WO2015179999A1 - Substrat de sonde fluorescente de type à rapport constitué d'une cytochrome oxydase cyp1a et son utilisation - Google Patents

Substrat de sonde fluorescente de type à rapport constitué d'une cytochrome oxydase cyp1a et son utilisation Download PDF

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WO2015179999A1
WO2015179999A1 PCT/CN2014/000958 CN2014000958W WO2015179999A1 WO 2015179999 A1 WO2015179999 A1 WO 2015179999A1 CN 2014000958 W CN2014000958 W CN 2014000958W WO 2015179999 A1 WO2015179999 A1 WO 2015179999A1
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cyp1a
substrate
probe substrate
cytochrome oxidase
fluorescent probe
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PCT/CN2014/000958
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English (en)
Chinese (zh)
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杨凌
戴子茹
崔京南
葛广波
冯磊
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
    • C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
    • C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
    • C07D221/04—Ortho- or peri-condensed ring systems
    • C07D221/06—Ring systems of three rings
    • C07D221/14—Aza-phenalenes, e.g. 1,8-naphthalimide
    • 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
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase

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  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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:
  • 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;
  • 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%;
  • 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 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.
  • 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.
  • 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 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:
  • 1,8-naphthylimide compounds can be highly specifically metabolized by the cytochrome oxidase CYP1A single enzyme into a metabolite, that is, an O-demethylated product.
  • 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.
  • Figure 5.14 is a metabolic diagram of HLM versus N-(3-carboxypropyl)-4-methoxy-1,8-naphthalimide;
  • CYP1A mediates the metabolic pathway of N-(3-carboxypropyl)-4-methoxy-1,8-naphthalimide
  • 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.
  • the nuclear magnetic resonance spectrum of the prepared product is as follows:
  • the nuclear magnetic resonance spectrum of the prepared product is as follows:
  • HLM human liver microsomes
  • the assay was performed on a microplate reader using a 96-well plate, N-(3-carboxypropyl)-4-methoxy-1,8-naphthylenimine 10 ⁇ M, 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 ⁇ L, 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.
  • the assay was performed on a microplate reader using a 96-well plate, N-(3-carboxypropyl)-4-methoxy-1,8-naphthalimide 10 ⁇ M, 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 ⁇ L, 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.

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Abstract

L'invention concerne un substrat de sonde fluorescente de type à rapport constitué d'une cytochrome oxydase CYP1A et son utilisation, qui appartiennent au domaine technique de la biomédecine. Le substrat de sonde spécifique a la structure d'un acide hydroxy-naphthalimide-alcanoïque, et peut être utilisé pour déterminer l'activité enzymatique de la CYP1A dans un biosystème. Le processus de flux permettant de déterminer l'activité enzymatique de CYP1A est le suivant : le choix de la déméthylation des acides hydroxy-naphthalimide-alcanoïques à titre de réaction de sonde ; et la détermination de l'activité enzymatique de CYP1A dans divers échantillons biologiques par détection quantitative de la quantité de produit métabolique de déméthylation générée par unité de temps. La présente invention peut être utilisée pour l'évaluation quantitative de l'activité enzymatique de CYP1A dans les échantillons biologiques provenant de divers individus, et pour la détermination quantitative de l'activité enzymatique de CYP1A dans des milieux de culture de cellules tissulaires animales et des préparations de cellules provenant de différentes sources, de façon à obtenir l'évaluation de l'aptitude d'élimination du médicament de l'importante enzyme CYP1A métabolisant le médicament. En outre, la réaction de sonde peut également être utilisée pour un criblage rapide de l'inhibiteur de CYP1A in vitro et l'évaluation de sa capacité d'inhibition.
PCT/CN2014/000958 2014-05-30 2014-10-31 Substrat de sonde fluorescente de type à rapport constitué d'une cytochrome oxydase cyp1a et son utilisation WO2015179999A1 (fr)

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CN110878050A (zh) * 2019-12-26 2020-03-13 安徽大学 一种可用于双光子光动力治疗的多功能生物探针及其制备方法和用途
CN111848543A (zh) * 2020-07-27 2020-10-30 吉林大学 一种用于检测二价铅离子的比率型荧光探针及其制备方法
CN116199678A (zh) * 2023-02-10 2023-06-02 兰州大学 萘酰亚胺化合物、荧光传感材料及其应用

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CN106147750B (zh) * 2015-04-17 2019-03-05 中国科学院大连化学物理研究所 一种双光子比率荧光探针及其在检测β-半乳糖苷酶中的应用
CN107022349B (zh) * 2016-01-29 2019-10-11 中国科学院大连化学物理研究所 细胞色素氧化酶cyp1a1特异性荧光探针及其制备方法与应用
CN107153051A (zh) * 2016-03-03 2017-09-12 中国科学院大连化学物理研究所 细胞色素氧化酶1a1酶检测试剂盒及其使用方法和应用
CN105646349B (zh) * 2016-03-04 2018-10-26 中南大学 一种有机磷农药分子探针、其制备方法、应用方法及无机/有机复合稀土上转换纳米材料
CN105968170B (zh) * 2016-06-12 2018-10-30 安阳师范学院 一种二肽基肽酶iv的荧光探针底物及制备方法及应用
CN111228518A (zh) * 2020-02-19 2020-06-05 无锡艾德美特生物科技有限公司 一种在体检测cyp1a活性的探针底物及其应用
CN113804666B (zh) * 2021-09-17 2022-07-19 大连理工大学 基于cyp3a4酶抑制法的生鲜果蔬中农药残留的快速检测方法
CN114105979B (zh) * 2021-11-27 2022-12-23 大连医科大学附属第二医院 一种检测细胞色素氧化酶cyp3a的广谱型荧光探针的应用

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CS185273B1 (en) * 1977-02-17 1978-09-15 Jaroslav Kroupa Compounds based on naphthalimide and process for making them
CN102993263A (zh) * 2011-09-19 2013-03-27 中国科学院大连化学物理研究所 一类细胞色素p450 3a4酶的特异性探针底物及其应用
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110878050A (zh) * 2019-12-26 2020-03-13 安徽大学 一种可用于双光子光动力治疗的多功能生物探针及其制备方法和用途
CN110878050B (zh) * 2019-12-26 2023-02-14 安徽大学 一种可用于双光子光动力治疗的多功能生物探针及其制备方法和用途
CN111848543A (zh) * 2020-07-27 2020-10-30 吉林大学 一种用于检测二价铅离子的比率型荧光探针及其制备方法
CN111848543B (zh) * 2020-07-27 2022-04-01 吉林大学 一种用于检测二价铅离子的比率型荧光探针及其制备方法
CN116199678A (zh) * 2023-02-10 2023-06-02 兰州大学 萘酰亚胺化合物、荧光传感材料及其应用
CN116199678B (zh) * 2023-02-10 2023-08-18 兰州大学 萘酰亚胺化合物、荧光传感材料及其应用

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