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Application of two-photon fluorescence probe for detecting cytochrome oxidase CYP3A4

A cytochrome and fluorescent probe technology, applied in the application field of two-photon fluorescent probes, can solve the problems of unclear central nervous system function, differences in cell structure distribution, etc., and achieve the effects of high sensitivity, sensitive detection, and easy detection.

Active Publication Date: 2019-06-25
DALIAN MEDICAL UNIVERSITY
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  • Abstract
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  • Claims
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Problems solved by technology

It is also distributed in bone marrow, lung, kidney and other organ tissues, and also found in brain tissue, but its role in the central nervous system is not yet clear
In addition, CYP3A4 is found in tumor cells, but there are differences in the distribution of cell structure. The application of CYP3A4 antibody detection shows that the cytoplasmic staining in normal breast tissue is 68%, while that in tumor cells is 100%, which may be related to the high metabolism of tumor cells. related

Method used

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  • Application of two-photon fluorescence probe for detecting cytochrome oxidase CYP3A4
  • Application of two-photon fluorescence probe for detecting cytochrome oxidase CYP3A4
  • Application of two-photon fluorescence probe for detecting cytochrome oxidase CYP3A4

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1. Synthesis of N-ethyl-1,8-naphthalimide

[0042] Add 5 mmol of 1,8-naphthalene anhydride and 5.5 mmol of ethylamine to 30 ml of ethanol, heat to reflux for 12 hours, cool, filter the solid, rinse the filter cake with ethanol, and dry to obtain a milky white solid (yield 63% ).

[0043]

[0044] 1 H NMR (500 MHz, CDCl3 ) δ 8.65 – 8.58 (m, 2H), 8.21 (d, J = 8.3 Hz,2H), 7.79 – 7.73 (m, 2H), 4.26 (q, J = 7.1 Hz, 2H), 1.34 (t, J = 7.1 Hz, 3H). 13 C NMR (125 MHz, CDCl 3 ) δ 163.93, 133.77, 131.52, 131.04, 128.05, 126.85, 122.72, 35.46, 13.34. HRMS calcd for [M+H] + 226.0863, found 226.0683.

[0045] Note: The compound N-ethyl-1,8-naphthalimide 1 H-NMR spectrum, 13 C-NMR spectra and high-resolution mass spectrograms such as figure 2 , 3 , 4 shown.

Embodiment 2

[0046] Example 2. Synthesis of N-benzyl-1,8-naphthalimide

[0047] Add 5 mmol of 1,8-naphthalene anhydride and 5.5 mmol of benzylmethylamine into 30 ml of ethanol, heat to reflux for 12 hours, cool, filter the solid, rinse the filter cake with ethanol, and dry to obtain a light milky white solid (yield 55%).

[0048]

[0049] 1 H NMR (500 MHz, CDCl 3 ) δ 8.61 (dd, J = 7.3, 0.9 Hz, 2H), 8.20 (d, J =8.3 Hz, 2H), 7.75 (t, J = 7.8 Hz, 2H), 7.55 (d, J = 7.2 Hz, 2H), 7.30 (t, J =7.4 Hz, 2H), 7.24 (d, J = 7.3 Hz, 1H), 5.39 (s, 2H). 13 C NMR (125 MHz, CDCl 3 )δ 164.13, 137.35, 133.96, 131.47, 131.32, 129.08, 128.45, 128.03, 127.52,126.88, 122.53, 43.54. HRMS calcd for [M+H] + 288.1019, found 288.1024.

[0050] Note: N-benzyl-1,8-naphthalimide 1 H-NMR spectrum, 13 C-NMR spectra and high-resolution mass spectrograms such as Figure 5 , 6 , 7 shown.

Embodiment 3

[0051] Example 3. In vitro determination of the selectivity of human recombinant CYP single enzyme

[0052] (1) Prepare 90 µL CYP metabolic reaction system in advance, including PBS buffer (100 mM) at pH 7.4, recombinant human CYP individual enzymes, and the final concentration of N-ethyl-1,8-naphthalimide is 10 μM, at 37 o Pre-incubation with shaking for 3 minutes under C condition;

[0053] (2) Add 10 µL of 10 mM NADP to the reaction system + initial reaction;

[0054] (3) After 40 minutes, add 50 µL of glacial acetonitrile and shake vigorously to terminate the reaction;

[0055] (4) Use a high-speed refrigerated centrifuge at 4 o C, 20,000 × g Under the conditions of high-speed centrifugation for 20 minutes, the supernatant was taken for fluorescence detection (E x = 450 nm, E m = 558 nm); the selectivity of recombinant human CYP3A4 enzyme is about 29 times higher than that of other single enzymes ( Figure 8 ).

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Abstract

The invention discloses application of a two-photon fluorescence probe for detecting cytochrome oxidase CYP3A4, and belongs to the technical field of biomedicine. The specific probe substrate can be used for measuring the enzyme activity of the CYP3A4 in a biological system. The procedure for determining CYP3A4 enzyme activity is as follows: Naphthalimide 4-position hydroxylation is selected as aprobe reaction, and the CYP3A4 enzyme activity in various biological samples can be determined by quantitatively detecting the amount of hydroxylated metabolites generated per unit time. The two-photon fluorescence probe can be used for quantitative evaluation of the CYP3A4 enzyme activity in the biological samples of different species and different individual sources, and quantitative determination of CYP3A4 activity in animal tissue cell culture fluids and cell preparations of different sources so as to realize evaluation of drug disposal capability of the important drug metabolizing enzymeCYP3A4. The two-photon fluorescence probe can also be used to rapidly screen inhibitors of the CYP3A4 in vitro, evaluate inhibitory ability of the inhibitors and detect the CYP3A4 activity in tumors,can detect the CYP3A4 activity in zebrafish, and can be used to detect drug-drug interaction of the CYP3A4 in vivo.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to the application of a two-photon fluorescent probe for detecting cytochrome oxidase CYP3A4. Background technique [0002] Cytochrome P450 is a superfamily of heme-thiolate proteins. It is a mixed-function oxidase system terminal oxidase expressed on the membrane of the endoplasmic reticulum. The metabolic activation of steroids and the in vivo metabolism of exogenous substances including drugs, carcinogens, and environmental pollutants all play an important role. [0003] CYP3A4 is an important subfamily of cytochrome P450, mainly distributed in hepatocytes, hepatic bile duct epithelial cells and jejunum villous columnar epithelial cells. Metabolizes about 90% of the drug. It is also distributed in bone marrow, lung, kidney and other organ tissues, and also found in brain tissue, but its role in the central nervous system is not yet clear. In addition, CYP3A4 is...

Claims

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Application Information

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IPC IPC(8): C07D221/14C09K11/06G01N21/64
Inventor 马骁驰冯磊宁静王超于振龙田象阁霍晓奎孙成鹏
Owner DALIAN MEDICAL UNIVERSITY
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