WO2017041403A1 - Fluorescent probe substrate for determining activity of dipeptidyl peptidase iv, and use thereof - Google Patents

Abstract

The present invention provides a fluorescent probe substrate for determining an activity of dipeptidyl peptidase IV (DPP-IV) and a use thereof. The probe substrate is a C-4 amide derivative of naphthalimides, GPAN, and can be used to determine an enzyme activity of DPP-IV in different biological systems.

Description

Fluorescent probe substrate for determining dipeptidyl peptidase IV activity and application thereof Technical field

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.

Background technique

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 α- and β-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 (SDF1-1α) and matrix P to ensure insulin concentration to provide a potential heart. Protection function (Diabetologia, 2012, 55, 2267-2275). In addition, DPP-IV is underexpressed in melanoma, lung cancer, and prostate cancer, in oral and rectal cancer. Low expression in serum, DPP-IV expression gradually decreased with the deterioration of endometrial malignant adenoma; however, the study found in primary lung cancer, prostate cancer, ovarian cancer, thyroid cancer, esophageal malignant adenoma The opposite result was obtained (Biotecnología Aplicada 2014, 31, 102-110). Therefore, the development of DPP-IV probe substrate with high sensitivity and specificity can not only better explore the important role of DPP-IV in related diseases, but also screen for DPP-IV targeted drugs. Quantitative determination of the activity of DPP-IV in biological systems provides strong technical support.

At present, 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. At this time, 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. Compared with traditional fluorescent probes, 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.

Summary of the invention

It is an object of the present invention to provide a specific fluorescent probe substrate for the determination of dipeptidyl peptidase IV (DPP-IV) and its use, the fluorescence emission of the ratiometric fluorescent probe substrate and the dedipeptided product 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:

Wherein 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. -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 ;

B. In PBS buffer, the reaction temperature is between 20 and 60 ° C; the incubation system pH is between 5.5 and 10.5;

C. 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%;

D. 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.

Among the specific measurement methods and conditions, the substrate concentration at the time of single point measurement is preferably K _m .

In the specific measurement method and conditions, the reaction temperature is preferably 37 ° C, and the 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.

As a highly specific DPP-IV single enzyme fluorescent probe substrate, 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. The enzyme activity assay of DPP-IV, as well as the activity calibration of DPP-IV in preparations of microsomes, S-9 and the like from various mammalian tissues and organs.

Detection of DPP-IV single enzyme in vitro by ratiometric fluorescent probe reaction of DPP-IV single enzyme according to the present invention Activity has the following outstanding advantages:

(1) High specificity: GPAN can be highly specifically metabolized by DPP-IV into a metabolite, that is, an N-dedipeptide product.

(2) Cheap and easy to obtain: GPAN can be obtained by chemical synthesis, and the synthesis process is simple and easy.

(3) Easy high-throughput detection: It can be measured on various fluorescent microplate readers and clinical biochemical analyzers in the laboratory. It can be tested in batches using 96 or 386 microplates.

(4) High sensitivity: Compounds with a 1,8-naphthalimide core structure have good fluorescence emission characteristics (450-700 nm), and the substrate and its N-de-peptidation metabolites are different. The characteristics of the fluorescence emission spectrum can be better distinguished and detected. At the same time, the quantitative determination of DPP-IV can be carried out by establishing the ratio standard curve. The detection limit is 10 ng/mL.

DRAWINGS

Figure 1. Structural formula of 4-(glycine-valine)-amino-1,8-naphthalimide-based compound;

Figure 2. Synthetic route of N-butyl-4-(glycine-valine)-amino-1,8-naphthalimide (GPAN);

Figure 3. ¹ H-NMR spectrum of N-butyl-4-(glycine-valine)-amino-1,8-naphthalimide (GPAN);

Figure 4. Selectivity of N-butyl-4-(glycine-valine)-amino-1,8-naphthalimide (GPAN);

Figure 5. Linear reaction time for DPP-IV catalyzed hydrolysis of GPAN;

Figure 6. Quantitative standard curve for DPP-IV;

Figure 7. Enzymatic kinetics of DPP-IV catalyzing the hydrolysis of GPAN;

Figure 8. Quantitative assessment of DPP-IV activity in human tissue microsomes;

Figure 9. Screening of DPP-IV inhibitors;

detailed description

The invention is further illustrated by the following examples, which are not intended to limit the invention.

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).

The structural formula of the 4-(glycine-valine)-amino-1,8-naphthalimide compound is shown in FIG.

Example 1

Synthesis of N-butyl-4-(glycine-valine)-amino-1,8-naphthalimide (GPAN)

The synthetic route of N-butyl-4-(glycine-valine)-amino-1,8-naphthalimide (GPAN) is shown in FIG.

(1) Synthesis of Compound 1

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.

(2) Synthesis of Compound 2

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] +.

