WO2016049998A1 - Substrat de sonde fluorescente pour détecter l'activité de la carboxylestérase intestinale humaine et ses utilisations - Google Patents

Substrat de sonde fluorescente pour détecter l'activité de la carboxylestérase intestinale humaine et ses utilisations Download PDF

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WO2016049998A1
WO2016049998A1 PCT/CN2015/000326 CN2015000326W WO2016049998A1 WO 2016049998 A1 WO2016049998 A1 WO 2016049998A1 CN 2015000326 W CN2015000326 W CN 2015000326W WO 2016049998 A1 WO2016049998 A1 WO 2016049998A1
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hce2
activity
reaction
substrate
human intestinal
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PCT/CN2015/000326
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Chinese (zh)
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杨凌
崔京南
冯磊
葛广波
刘兆明
宁静
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中国科学院大连化学物理研究所
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/68—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase

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  • 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.
  • Carboxylesterase 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.
  • CYP450 cytochrome P450
  • UHTs uridine diphosphate glucuronyltransferase
  • 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.
  • hCE2 carboxylesterase 1
  • hCE2 carboxylesterase 2
  • 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.
  • 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:
  • 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.
  • 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.
  • 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.
  • reaction temperature is between 20 ° C and 60 ° C; the incubation system pH is between 5.5 and 10.5;
  • 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%;
  • reaction temperature is between 20 ° C and 60 ° C; the incubation system pH is between 5.5 and 10.5;
  • 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.
  • fluorescence detection conditions are: excitation wavelength 560nm, fluorescence emission spectrum detection at 590 ⁇ 675nm.
  • 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.
  • 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:
  • 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.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Toxicology (AREA)
  • Materials Engineering (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

La présente invention concerne un substrat de sonde fluorescente permettant de détecter l'activité de la carboxylestérase intestinale humaine et ses utilisations, qui concernent le domaine technique des produits pharmaceutiques. Le substrat de sonde spécifique est un ester aromatique (TCFE en abrégé) de (E) -2- (4- (4-hydroxybenzène de vinyle) -3-cyan -5,5-diméthylfuranne -2 (5H)-sous-unité) malononitrile (TCF en abrégé). Le substrat peut être utilisé pour la détection quantitative rapide de l'activité carboxylestérase intestinale humaine et pour le criblage et l'estimation rapides d'un inhibiteur de la carboxylestérase intestinale humaine. Le procédé de détection quantitative rapide de l'activité de la carboxylestérase intestinale humaine consiste à : sélectionner une réaction d'hydrolyse TCFE comme sonde réactionnelle, ajouter un substrat approprié dans un système tampon comprenant hCE2, et déterminer l'activité réelle de l'enzyme hCE2 dans chaque échantillon d'organisme in vitro par détection quantitative de la production d'un produit du métabolisme d'hydrolyse TCF dans une unité de temps dans un intervalle de réaction linéaire. La sonde a une bonne sélectivité, le produit d'hydrolyse TCF a une longue longueur d'onde d'émission de fluorescence (612 nm), et la sonde peut réduire une fluorescence de fond de l'échantillon d'organisme et augmenter la sensibilité, et a une bonne possibilité d'exécution et de bonnes perspectives d'application.
PCT/CN2015/000326 2014-09-29 2015-05-13 Substrat de sonde fluorescente pour détecter l'activité de la carboxylestérase intestinale humaine et ses utilisations WO2016049998A1 (fr)

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CN201410513296.7 2014-09-29
CN201410513296.7A CN105441062B (zh) 2014-09-29 2014-09-29 一种人肠道羧酸酯酶活性检测的荧光探针底物及其应用

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CN106478576A (zh) * 2016-09-29 2017-03-08 华南理工大学 一种用于检测羧酸酯酶的荧光探针及其制备方法与应用
CN115745843A (zh) * 2022-11-10 2023-03-07 大连理工大学 一种cyp 1a1酶激活反应型荧光探针及其制备方法与应用

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CN107271432B (zh) * 2016-04-08 2019-10-11 中国科学院大连化学物理研究所 人羧酸酯酶1的生物发光检测试剂盒及其使用方法和应用
CN107298664A (zh) * 2017-07-11 2017-10-27 济南大学 一种分析汞离子的比色荧光探针、制备方法及应用
CN108558801A (zh) * 2018-05-30 2018-09-21 济南大学 一种长波长超灵敏一氧化碳比色荧光探针
CN114136941B (zh) * 2020-11-27 2024-04-16 北京大学深圳研究生院 一种sarm1酶抑制剂及其筛选方法和应用
CN114957380A (zh) * 2022-04-26 2022-08-30 青岛科技大学 一种测定谷氨酰转肽酶的水溶性荧光探针及其合成方法与应用

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Publication number Priority date Publication date Assignee Title
CN106478576A (zh) * 2016-09-29 2017-03-08 华南理工大学 一种用于检测羧酸酯酶的荧光探针及其制备方法与应用
CN106478576B (zh) * 2016-09-29 2019-01-18 华南理工大学 一种用于检测羧酸酯酶的荧光探针及其制备方法与应用
CN115745843A (zh) * 2022-11-10 2023-03-07 大连理工大学 一种cyp 1a1酶激活反应型荧光探针及其制备方法与应用
CN115745843B (zh) * 2022-11-10 2024-04-02 大连理工大学 一种cyp 1a1酶激活反应型荧光探针及其制备方法与应用

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