WO2017041403A1 - Substrat de sonde fluorescente pour déterminer l'activité de la dipeptidyl-peptidase iv et utilisation de ce substrat - Google Patents

Substrat de sonde fluorescente pour déterminer l'activité de la dipeptidyl-peptidase iv et utilisation de ce substrat Download PDF

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WO2017041403A1
WO2017041403A1 PCT/CN2016/000476 CN2016000476W WO2017041403A1 WO 2017041403 A1 WO2017041403 A1 WO 2017041403A1 CN 2016000476 W CN2016000476 W CN 2016000476W WO 2017041403 A1 WO2017041403 A1 WO 2017041403A1
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substrate
dpp
probe substrate
alkylene
dipeptidyl peptidase
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PCT/CN2016/000476
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English (en)
Chinese (zh)
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杨凌
邹立伟
葛广波
王平
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中国科学院大连化学物理研究所
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Publication of WO2017041403A1 publication Critical patent/WO2017041403A1/fr

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    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase

Definitions

  • the invention belongs to the technical field of biomedicine, and particularly relates to a specific fluorescent probe substrate for determining dipeptidyl peptidase IV and application thereof.
  • Dipeptidyl peptidase IV (DPP-IV, EC 3.4.14.5) is a transmembrane serine protease present in dimeric form, widely distributed in mammalian kidney, liver, gastrointestinal, pancreatic epithelial cells and Vascular endothelial cells can also be present in dissolved form in plasma and cerebrospinal fluid.
  • DPP-IV can specifically catalyze the hydrolytic cleavage of the amino acid residue proline (Pro) or alanine (Ala) peptide bond at the 2nd position of the N-terminus of the polypeptide chain, ie, hydrolyze two amino acid residues Xa-Pro and Xa -Ala (Xa is any amino acid other than proline), thereby participating in the activation of various biologically active polypeptides in the body, and partially or completely inactivating various active polypeptides in the body, such as incretin, neuropeptide Y (neuropeptide Y), gastrin-releasing peptide (GRP), growth hormone-releasing hormone (GHRH), and the like.
  • a new inducible insulin-based DPP-IV inhibitor can increase glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) in vivo. Concentration, prolonged the action time, improved ⁇ - and ⁇ -cell dysfunction, and also has the effect of increasing insulin sensitivity, and has a low incidence of hypoglycemia, does not affect the characteristics of gastric emptying. Therefore, DPP-IV has become a new target for the treatment of type 2 diabetes. Studies have speculated that DPP-IV inhibitors may inhibit the development of atherosclerosis by inhibiting the formation of foam cells, and also inhibit the degradation of stromal cell-derived factor (SDF1-1 ⁇ ) and matrix P to ensure insulin concentration to provide a potential heart.
  • GLP-1 glucagon-like peptide-1
  • GIP glucose-dependent insulinotropic polypeptide
  • DPP-IV is underexpressed in melanoma, lung cancer, and prostate cancer, in oral and rectal cancer.
  • methods for measuring DPP-IV activity mainly include p-nitroaniline derivative substrate color development method and 4-methyl-7-aminocoumarin derivative fluorescent probe method.
  • the known fluorogenic substrates are all off-on probes, the single-enzyme selectivity is not high and is susceptible to interference by biological matrices, and the quantitative error is large. It is easy to get false when applied (such as screening of DPP-IV inhibitors). Positive or false negative results.
  • the blue shift/red shift of the emission spectrum of the ratio probe can be used for ratio detection, and the enzyme activity detection is performed by the fluorescence ratio method. The ratio is used as the signal parameter based on the fluorescence intensity at two different wavelengths of the enzyme substrate and the product.
  • the probe molecular prototype can be used as internal calibration to reduce the influence of illumination intensity, probe concentration, sample unevenness, instrument parameters, etc. on quantitative analysis.
  • this ratio type fluorescent probe Has better selectivity, sensitivity and dynamic response range. Therefore, the development of highly selective DPP-IV ratio type fluorescent probe reaction and its associated high-throughput detection method have important practical value.
  • the wavelengths are significantly different and the fluorescence quantum yield of the product is higher and easier to detect.
  • the probe reaction can be used to quantitatively evaluate the distribution and function of DPP-IV in various biological systems.
  • the invention provides a specific fluorescent probe substrate of dipeptidyl peptidase IV (DPP-IV), the probe
  • the needle substrate can be specifically catalyzed by DPP-IV to produce products with different fluorescent properties and the corresponding 4-aminonaphthalimide is formed.
  • the substrate has the following structural formula:
  • R is selected from C 2 -C 10 alkyl, -(C 1 -C 8 alkylene)-carboxyl, -(C 1 -C 8 alkylene)-ester, -(C 1 -C 8 Alkyl)-amino, -(C 1 -C 8 alkylene)-cyano, -(C 1 -C 8 alkylene)-nitro, -(C 1 -C 3 alkylene)-O- (C 1 -C 3 alkyl), carbocyclyl, -(C 1 -C 3 alkylene)-carbocyclyl, aryl, -(C 1 -C 3 alkylene)-aryl, heteroaryl a group, -(C 1 -C 3 alkylene)-heteroaryl, heterocyclic or -(C 1 -C 3 alkylene)-heterocyclyl.
  • the invention also provides an application for determining a specific fluorescent probe substrate of dipeptidyl peptidase IV (DPP-IV), which uses the dipeptidyl peptidase IV (DPP-IV) specific substrate, and
  • the peptidyl peptidase IV (DPP-IV) biological sample is mixed and subjected to an enzymatic reaction to quantitatively determine the DPP in different biological systems by quantitatively detecting the substrate elimination rate per unit time or the rate of formation of the dipeptidation product.
  • DPP-IV activity, specific determination methods and conditions are as follows:
  • A. GPAN is used as the ratio probe substrate in the system; the substrate concentration is selected from 1/10 to 10K m ;