(3) Synthesis of Compound 3

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. After 36 h of reaction, the reaction system was cooled to 0-5 ° C, water (25 mL) was added, and ethyl acetate was extracted three times (30 mL×3), and the organic phase was washed three times (15 mL×3), and washed with 5% sodium hydrogen carbonate solution (20 mL× 1), washed with water (15 mL × 1), saturated sodium chloride solution (20 mL × 1), dried over anhydrous sodium sulfate, evaporated to give a solvent. 3, a yellow oil, a yield of 65-75%, ESI-MS m / z 521.1 [MH] -.

(4) Synthesis of Compound 4

Compound 1 (128 mg, 0.23 mmol) was added to a mixed solution of dichloromethane (8 mL) and trifluoroacetic acid (2 mL) at room temperature, and the reaction was monitored by thin-plate chromatography (TLC). After 6 h of reaction, the reaction solvent was evaporated, the crude product was added to water (15 mL), and the mixture was adjusted to pH=7-8 with saturated sodium hydrogen carbonate solution and extracted three times with dichloromethane (25 mL×3), and the organic phase was washed with water (15 mL×1). saturated sodium chloride solution (20mL × 1), dried over anhydrous sodium sulfate, the solvent was evaporated, the crude product by column chromatography (dichloromethane / methanol / water = 20/5 / 0.5) to give compound 4 which ¹ H- The NMR spectrum is shown in Figure 3. The product was a pale yellow solid with a yield of 55-65%.

The nuclear magnetic resonance spectrum of the prepared product is as follows:

^{1 H NMR (400MHz, DMSO)} δ10.74 (s, 1H), 8.71 (d, J = 8.5Hz, 1H), 8.54 (d, J = 7.1Hz, 1H), 8.50 (d, J = 8.1Hz, 1H), 8.21 (brs, 2H), 8.17 (d, J = 8.1 Hz, 2H), 7.91 (t, J = 7.9 Hz, 1H), 4.99 - 4.71 (m, 1H), 4.05 (t, J = 7.3) Hz, 2H), 3.90 (s, 2H), 3.70-3.57 (m, 2H), 2.36-2.36 (m, 1H), 2.7-1.87 (m, 3H), 1.70-1.53 (m, 2H), 1.42 1.28 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H); ESI-MS m/z 423.1 [M+H] ⁺ .

Example 2.

Determination of the selectivity of human recombinant DPP-IV single enzyme in vitro

(1) Prepare a 198μLDPP-IV metabolic reaction system in advance, including PBS buffer pH 7.4 (50) mM), recombinant human DPP-IV monozyme (1 μg/mL), carbonic anhydrase (CA, 10 μg/mL), trypsin (trypsin, 10 μg/mL), pepsin (pepsin, 10 μg/mL), butyrylcholine Alkaline esterase (BChE, 10 μg/mL), acetylcholinesterase (AChE, 10 μg/mL), carboxylesterase (hCE1, hCE2, 10 μg/mL), bovine serum albumin (BSA, 10 μg/mL), human serum Albumin (HAS, 10 μg/mL) was pre-incubated for 3 minutes at 37 °C;

(2) adding 2 μL of a GPAN concentration reaction of 10 mM to the reaction system;

(3) After 30 minutes, 200 μL of ice acetonitrile was added, and after violent shaking, the reaction was terminated;

(4) After high-speed centrifugation at 20,000 × g for 20 minutes at 4 ° C in a high-speed refrigerated centrifuge, the supernatant was taken for fluorescence detection (Ex = 400 nm, Em = 535 nm); selection of recombinant human DPP-IV enzyme The highest sex is about 50 times that of other single enzymes (Fig. 4).

Example 3.

DPP-IV time standard curve determination

The assay was performed on a microplate reader using a 96-well plate, GPAN 100 μM, DPP-IV single enzyme 0.1 μg, pH 7.4 PBS buffer 50 mM, total volume 200 μL, incubation at 37 ° C for 30 min, every 5 minutes. Analysis, the ratio of the fluorescence intensity of the product to the fluorescence intensity of the substrate and the incubation time as a standard curve, each standard curve R ² > 0.99, indicating that the linear range of the standard curve is broad, can accurately quantify the DPP-IV content (Figure 5) .

Example 4.

Determination of the lower limit of detection of DPP-IV in vitro

The assay was performed on a microplate reader using a 96-well plate, GPAN 100 μM, DPP-IV single enzyme 0.5 μg/mL to 1.2 μg/mL, pH 7.4 PBS buffer 50 mM, total volume 200 μL, 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).

Example 5.

DPP-IV enzymatic kinetic determination

The assay was performed on a microplate reader using a 96-well plate, substrate 1-500 μM, 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 μL, 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).

Example 6.

Quantitative assessment of DPP-IV activity in human liver microsomes and human intestinal microsomes

(1) Dilute human liver microsomes (HLM) and human intestinal microsomes (HIM) to 2 mg/mL to prepare DPP-IV metabolic reaction system, including pH 7.4 PBS buffer (50 mM) and human liver microsomes (0.2). Mg/mL) and human intestinal microsomes (0.2mg/mL), pre-incubated for 3 minutes at 37 °C;

(2) adding 2 μL of a GPAN concentration reaction of 10 mM to the reaction system;

(3) After 30 minutes, 200 μL of ice acetonitrile was added, and after violent shaking, the reaction was terminated;

(4) After high-speed centrifugation at 20,000 × g for 20 minutes at 4 ° C in a high-speed refrigerated centrifuge, the supernatant was taken for fluorescence detection (Ex = 400 nm, Em = 535 nm), and the obtained fluorescence intensity was substituted into a standard curve. The metabolic rate of GPAN to human liver microsomes (HLM) and human intestinal microsomes (HIM) was obtained (Fig. 8).

