CN103149186B - A kind of arimedex high flux screening model based on fluoroscopic examination principle - Google Patents
A kind of arimedex high flux screening model based on fluoroscopic examination principle Download PDFInfo
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- CN103149186B CN103149186B CN201310045917.9A CN201310045917A CN103149186B CN 103149186 B CN103149186 B CN 103149186B CN 201310045917 A CN201310045917 A CN 201310045917A CN 103149186 B CN103149186 B CN 103149186B
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Abstract
A kind of aromatic enzyme-tion suppressioning activity high flux screening utilizing homogeneous phase time discrimination fluorescence technology to realize, by detecting fluorescence intensity ratio (665nm/610nm) in the postradiation reaction system of exciting light, through formula scales, the inhibit activities of testing sample to aromatizing enzyme can be judged, utilize high flux screening new model of the present invention, the experimental technique that uses based on radioactive ligand combination principle can be evaded to the harm of experimenter and environment, and there is efficient, quick, accurate, cheap advantage.
Description
Technical field
The invention belongs to area of pharmacology, utilize homogeneous phase time discrimination fluorescence detection technique, construct the high flux screening model of external arimedex, for testing sample, the high flux of aromatic enzyme-tion suppressioning activity is detected.
Background technology
Steroidal A ring aromatizing enzyme is mainly present in ovary and placenta, also sees other tissue, as brain, adrenal gland, testis, marrow, fat, skin, muscle and some tumor tissues.Aromatizing enzyme take androgen as substrate, and under oxygen and NADPH participate in, 19 in substrate are carried out twice hydroxylation, finally makes the sweetening treatment of A cyclophane generate estrogen product.Suppress the activity of aromatizing enzyme or make its inactivation that body inner estrogen level can be caused to reduce, thus the physiology course relevant with estrogen such as ovulation, implantation etc. are interfered, the disease that estrogen also can be made to maintain such as some breast cancer, carcinoma of endometrium, woman's sex premature, gynecomastia etc. are cured or are alleviated.Reaction due to the catalysis of aromatizing enzyme institute is the biosynthetic final step of all kinds of steroid hormone, thus this step is stoped to react the secretion level that will do not reduce other hormone, if arimedex itself does not have obvious hormonal activity, so it is expected to, arimedex will have less toxic and side effect for clinical.Therefore, study arimedex to be significant.
In recent years, can disturb or damage the mankind and wild animal internal system in a large number and cause swollen neoplastic Enviromental pollutants to cause the extensive concern of people, 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, thus affect male and growth that is female repro ductive system, therefore screen the plan hormonal activity of these chemical substances, the detection system setting up evaluating chemical material endocrine disruption vigor is quite important.Therefore the aromatizing enzyme screening model setting up quickly, efficiently and accurately is significant.
Time-resolved fluorescence technology (time-resolved fluorescence, TRF) is that the feature having longer fluorescence lifetime as europium (Eu), samarium (Sm), dysprosium (Dy) etc. based on lanthanide series develops.When the spacing of Europium chelate donor and acceptor is less than 10nm, and donor emission is when having overlapping with acceptor excitation spectrum, then there is FRET (fluorescence resonance energy transfer), 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 material is very short (being generally several milliseconds), in order to avoid the interference of of short duration fluorescence, Cisbio company utilizes the lanthanide chelate of longer fluorescence lifetime as fluorescent energy donor, acceptor is modified through allophycocyanin (allophycocyanin) or fluorescein, and donor just can make acceptor also have longer fluorescence lifetime when energy trasfer.Therefore, during energy trasfer, acceptor emission light die-out time is directly proportional to donor-emitted light die-out time, and and be inversely proportional to for the distance between acceptor, this method extends the fluoroscopic examination time, reduces the background interference that of short duration fluorescence causes.
Homogeneous phase resolved fluorometric technology is used to set up the quantitative analysis method of estradiol, and estradiol is the catalysate of aromatizing enzyme, by the content of estradiol in detection reaction system, thus reach the object measuring testing sample and activity of aromatizing enzyme is affected, eventually through to enzyme and the optimization of reaction conditions and the milligram ammonia of reaction system such as concentration of substrate, antibody incubation time, the foundation of the high flux screening model to arimedex can be realized.
At present, the screening technique of existing multiple arimedex, such as isotope Binding experiment, comprise TLC thin layer chromatography and [3H] incorporation methods, but isotope detection is very high for requirement for experiment condition, and the health of meeting welding and infringement experimenter; Also ELISA method or Syrups by HPLC reaction product can be utilized 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, there is signal to noise ratio (S/N ratio) high, use safety, the feature that sample consumption is little.
Technical scheme of the present invention: fluorescence intensity ratio (665nm/610nm) in the reaction system after using the method for homogeneous phase time discrimination fluorescence detection aromatization enzyme-to-substrate and coenzyme to hatch, this ratio can reflect the content of estradiol in end reaction system, thus reach the object measuring activity of aromatizing enzyme, finally realize the high flux screening of testing sample to aromatic enzyme-tion suppressioning activity.
The present invention utilizes a kind of arimedex high flux screening model of the method establishment of homogeneous phase time discrimination fluorescence.
Step one: the Establishment and optimization of arimedex screening model.
Step 2: positive drug verification model reliability.
Step 3: high flux screening model is verified.
Accompanying drawing illustrates:
Fig. 1: aromatizing enzyme concentration gradient Optimal Experimental result.(n＝3，
)
Fig. 2: aromatizing enzyme temperature incubates 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: positive drug Letrozole is to the suppression curve map of aromatizing enzyme.(n＝3，
)
Fig. 6: arimedex high flux screening model detection window signal.
Fig. 7: high flux screening Z ' