Aromatase inhibitor high-throughput screening model based on fluorescence detection principle

An aromatase, high-throughput technology, applied in the field of pharmacology, can solve the problems of time-consuming and laborious, high experimental conditions, and difficulty in high-throughput screening.

Inactive Publication Date: 2013-06-12
CHINA PHARM UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] At present, there are many screening methods for aromatase inhibitors, such as isotope binding experiments, including TLC thin layer chromatography and [3H] incorporation method, however, isotope detection requires high experimental conditions and will da

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  • Aromatase inhibitor high-throughput screening model based on fluorescence detection principle
  • Aromatase inhibitor high-throughput screening model based on fluorescence detection principle
  • Aromatase inhibitor high-throughput screening model based on fluorescence detection principle

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Experimental program
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specific Embodiment approach

[0021] 1. Detection of aromatase activity

[0022] 1) Experimental materials

[0023] Estradiol detection kit (Cisbio, France), testosterone (Sigma-aldrich, USA), NADPH (Roche, USA), letrozole (Meilon, China), 384 low volume white plate (Corning, USA), human source Aromatase (BD, USA), pipette tip (Axygen, USA).

[0024] 2) Experimental steps

[0025] ●Carry out experiments on aromatase concentration gradient, incubation time, substrate concentration, and NADPH concentration, see Figure 1-4 .

[0026] Precisely weigh the compound to be tested, add DMSO solvent to form a mother solution, and then use the detection buffer to prepare the solution of the compound to be tested to the required concentration, the initial screening concentration is about 1×10 -3 mol / L.

[0027] ● Add 2 μl of aromatase solution, 2 μl of substrate solution, 4 μl of buffer or compound to be screened, and 2 μl of NADPH to each well of the reaction vessel. React at 37°C for 1 hour.

[0028] ●Add Es...

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Abstract

The invention relates to aromatase inhibitory activity high-throughput screening realized by means of a homogeneous time-resolved fluorescence technique. By detecting the fluorescence intensity ratio (665nm/610nm) of an excitation light irradiated reaction system and performing formula conversion, the inhibitory activity of a to-be-detected sample on aromatase can be determined. By means of high-throughput screening new model, the harm of a radioligand binding principle-based experimental method to laboratory personnel and environment can be avoided. And the high-throughput screening model provided in the invention has the advantages of high efficiency, rapidity, accuracy, and inexpensiveness.

Description

technical field [0001] The invention belongs to the field of pharmacology. A high-throughput screening model of aromatase inhibitors in vitro is constructed by using a homogeneous time-resolved fluorescence detection technology, which is used for high-throughput detection of aromatase inhibitory activity of test samples. Background technique [0002] Steroid A-ring aromatase mainly exists in ovary and placenta, but also in other tissues, such as brain, adrenal gland, testis, bone marrow, fat, skin, muscle and some tumor tissues. Aromatase takes androgen as a substrate, and with the participation of oxygen and NADPH, undergoes two hydroxylations at the 19-position of the substrate, and finally aromatizes the A ring to generate estrogen products. Inhibition or inactivation of aromatase activity can lead to a decrease in estrogen levels in the body, thereby interfering with estrogen-related physiological processes such as ovulation, implantation, etc., and can also cause estrog...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 严明吉金子苗靖姗向华张陆勇
Owner CHINA PHARM UNIV
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