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Application of broad-spectrum fluorescent probe for detecting cytochrome oxidase CYP3A

A cytochrome and fluorescent probe technology, applied in the field of two-photon fluorescent probes, can solve the problems of simultaneous detection of fluorescent probes, inability to effectively evaluate CYP3A inhibitory behavior, and inability to accurately reveal the inhibitory effect, and achieve the effect of sensitive detection

Active Publication Date: 2022-03-01
THE SECOND HOSPITAL OF DALIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, fluorescent probes that detect CYP3A4 or CYP3A5 alone, but not both, cannot effectively assess the mechanism-based inhibitory behavior of CYP3A
In other words, measurements of CYP3A4 or CYP3A5 activity alone may not accurately reveal true mechanism-based inhibitory effects in the physiological context

Method used

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  • Application of broad-spectrum fluorescent probe for detecting cytochrome oxidase CYP3A
  • Application of broad-spectrum fluorescent probe for detecting cytochrome oxidase CYP3A
  • Application of broad-spectrum fluorescent probe for detecting cytochrome oxidase CYP3A

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1. the synthesis of compound BN-1

[0039] 1,8-Naphthalic anhydride (990.85 mg, 5.0 mmol) was dissolved in 20 mL of glacial acetic acid, o-phenylenediamine (648.84 mg, 6.0 mmol) was added, and the reaction solution was stirred and refluxed for 4 h. TLC traced the end of the reaction, and the reaction solution was allowed to stand and cooled to room temperature, and a large amount of solids were precipitated, filtered, and the filter cake was washed with ethanol to obtain a crude product, which was further separated by silica gel column (developing agent was dichloromethane) to obtain compound BN-1 (1272.07 mg, yield 94.2%) as a yellow-green solid.

[0040] BN-1: 1 H NMR (500MHz, CDCl 3 )δ8.76–8.71(m,1H),8.68(dd,J=7.3,0.9Hz,1H),8.54–8.45(m,1H),8.17(d,J=8.1Hz,1H),8.04(d ,J=8.1Hz,1H),7.87–7.81(m,1H),7.72(dd,J=15.9,8.3Hz,2H),7.49–7.41(m,2H). 13 C NMR (125MHz, CDCl 3 )δ160.58, 149.22, 143.74, 135.19, 132.15, 131.82, 131.75, 131.56, 127.27, 127.06, 126.80, 125.7...

Embodiment 3

[0042] Embodiment 3. In vitro determination of the selectivity of human recombinant CYP single enzyme

[0043] Prepare 180 μL CYP metabolic reaction system in advance, including Buffer buffer (100mM) at pH 7.4, glucose-6-phosphate (10mM), glucose-6-phosphate dehydrogenase (1unit / mL), MgCl 2 (4mM), recombinant human CYP single enzyme, the final concentration of BN-1 is 10μM, pre-incubated with shaking at 37°C for 3 minutes; add 20μL of 10mM NADP to the reaction system + Initiate the reaction; after 60 minutes, add 100 μL of glacial acetonitrile, shake vigorously, and terminate the reaction; use a high-speed refrigerated centrifuge to centrifuge at 20,000×g for 20 minutes at 4°C, take the supernatant, and perform fluorescence detection ( E. x =470nm,E m = 526nm).

[0044] From Figure 5 It can be seen that the probe has good selectivity to recombinant human CYP3A4 and CYP3A5 enzymes, and the fluorescence response to CYP3A4 and CYP3A5 is close, while other enzymes of the CYP ...

Embodiment 4

[0045] Example 4. Determination of CYP3A protein concentration standard curve

[0046] The experiment was carried out on a microplate reader using a 96-well plate, 10 μM BN-1, 6-phosphate glucose (10 mM), glucose-6-phosphate dehydrogenase (1 unit / mL), MgCl 2 (4mM)NADP + (1mM), CYP3A4 and CYP3A5 single enzyme (0-15nM), pH 7.4 Buffer buffer 100mM, total volume 200μL, incubated at 37℃ for 30min, the ratio of the fluorescence intensity of the product to the fluorescence intensity of the substrate and the protein concentration standard curve line.

[0047] Figure 6 Middle a) is the fluorescence intensity curve catalyzed by different concentrations of CYP3A4, b) is the linear relationship between CYP3A4 protein concentration and fluorescence intensity; c) is the fluorescence intensity curve catalyzed by different concentrations of CYP3A5, d) is the linear relationship between CYP3A5 protein concentration and fluorescence intensity . The results indicated that the probe substrat...

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Abstract

The invention discloses application of a broad-spectrum fluorescent probe for detecting cytochrome oxidase CYP3A, and belongs to the technical field of biological medicines. The specific probe substrate can be used for determining the enzymatic activity of CYP3A in a biological system. The CYP3A enzyme activity determination process comprises the following steps: selecting a BN compound 4-site hydroxylation reaction as a probe reaction, and determining the CYP3A enzyme activity in various biological samples by quantitatively detecting the generation amount of hydroxylation metabolites in unit time. The BN series probe can be used for simultaneously detecting the activity of CYP3A4 and CYP3A5, and is a broad-spectrum probe for detecting the activity of CYP3A, so that efficient discovery and deep research related to CYP3A based on a mechanism inhibitor can be realized. The probe can also be used for rapidly screening reversible and irreversible inhibitors, activators and inducers of CYP3A in vitro, and can be used for detecting drug-drug interaction of CYP3A on a living body level.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a two-photon fluorescent probe for detecting cytochrome oxidase CYP3A and an application thereof. Background technique [0002] Cytochrome P450 is a superfamily of heme-thiolate proteins, which play an important role in the metabolism of various endogenous and exogenous substances such as drugs, carcinogens, and environmental pollutants. CYP3A is an important subfamily of cytochrome P450. CYP3A4 and CYP3A5 are the core members of the human CYP3A subfamily. The expression of the two accounts for more than 30% of the total P450 content in the liver, and participates in the phase I metabolism of more than 50% of clinical drugs. , is one of the key metabolic targets that mediate drug metabolism in the human body. [0003] CYP3A4 and CYP3A5 have large substrate cavities, have a good tolerance for substrates with various structural skeletons, and have a wide spectrum of...

Claims

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Application Information

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IPC IPC(8): C07D471/04C09K11/06C12Q1/26G01N21/64
CPCC07D471/04C09K11/06G01N21/6428G01N21/6456C12Q1/26C09K2211/1044G01N2333/90245
Inventor 马骁驰宁静冯磊崔京南田镇豪霍晓奎田象阁于振龙王博
Owner THE SECOND HOSPITAL OF DALIAN MEDICAL UNIV
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