  • reaction temperature is between 20 and 60 ° C; the incubation system pH is between 5.5 and 10.5;
  • the reaction time is 5 to 120 minutes, and the reaction is terminated when the corresponding N-dedipeptided product of the above substrate reaches the limit of quantitation and the substrate conversion rate does not exceed 20%;
  • the amount of substrate reduction per unit time or the amount of N-de-dipeptidation product produced was determined as an evaluation index of DPP-IV activity.
  • the substrate concentration at the time of single point measurement is preferably K m .
  • reaction temperature is preferably 37 ° C
  • incubation system pH is preferably pH 7.4.
  • the biological system is any one of recombinant expression of DPP-IV single enzyme, human or animal tissue preparation solution or various types of mammalian tissue cells and preparations thereof.
  • the fluorescent signal of the probe substrate and its dedipeptide-based product needs to be detected by different detection wavelengths, and the fluorescence detection conditions of the dipeptide-based product and the substrate are: excitation wavelength 430, 360 nm, maximum emission wavelength respectively It is 535, 455 nm.
  • the probe substrate can also be used for rapid screening of DPP-IV inhibitors and quantitative evaluation of inhibitory capacity.
  • the probe substrate can also be used as a probe substrate for the in vivo and overall DPP-IV of experimental animals to assess individual and species differences in the metabolic enzyme DPP-IV.
  • the invention provides a DPP-IV ratio type fluorescent probe reaction, wherein the probe substrate and the N-dedipeptided product thereof have fluorescent properties, and the two have different optical properties, and the fluorescence detection can be adopted. At the same time, the substrate and the product were detected rapidly and sensitively.
  • the fluorescence detection conditions of the N-dedipeptided product and the substrate were: excitation wavelength 430, 360 nm, and the maximum emission wavelength was 535, 455 nm, respectively.
  • 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 DPP-IV activity, and can be used for various recombinant DPP-IV, human and animal tissue preparation liquids and various types. Quantitative determination of DPP-IV activity in tissue cells; also as a probe substrate for DPP-IV in vivo and in vivo, to assess individual and species differences in the metabolic enzyme DPP-IV.
  • the probe substrate and the fluorescence detection method of the N-dedipeptide-based metabolite can also be used for rapid screening of DPP-IV inhibitors and quantitative evaluation of inhibitory ability.
  • this compound can be used to detect the activity of DPP-IV, especially suitable for the production of bacterial, insect cells, mammalian cells and yeast cloning expression systems.
  • GPAN can be highly specifically metabolized by DPP-IV into a metabolite, that is, an N-dedipeptide product.
  • the apparatus and model of the invention are: fluorescence emission/excitation spectroscopy is completed by Synergy H1 full-function microplate detector; 1 H-NMR spectrum is detected by nuclear magnetic resonance spectrometer (Avance II 400 MHz).
  • N-butylamine (1.10 g, 15 mmol) was added to a solution of 4-nitro-1,8-naphthalic anhydride (2.43 g, 10 mmol) in acetic acid (50 mL) at room temperature, and reacted at 100-110 ° C overnight.
  • the filter cake was washed with acetic acid and dried in vacuo to give compound 1 as a beige solid.
  • N-butyl-4-nitro-1,8-naphthalimide (1.49 g, 5 mmol) and tin dichloride dihydrate (6.77 g, 30 mmol) were added to a solution of ethanol (50 mL) and stirred at room temperature. After homogenization, concentrated hydrochloric acid (10 mL) was slowly added dropwise, and the reaction was carried out for 30 min at room temperature. 10% sodium carbonate solution to quench the reaction (40 mL), filtered, cake washed with water, and dried in vacuo to give compound 2, orange solid, a yield of 70-80%, ESI-MS m / z 269.1 [M + H] +.
  • N-butyl-4-amino-1,8-naphthalimide (100 mg, 0.37 mmol), Boc-Gly-Pro-OH (304.5 mg, 1.12 mmol), N-hydroxybenzotriazole at room temperature (151.3 mg, 1.12 mmol), 1-ethyl-(3-dimethylaminopropyl)carbonyldiimide hydrochloride (214.7 mg, 1.12 mmol), 2-(7-azobenzotriazine) Azole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (281.4 mg, 1.12 mmol) was added to a solution of N,N-dimethylformamide (5 mL) in sequence, thin-plate chromatography (TLC) monitors the reaction.
  • TLC thin-plate chromatography
  • the nuclear magnetic resonance spectrum of the prepared product is as follows:
  • the assay was performed on a microplate reader using a 96-well plate, GPAN 100 ⁇ M, DPP-IV single enzyme 0.1 ⁇ g, pH 7.4 PBS buffer 50 mM, total volume 200 ⁇ L, incubation at 37 ° C for 30 min, every 5 minutes.
  • the assay was performed on a microplate reader using a 96-well plate, GPAN 100 ⁇ M, DPP-IV single enzyme 0.5 ⁇ g/mL to 1.2 ⁇ g/mL, pH 7.4 PBS buffer 50 mM, total volume 200 ⁇ L, and incubation at 37 ° C for 1 h.
  • the average value of each group was compared with the control group without DPP-IV by enzyme microplate analysis.
  • the results showed that DPP-IV of 300 ng/mL was statistically significant (P ⁇ 0.05), so the detection of DPP-IV was determined.
  • the lower limit is 10 ng/mL ( Figure 6).
  • the assay was performed on a microplate reader using a 96-well plate, substrate 1-500 ⁇ M, DPP-IV single enzyme 0.25 mg/mL or intestinal microsome 2.5 mg/mL, pH 7.4 PBS buffer 100 mM, total volume 200 ⁇ L, 37
  • the incubation was carried out at ° C for 1 h by a microplate reader and measured every 1 minute.
  • Detection conditions excitation wavelength 430 nm, maximum emission wavelength was 535 nm.
  • the obtained fluorescence intensity was substituted into a standard curve to obtain Vmax and Km of DPP-IV single enzyme and human intestinal microsomal (HIM) to GPAN, respectively (Fig. 7).
  • HMM human liver microsomes
  • HIM human intestinal microsomes
  • HIM human intestinal microsomes
  • the substrate was added to the reaction system (final concentration 100 ⁇ M) to initiate the reaction; after reacting at 37 ° C for 30 minutes, 200 ⁇ l of acetonitrile was added, and the reaction was terminated after vigorous shaking;

