figure BDA0002847780440000022
preferably, the EWG group is TCFC (3-cyano-5, 5-dimethylmalononitrile furan).
The application of the near-infrared fluorescent probe substrate of COMT in the determination of COMT enzyme activity.
Preferably, the system source of the COMT comprises recombinant expression of COMT single enzyme, animal or human source cell, animal or human source tissue preparation.
Further, the application method is as follows:
step 1, placing COMT to be detected in 0.1mol/L Tris-HCl buffer solution, wherein the pH value of the buffer solution is 5.5-10.5; adding magnesium ions, S-adenosylmethionine and dithiothreitol into buffer solution, mixing uniformly, and performing incubation reaction for 3-5min at 20-60 deg.C;
step 2, adding a probe substrate into the reaction system in the step, wherein the substrate concentration is 1/10-10KmContinuing to react for 5-120min, and stopping the reaction when the 8-O-methylated products corresponding to the substrates reach the quantitative limit and the substrate conversion rate does not exceed 20%;
and 3, measuring the generation amount of the 8-O-methylation product of the probe substrate in unit time as an evaluation index of COMT activity.
Preferably, in the reaction system, the ratio of the amounts of the probe substrate, the magnesium ion and the S-adenosylmethionine is 1:1000: 40; the concentration of dithiothreitol in the system is 40 mmol/L.
Preferably, the substrate concentration in step 2 is 1-2Km。
Preferably, the reaction temperature is 37 ℃ and the reaction pH is 7.4.
Preferably, the amount of the 8-O-methylated product is measured by a fluorescence detector under the following conditions: the excitation wavelength is 470nm, and the maximum emission wavelength is 620 nm.
The near-infrared fluorescent probe substrate of the COMT is applied to screening of COMT enzyme inducer or activator and quantitative evaluation of inhibition or induction capability.
In addition, the probe substrate can also be used for evaluating the catalytic activity of COMT of different species and COMT mutants with different amino acid sequences, and further evaluating the capability of the COMT to metabolize catechol drugs.
The probe substrate can also be used for activity evaluation of metabolic enzyme COMT in human erythrocytes and individual or species difference evaluation, and can also be used for study on the time-space distribution and related biological functions of COMT by COMT activity imaging in living cells and tissues.
The COMT probe substrate is an OFF-ON type near infrared fluorescent probe, and is not easily interfered by a biological system matrix and impurities in the COMT activity detection process.
The reaction principle of the probe substrate of the present invention is as follows: the probe substrate leads the fluorescence emission wavelength to reach a near infrared region by introducing an electron-withdrawing group, so as to obtain a series of near infrared fluorescent probe molecules. The probe substrate was selectively catalyzed by COMT to the corresponding 8-O-methoxy metabolite and showed a fluorescent signal at 620 nm. The near-infrared fluorescent probe substrate is mixed with a biological sample containing COMT to carry out enzymatic reaction, and the activity of COMT in different samples is quantitatively determined by quantitatively detecting the generation rate of methylated metabolites in unit time. On the contrary, the probe substrate does not have the fluorescence property, so the fluorescence property of the 8-O-methoxy metabolite can realize the rapid and sensitive detection of the product through a fluorescence detector, thereby reflecting the activity of COMT enzyme. In addition, the probe substrate is used for quantitatively measuring the residual activity of the COMT enzyme in the presence of an inducer or an activator, so that the rapid screening of the inhibitor and the quantitative evaluation of the inhibition capacity are realized.
Has the advantages that: (1) high specificity and sensitivity: the probe substrate can be specifically metabolized by COMT, and the product has good fluorescence emission characteristics. (2) Easy high-throughput detection: the kit can be used for measuring on various common fluorescent enzyme labeling instruments and clinical large biochemical instruments in laboratories, and can be used for batch detection by utilizing 96 or 386 microporous plates. (3) The anti-interference capability is strong: the methylation product of the probe substrate can emit fluorescence in a near infrared region, has better capability of resisting autofluorescence of biological matrix, and improves the accuracy of enzyme activity method quantification.
Drawings
FIG. 1 is a high resolution mass spectrum of probe substrate TCFC (3-cyano-5, 5-dimethylmalononitrile furan-7, 8-dihydroxycoumarin) and its methylation product (3-cyano-5, 5-dimethylmalononitrile furan-7-hydroxy-8-methoxycoumarin);
FIG. 2 is a graph showing absorption spectra of TCFC (3-cyano-5, 5-dimethylmalononitrile furan-7, 8-dihydroxycoumarin) and its methylation product TCMFC (3-cyano-5, 5-dimethylmalononitrile furan-7-hydroxy-8-methoxycoumarin);
FIG. 3 is a graph of the emission spectra of TCFC and its methylated product, TCMFC;
FIG. 4 shows the quantitative standard curve (A) and the lower limit of detection (B) of COMT;
FIG. 5 shows the enzymatic kinetics of COMT catalyzed methylation of TCFC (3-cyano-5, 5-dimethylmalononitrile furan-7, 8-dihydroxycoumarin);
fig. 6 is a quantitative evaluation of COMT activity in different biological samples.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and substance of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
The equipment adopted by the invention and the types thereof are as follows: fluorescence emission/excitation Spectra were detected by Spectra Max I8 full-function microplate detector.
