CN103149186A

CN103149186A - Aromatase inhibitor high-throughput screening model based on fluorescence detection principle

Info

Publication number: CN103149186A
Application number: CN2013100459179A
Authority: CN
Inventors: 严明; 吉金子; 苗靖姗; 向华; 张陆勇
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2013-02-06
Filing date: 2013-02-06
Publication date: 2013-06-12
Anticipated expiration: 2033-02-06
Also published as: CN103149186B

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

A kind of arimedex high flux screening model based on the fluoroscopic examination principle

Technical field

The invention belongs to area of pharmacology, utilize the homogeneous phase time discrimination fluorescence detection technique, built the high flux screening model of external arimedex, be used for testing sample to the high flux detection of aromatic enzyme-tion suppressioning activity.

Background technology

Steroidal A ring aromatizing enzyme mainly is present in ovary and placenta, also sees other tissue, as brain, adrenal gland, testis, marrow, fat, skin, muscle and some tumor tissues.Aromatizing enzyme is take androgen as substrate, under oxygen and NADPH participate in, carries out hydroxylation twice in 19 of substrate, finally makes the sweetening treatment of A cyclophane generate the estrogen product.Suppress the active of aromatizing enzyme or make its inactivation can cause body inner estrogen level to reduce, thereby the physiology course relevant with estrogen is interfered as ovulation, implantation etc., also can makes disease that estrogen keeps such as some breast cancer, carcinoma of endometrium, woman's sex premature, gynecomastia etc. be cured or alleviate.Because the reaction of aromatizing enzyme institute catalysis is the biosynthetic final steps of all kinds of steroid hormones, thereby stop this step reaction will not reduce the secretion level of other hormone, if arimedex itself does not have obvious hormonal activity, can expect so, arimedex will have less toxic and side effect for clinical.Therefore, the research arimedex is significant.

In recent years, can disturb or damage in a large number the mankind and wild animal internal system and cause swollen neoplastic Enviromental pollutants to cause people's extensive concern, comprise some exogenous estrogens and antiandrogen and antithyroid chemical substance, as pesticide, can block or activate steroid receptor, affect sex hormone level and disturbance endocrine, thereby affect male and growth female repro ductive system, therefore the plan hormonal activity of these chemical substances screened, set up the endocrine of evaluating chemical material and disturb the detection system of vigor quite important.Therefore it is significant to set up the aromatizing enzyme screening model of quickly, efficiently and accurately.

Time-resolved fluorescence technology (time-resolved fluorescence, TRF) is based on that characteristics that lanthanide series such as europium (Eu), samarium (Sm), dysprosium (Dy) etc. have longer fluorescence lifetime develop.Distance is less than 10nm between europium chelate donor and acceptor, and donor emission spectrum and acceptor excitation spectrum have when overlapping, FRET (fluorescence resonance energy transfer) occurs, homogeneous phase time discrimination fluorescence (homogeneous time-resolved fluorescence, HTRF) technology is that French Cisbio company utilizes this principle to carry out the product of deep exploitation.The fluorescence lifetime of most of fluorescent materials very short (being generally several milliseconds), disturb for fear of of short duration fluorescence, Cisbio company utilizes the lanthanide chelate of longer fluorescence lifetime as the fluorescent energy donor, acceptor is modified through allophycocyanin (allophycocyanin) or fluorescein, and donor just can make acceptor also have long fluorescence lifetime when energy shifts.Therefore, when energy shifts, acceptor utilizing emitted light die-out time is directly proportional to donor utilizing emitted light die-out time, and and be inversely proportional to for the distance between acceptor, this method has extended the fluoroscopic examination time, has reduced the background interference that of short duration fluorescence causes.

Use homogeneous phase to differentiate the quantitative analysis method that fluorescent technique is set up estradiol, and estradiol is the catalysate of aromatizing enzyme, content by estradiol in the detection reaction system, measure testing sample to the purpose of activity of aromatizing enzyme impact thereby reach, final by to the optimization of the reaction conditionss such as enzyme and concentration of substrate, antibody incubation time and the trace of reaction system, can realize the foundation to the high flux screening model of arimedex.

At present, the screening technique of existing multiple arimedex, for example isotope in conjunction with experiment, comprise that TLC thin layer chromatography and [3H] mix method, yet isotope detection is very high for requirement for experiment condition, and meeting welding and infringement experimenter's health; Also can utilize ELISA method or Syrups by HPLC reaction product to screen arimedex, but the method waste time and energy, and is difficult to accomplish high flux screening.Therefore, set up inactive detection method, particularly external functional detection more and more comes into one's own in drug screening.

Summary of the invention

The object of the invention is to set up a kind of arimedex high flux screening model based on fluorescence, have signal to noise ratio (S/N ratio) high, use safety, the characteristics that the sample consumption is little.