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Abstract

La présente invention concerne un substrat de sonde fluorescente pour déterminer une activité de la dipeptidyl-peptidase IV (DPP-IV) et une utilisation de ce substrat. Ledit substrat de sonde est un dérivé amide C-4 de naphtalimides, GPAN, et peut être utilisé pour déterminer une activité enzymatique de la DPP-IV dans différents systèmes biologiques.
PCT/CN2016/000476 2015-05-14 2016-08-23 Substrat de sonde fluorescente pour déterminer l'activité de la dipeptidyl-peptidase iv et utilisation de ce substrat WO2017041403A1 (fr)

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CN201510572172.0A CN106146611B (zh) 2015-05-14 2015-09-10 一种测定二肽基肽酶iv活性的荧光探针底物及其应用

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CN115046977A (zh) * 2022-06-30 2022-09-13 重庆工商大学 一种荧光检测碳酸酐酶活性的方法
CN116693610A (zh) * 2023-06-16 2023-09-05 安阳师范学院 用于测定多肽连接酶的探针分子、测定转肽酶a的方法及应用

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WO2019117812A1 (fr) * 2017-12-12 2019-06-20 Nanyang Technological University Sondes moléculaires en proche infrarouge destinées à être utilisées dans le diagnostic de troubles fibrotiques et le criblage de médicaments anti-fibrotiques
CN110286105B (zh) * 2019-03-08 2021-10-19 华南农业大学 一种荧光探针及其制备方法
CN112574030B (zh) * 2020-12-01 2022-07-15 北京化工大学 一种用于检测生物酶的比率型单苯环荧光探针及其制备方法和应用
CN113218928A (zh) * 2021-05-21 2021-08-06 宁德师范学院 一种基于荧光探针的快速测定抑菌活性的比色方法

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WO2014018350A1 (fr) * 2012-07-23 2014-01-30 Merck Sharp & Dohme Corp. Traitement du diabète par administration d'inhibiteurs de dipeptidyl peptidase-iv

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115046977A (zh) * 2022-06-30 2022-09-13 重庆工商大学 一种荧光检测碳酸酐酶活性的方法
CN116693610A (zh) * 2023-06-16 2023-09-05 安阳师范学院 用于测定多肽连接酶的探针分子、测定转肽酶a的方法及应用
CN116693610B (zh) * 2023-06-16 2024-05-03 安阳师范学院 用于测定多肽连接酶的探针分子、测定转肽酶a的方法及应用

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