Example 1
Synthesis of TCFC:
3-aldehyde-7, 8-dihydroxycoumarin (0.41g,2mmol) and 2- (3-cyano-4, 5, 5-trimethylfuran-2 (5H) -alkylidene) malononitrile (TCF) (0.45g,2.3mmol) were dissolved in methanol (40ml), piperidine (100. mu.l) was added, the reaction leaves were heated under reflux for 2H, cooled and filtered, the cake was recrystallized from methanol, and the crude product was column chromatographed (dichloromethane/methanol) to give TCFC, 110mg of red solid, 15% yield. HRMS (TOF-ESI) m/z C21H13N3O5387.0855, mass spectrum shows M/z 386.0783[ M-H ]]-。
2. Optical properties of probe substrate TCFC and methylation product TCFMC:
(1) the probe substrate TCFC and its methylation product TCFMC were dissolved in a mixture of pH 7.4Tris-HCl buffer (50mM) and 1% formic acid-acetonitrile (v/v 1: 1).
(2) 200 μ L of the sample was placed in a transparent 96-well plate, and the absorption spectrum was scanned from 200nm to 800 nm. Then, 200. mu.L of the sample was placed in a black opaque 96-well plate, and the fluorescence emission spectrum from 500nm to 850nm was scanned with 470nm as the excitation wavelength, as shown in FIGS. 2 and 3.
3. Recombinant COMT single enzyme activity assay:
(1) the measurement system prepared in advance includes: pH 7.4Tris-HCl buffer (50mM), MgCl2(5mM), DTT (1mM), SAM (0.5mM) and human recombinant COMT monoose (6.5. mu.g/mL);
(2) after preincubation at 37 ℃ for 3 minutes, 2. mu.L of probe substrate TCFC (0.1,0.25,0.5,1.0,2.5 and 5.0. mu.M) with different concentrations were added to start the enzyme reaction, and the reaction was carried out at 37 ℃ for 6 minutes;
(3) adding 200 mu L of 1% glacial acetic acid-acetonitrile, and terminating the reaction after violent shaking;
(4) after centrifugation at 20,000 Xg for 10 minutes at 4 ℃ in a high-speed refrigerated centrifuge, 200. mu.L of the supernatant was put in a black opaque 96-well plate and subjected to fluorescence detection (Ex. at 470nm/Em. at 620 nm).
(5) The O-methylation rate of TCFC is expressed as the molar amount of methylation product formed per minute per mg of COMT protein, abbreviated to nmol/min/mg protein, based on the intensity of the fluorescence signal described above. The enzyme activity was determined by fitting a Mie's curve with GraphPad Prism 5.0 software.
4. In vitro determination of anti-interference Capacity of Probe substrate TCFC:
(1) a198. mu.L COMT metabolic reaction system was prepared in advance, which included Tris-HCl buffer (50mM) pH 7.4, MgCl2(5mM), SAM (0.5mM), human recombinant COMT monoose (6.5. mu.g/mL) and common amino acids at concentrations of 0.1mM each: glutamic acid (Glu), lysine (Lys), alanine (Ala), proline (Pro), tyrosine (Tyr), phenylalanine (Phe), valine (Val), Asn (asparagine), serine (Ser), threonine (Thr), arginine (Arg), tryptophan (Trp), isoleucine (Ile), histidine (His), glycine (Gly) pre-incubated for 3 minutes at 37 ℃ with shaking;
(2) 2 μ L of TCFC with a concentration of 250 μ M was added to the reaction system to initiate the reaction;
(3) after 6 minutes, adding 200 mu L of 1% glacial formic acid-acetonitrile, and terminating the reaction after violent shaking;
(4) after high-speed centrifugation at 20,000 Xg for 10 minutes at 4 ℃ in a high-speed refrigerated centrifuge, the supernatant was subjected to fluorescence detection (Ex. at 470nm/Em. at 620 nm).
TCFCs are specifically catalyzed by COMT and do not interfere with the common amino acids in the receptor.
