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Method for screening I type 17 beta-hydroxysteroid dehydrogenase inhibitor through immobilized enzyme

A technology of dehydrogenase inhibitor and immobilized enzyme is applied in the field of screening I type 17β hydroxysteroid dehydrogenase inhibitors, which can solve the problems of failure of screening test, difficulty in obtaining raw materials, and easy aggregation of free aromatase.

Active Publication Date: 2015-06-10
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the method for measuring the enzyme activity of 17β-HSD1 often adopts the substrate radiolabeling method (Insulin-like growth factor type I and insulin-like growth factor type II stimulate oestradiol-17βhydroxysteroid dehydrogenase (reductive) activity in breast cancer cells[J] .Singh A, Reed MJ, JEndocrinol, 1991,129: R5-R8.), but this method uses radioactive substances, which brings hidden dangers to post-processing and personnel protection
In addition to the above disadvantages, the free aromatase model also has the following obvious defects: (1) The enzyme source is not easy to come by. Since there is no commercial aromatase supply, each screening test needs to find fresh human placenta to prepare microparticles As the enzyme source of aromatase, it is difficult to obtain raw materials; (2) the artificially extracted free enzyme is stored under harsh conditions and is easily inactivated; (3) free aromatase is insoluble in organic solvents, and many small molecules to be screened The compound is not soluble in water, and organic solvent or emulsifier must be added, thereby affecting the enzyme activity; (4) free aromatase is easy to aggregate during the reaction, so that the enzyme molecule and the substrate molecule cannot be fully contacted, and the catalytic efficiency is low, resulting in screening The test failed; (5) The activity of free aromatase is unstable, and it is easily inactivated by environmental factors such as temperature, pH, etc.
(6) It is difficult to separate the enzyme and product after the reaction, and it cannot be recycled and reused
So far, there is no research report on the immobilization of 17β-HSD1 at home and abroad

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: the preparation of immobilized enzyme

[0034] 1) Extraction of 17β-HSD1

[0035] All steps were carried out on ice at 0-4°C. Remove the chorion and large blood vessels from the placenta, wash it with 50mM PBS (pH=7.4, containing 1% KCl), cut it with scissors, and homogenize it with 50mM PBS (containing 0.25M sucrose). Centrifuge at 2500×g for 30min, discard the precipitate, and centrifuge the supernatant at 60000×g for 60min to precipitate mitochondrial components. Discard the precipitate again, add a certain amount of recrystallized ammonium sulfate solid to the supernatant, the precipitate accumulates, discard the supernatant after centrifugation, and resuspend the precipitate in 50mM PBS (containing 1mM EDTA and 1mM cysteine ​​hydrochloride) , and stored at -80°C.

[0036] 2) Preparation of amino-modified silicon spheres

[0037] Take 200 mg of silica gel with a particle size of 5 μm, add 20 mL of 10% hydrochloric acid, reflux at 90° C. for 12 hour...

example 2

[0040] Example 2: Enzymatic Properties of Immobilized Enzymes

[0041] 1) Optimum reaction conditions for enzymatic reactions

[0042] Take an appropriate amount of immobilized enzyme and free enzyme, add 150 μL of androstenedione with a concentration of 35 μM, and 20 μL of NAPDH with a concentration of 25 mg / mL, and incubate at 37 ° C for 8 h to determine the content of the product testosterone. The optimum reaction pH is 8.0, and the optimum reaction temperature is 40°C. After repeated use 5 times, the immobilized enzyme was inactivated.

[0043] 2) Study on the kinetics of enzymatic reactions

[0044] Take 20 mg of the immobilized enzyme, add 200 μL of NAPDH with a concentration of 25 mg / mL, and 1500 μL of androstenedione with a concentration of 35 μM in sequence. After shaking and mixing, the mixture is evenly divided into 10 tubes. Under the phosphate buffer system of pH=8.0, Incubate at 37°C for 0min, 15min, 30min, 1h, 2h, 4h, 6h, 10h, 12h, 24h, centrifuge to take the...

example 3

[0046] Example 3: Research of apigenin on immobilized enzyme inhibition

[0047] 1) Apigenin enzymatic inhibition curve

[0048] Take 20 mg of the immobilized enzyme, add 200 μL of NAPDH with a concentration of 25 mg / mL, 1500 μL of androstenedione with a concentration of 35 μM, and 100 μL of apigenin with a concentration of 400 nM in sequence. Under the phosphate buffer system, incubate at 37°C for 0min, 15min, 30min, 1h, 2h, 4h, 6h, 10h, 12h, 24h, centrifuge to take the supernatant, and use high performance liquid chromatography to determine the content of the product at each time point (deduct corresponding blank), prepare enzymatic curve, calculate enzymatic reaction kinetic coefficient V m and K m .

[0049] 2) Apigenin IC 50 Determination of value

[0050] Take 160 mg of the immobilized enzyme, add 160 μL of NAPDH with a concentration of 25 mg / mL, and 1200 μL of androstenedione with a concentration of 35 μM in sequence, shake and mix well, and divide into 8 tubes, re...

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Abstract

The invention provides a method for screening a I type 17 beta-hydroxysteroid dehydrogenase inhibitor through an immobilized enzyme, and belongs to the technical field of enzymology and enzyme engineering. I type 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD1) has the important function in treating hormone-dependent diseases. At present, a substrate radiolabelling method is mainly adopted in studying the activity of 17 beta-HSD1, due to the fact that the free enzyme of 17 beta-HSD1 is likely to be inactivated, 17 beta-HSD1 is hard to prepare, a fresh placenta is needed for preparing an enzyme source in a new medicine screening experiment of every time, raw materials are hard to obtain, the price is high, and difficulty is brought to the screening work of new medicine. According to the method, an amino-modified silicon ball is adopted as a carrier, a glutaraldehyde crosslinking method is utilized, 17 beta-HSD1 extracted from the placenta is fixed, an external immobilization 17 beta-HSD1 enzyme model is built, androstenedione is adopted as a substrate, a high performance liquid chromatography method is used for detecting products, and the potential 17 beta-HSD1 inhibitor is screened. According to the method, instability of the free enzyme is overcome, operation is easy, the manufacturing cost is low, and repeated using is achieved.

Description

technical field [0001] The invention relates to a method for screening type I 17β hydroxysteroid dehydrogenase inhibitors by using immobilized enzymes, in particular to a method for establishing in vitro drug screening by using immobilized enzymes, which belongs to the technical field of enzymology and enzyme engineering. Background technique [0002] Type I 17β-hydroxysteroid dehydrogenase (17β-HSD1) is a type of enzyme with redox function, a key enzyme involved in the metabolism of estrogen and androgen, and the redox between the ketone group and the alcohol group at the C17 position The reaction plays a regulatory role, catalyzing the interconversion between highly active hormones and low active hormones, that is, the conversion between testosterone, estradiol and androstenedione and estrone. 17β-HSD1 is an enzyme that catalyzes the final step in the synthesis of sex hormones. It is closely related to breast cancer, endometrial cancer, and endometriosis, so it plays an im...

Claims

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Application Information

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IPC IPC(8): G01N30/02
Inventor 苏梦翔周雯迪狄斌
Owner CHINA PHARM UNIV
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