Example 7.

DPP-IV inhibitor screening

The catalysis of GPAN was used as a probe reaction, and the in vitro incubation system of human intestinal microsomes was used to determine the pentacyclic triterpenoid glycyrrhetinic acid, 11-carbonyl-β-boswellic acid, ursolic acid, oleanolic acid, and betulinic acid. , IC _{50 of} inhibition of DPP-IV by betulin:

a. 200 microliter in vitro metabolic reaction system containing phosphate buffer with pH 7.4, human intestinal microsomal protein concentration of 10μg / ml, inhibitor final concentration range of 0.01μM-100μM, shock pre-incubation at 37 ° C 10 minutes;

b. The substrate was added to the reaction system (final concentration 100 μM) to initiate the reaction; after reacting at 37 ° C for 30 minutes, 200 μl of acetonitrile was added, and the reaction was terminated after vigorous shaking;

c. Using a high-speed refrigerated centrifuge, centrifuge the system at 20,000 × g for 5 minutes, then take the supernatant for detection and analysis by a microplate reader; quantitatively detect the metabolic hydrolysate.

It can be seen from the experimental data that betulinic acid and betulin have certain inhibitory activities against DPP-IV.

Table 1 IC _{50 of} pentacyclic triterpenoids against DPP-IV inhibition

Claims (8)

1. A specific fluorescent probe substrate for determining dipeptidyl peptidase IV, characterized in that the probe substrate can be specifically catalyzed by DPP-IV to undergo peptide bond hydrolysis reaction to form the corresponding 4-aminonaphthoquinone The structure of the probe substrate is as follows:

   

   Wherein R is selected from C ₂ -C ₁₀ alkyl, -(C ₁ -C ₈ alkylene)-carboxy, -(C ₁ -C ₈ alkylene)-ester, -(C1-C ₈ alkylene -amino, -(C ₁ -C ₈ alkylene)-cyano, -(C ₁ -C ₈ alkylene)-nitro, -(C ₁ -C ₃ alkylene)-0-( C ₁ -C ₃ alkyl), carbocyclyl, -(C ₁ -C ₃ alkylene)-carbocyclyl, aryl, -(C ₁ -C ₃ alkylene)-aryl,heteroaryl , -(C ₁ -C ₃ alkylene)-heteroaryl, heterocyclic or -(C ₁ -C ₃ alkylene)-heterocyclyl.
2. Use of a specific fluorescent probe substrate for assaying dipeptidyl peptidase IV according to claim 1, characterized by using a specific substrate of the dipeptidyl peptidase IV and a dipeptidyl-containing peptide The biological sample of the enzyme IV is mixed and subjected to an enzymatic reaction, and the activity of DPP-IV in different biological systems is quantitatively determined by quantitatively detecting the substrate elimination rate per unit time or the rate of formation of the dipeptide-based product, and the specific measurement method and The conditions are as follows:

   A. The probe substrate GPAN is added to the phosphate buffer system; the substrate concentration is 1/10 to 10 K _m ;

   B. The reaction temperature is between 20 and 60 ° C; the incubation system pH is between 5.5 and 10.5;

   C. 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%;

   D. 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.
3. Use according measured dipeptidyl peptidase IV of claim 2 specific fluorescent probe substrate of claim, wherein: the particular assay method and conditions: Substrate concentration is preferably K _m.
4. The use of a specific fluorescent probe substrate for assaying dipeptidyl peptidase IV according to claim 2, wherein the specific measurement method and conditions are: the reaction temperature is preferably 37 ° C, and the incubation system pH is preferably pH 7.4.
5. The use of a specific fluorescent probe substrate for assaying dipeptidyl peptidase IV according to claim 2, wherein said biological system is a recombinant single enzyme, human or animal tissue preparation solution containing DPP-IV, Any of various mammalian tissue cells and preparations thereof.
6. The use of a specific fluorescent probe substrate for assaying dipeptidyl peptidase IV according to claim 2, wherein the probe substrate and its dedipeptide-based product have different fluorescence emission spectra, which are different The detection wavelength was detected, and the fluorescence detection conditions of the product and the substrate were: excitation wavelength 430, 360 nm, and maximum emission wavelength were 535, 455 nm, respectively.
7. Use of a ratiometric fluorescent probe substrate for assaying dipeptidyl peptidase IV according to claim 1, wherein the probe substrate is also useful as a DPP in a cell or tissue sample of a different species. Quantitative detection of -IV enzyme activity.
8. Use of a specific fluorescent probe for assaying dipeptidyl peptidase IV according to claim 1, characterized in that the probe substrate can also be used for rapid screening of DPP-IV inhibitors or inducers, and Quantitative assessment of inhibition or induction capacity.

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