5. Incubation time Standard Curve determination
The experiment is measured on a microplate reader by using a 96-well plate, the TCFC is 250 mu M, the COMT single enzyme is 6.5 mu g/mL, the Tris-HCl buffer solution with the pH value of 7.4 is 50mM, the total volume is 200 mu L, the Tris-HCl buffer solution is incubated at 37 ℃ for 20min, the measurement is carried out at intervals of 2min, when the reaction time is within 10min, the fluorescence intensity of the product and the incubation time present a good linear relation, and the R is2＞0.9。
Linear Range and lower detection limits for COMT Activity evaluation
The experiment is measured on an enzyme-linked immunosorbent assay (ELISA) instrument by using a 96-well plate, the TCFC is 500 mu M, the COMT single enzyme is 0.5 mu g/mL-14 mu g/mL, the Tris-HCl buffer solution with the pH value of 7.4 is 50mM, the total volume is 200 mu L, the mixture is incubated at 37 ℃ for 6min and then analyzed by the ELISA instrument, the background fluorescence when the COMT is not added is deducted from the fluorescence response value of each group, the result shows that the COMT concentration is in a range of 0.5 mu g/mL-14 mu g/mL, the good linear relation is presented with the fluorescence response value, and the R is20.99, and the lower detection limit of COMT was determined to be 0.5 μ g/mL (fig. 4).
COMT enzymatic kinetic assay
The assay was performed on a microplate reader using 96-well plates with substrate 0.1-5. mu.M, COMT-enzyme 6.5. mu.g/mL, Tris-HCl buffer 50mM pH 7.4, total volume 200. mu.L, incubation at 37 ℃ for 6min, addition of 200. mu.L of 1% glacial acetic acid-acetonitrile, and termination of the reaction after vigorous shaking. Detection conditions are as follows: the excitation wavelength is 470nm, and the maximum emission wavelength is 620 nm. The measured fluorescence intensity is converted into reaction rate by using the product marker, and the V of COMT single enzyme to the substrate TCFC can be obtainedmaxAnd Km(FIG. 5).
8. Quantitative evaluation of the activity of COMT in human liver S9 of different individuals
(1) Selecting 7 cases of human livers S9 of different individuals as COMT enzyme source, and oscillating for 3min at 37 ℃ according to the same metabolic reaction system;
(2) 2 μ L of TCFC with a concentration of 250 μ M was added to the reaction system to initiate the reaction;
(3) after 6min, adding 200 mu L of ice 1% formic acid-acetonitrile, and terminating the reaction after violent shaking;
(4) centrifuging at high speed at 20,000 Xg for 10min at 4 deg.C with a high-speed refrigerated centrifuge, collecting supernatant, and performing fluorescence detection (Ex.at 470nm, Em.at 620 nm). The metabolic rate of TCFC by human liver S9 was finally obtained for 7 individuals (fig. 6).

Claims (10)

  1. The near-infrared fluorescent probe substrate of COMT is characterized in that the probe substrate takes 7, 8-dihydroxycoumarin as a mother nucleus, a conjugated double bond is introduced at the C3 position of the probe substrate to serve as a connecting group, and an electron-withdrawing group EWG with hydrophilic/hydrophobic properties is connected, and the structural formula of the probe substrate is as follows:
    Figure FDA0002847780430000011
    wherein the EWG group is DMIC, TCFC, NIFC or BTC.
  2. 2. The COMT near-infrared fluorescent probe substrate of claim 1, wherein the EWG group is TCFC.
  3. 3. Use of a near-infrared fluorogenic probe substrate of COMT as defined in claim 1 for determining the activity of COMT enzyme.
  4. 4. Use according to claim 3, wherein the systemic source of COMT comprises recombinant expression of COMT single enzymes, animal or human cells, animal or human tissue preparations.
  5. 5. The use according to claim 3, characterized in that the method of application is as follows:
    step 1, placing COMT to be detected in 0.1mol/L Tris-HCl buffer solution, wherein the pH value of the buffer solution is 5.5-10.5; adding magnesium ions, S-adenosylmethionine and dithiothreitol into buffer solution, mixing uniformly, and performing incubation reaction for 3-5min at 20-60 deg.C;
    step 2, the above stepAdding a probe substrate into the reaction system of the step (C), wherein the substrate concentration is 1/10-10KmContinuing to react for 5-120min, and stopping the reaction when the 8-O-methylated products corresponding to the substrates reach the quantitative limit and the substrate conversion rate does not exceed 20%;
    and 3, measuring the generation amount of the 8-O-methylation product of the probe substrate in unit time as an evaluation index of COMT activity.
  6. 6. The use according to claim 5, wherein the ratio of the amounts of the probe substrate, magnesium ion, and S-adenosylmethionine in the reaction system is 1:1000: 40; the concentration of dithiothreitol in the system is 40 mmol/L.
  7. 7. Use according to claim 5, wherein the substrate concentration in step 2 is 1-2Km。
  8. 8. Use according to claim 5, wherein the reaction temperature is 37 ℃ and the reaction pH is 7.4.
  9. 9. The use according to claim 5, wherein the amount of 8-O-methylated products is determined by detecting with a fluorescence detector under the following conditions: the excitation wavelength is 470nm, and the maximum emission wavelength is 620 nm.
  10. 10. The COMT near-infrared fluorescent probe substrate of claim 1, in screening COMT enzyme inducer or activator and quantitatively evaluating the inhibition or induction ability.
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CN106467739A (en) * 2015-08-18 2017-03-01 中国科学院大连化学物理研究所 The specificity fluorescent probe of catechol O-methyltransferase and its application

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