Technical scheme of the present invention: fluorescence intensity ratio (665nm/610nm) in the reaction system after the method detection aromatization enzyme-to-substrate of utilization homogeneous phase time discrimination fluorescence and coenzyme are hatched, this ratio can reflect the content of estradiol in the end reaction system, thereby reach the purpose of measuring activity of aromatizing enzyme, realize that finally testing sample is to the high flux screening of aromatic enzyme-tion suppressioning activity.

The present invention utilizes the method for homogeneous phase time discrimination fluorescence to set up a kind of arimedex high flux screening model.

Step 1: the Establishment and optimization of arimedex screening model.

Step 2: positive drug verification model reliability.

Step 3: high flux screening model checking.

Description of drawings:

Fig. 1: aromatizing enzyme concentration gradient Optimal Experimental result.(n＝3，

)

Fig. 2: the aromatizing enzyme temperature is incubated time-optimized experimental result.(n＝3，

)

Fig. 3: aromatizing enzyme concentration of substrate Optimal Experimental result.(n＝3，

)

Fig. 4: aromatizing enzyme NADPH concentration optimization experimental result.(n＝3，

)

Fig. 5: the positive drug Letrozole is to aromatization enzymeinhibition curve map.(n＝3， )

Fig. 6: arimedex high flux screening model detection window signal.

Fig. 7: high flux screening Z ' value distributes.

Embodiment

Below in conjunction with description of drawings the specific embodiment of the present invention:

1. activity of aromatizing enzyme detects

1) experiment material

Estradiol detection kit (Cisbio, France), testosterone (Sigma-aldrich, the U.S.), NADPH (Luo Shi, the U.S.), Letrozole (U.S. logical sequence, China), 384 low volume blank (Corning, the U.S.), people source aromatizing enzyme (BD, the U.S.), rifle head (Axygen, the U.S.).

2) experimental procedure

● carry out aromatizing enzyme concentration gradient, temperature and incubate time, concentration of substrate, the experiment of NADPH concentration, see Fig. 1-4.

● the testing compound accurate weighing, add the DMSO solvent to become mother liquor, then to use and detect damping fluid preparation testing compound solution to desired concn, primary dcreening operation concentration is about 1 * 10 ^-3Mol/L.

● every hole adds aromatizing enzyme solution 2 μ l in reaction vessel, substrate solution 2 μ l, damping fluid or compound to be sieved 4 μ l, NADPH2 μ l.37 ℃ were reacted 1 hour.

● every hole adds Estradiol-XL6655 μ l, Anti-Estradiol-cryptate5 μ l, incubated at room 2 hours.

● utilize U.S. Beckman Ku Erte (Beckman Coulter) the detection platform HTRF of company module to detect respectively the fluorescence intensity at 665nm and 610nm place.

● draw positive drug Letrozole amount effect curve and measure its IC ₅₀Value is seen Fig. 5.

● the acquisition testing signal is also drawn, and determines the reliability of high flux screening model by signal window and Z ' value, sees Fig. 6,7.

2. data are processed

1) calculate the ratio (Ratio665/610) of each hole 665nm and 610nm place fluorescence intensity according to formula;

2) calculate the relative inhibition in each hole according to formula

3) active sample is carried out the relative inhibition value that detects after concentration dilution, making to ask as the mapping of Graphpad software to calculate half inhibiting rate IC50.

Experimental result

Aromatizing enzyme screening model optimum results: the required aromatizing enzyme of optimum response is 0.15pmol (seeing Fig. 1), the best temperature time of incubating is 1 hour (seeing Fig. 2), best concentration of substrate is 30nM (seeing Fig. 3), and optimum N ADPH concentration is 6 μ M (seeing Fig. 4).With positive drug Letrozole half inhibiting rate IC ₅₀Be 40nM (seeing Fig. 5), and (see Fig. 6 by signal to noise ratio (S/N ratio) and Z ' value, 7) checking shows that the arimedex in-vitro screening model that adopts this method to set up has reached the requirement of high flux screening, experimental result is reliable and stable, can be used for substituting the aromatizing enzyme high flux screening model based on the isotope detection technology.

Claims

1. aromatizing enzyme high flux screening model, the screening technique that it is characterized in that this model is fluorescence intensity ratio (665nm/610nm) in reaction system after using the method for homogeneous phase time discrimination fluorescence to detect aromatization enzyme-to-substrate and coenzyme to hatch, this ratio can reflect the content of estradiol in the end reaction system, thereby reach the purpose of measuring activity of aromatizing enzyme, realize that finally testing sample is to the high flux screening of aromatic enzyme-tion suppressioning activity.

2. aromatizing enzyme high flux screening model according to claim 1, is characterized in that the required aromatizing enzyme of optimum response is 0.15pmol, and the best temperature time of incubating is 1 hour, and best substrate is that estradiol and concentration are 30nM, and optimum N ADPH concentration is 6 μ M.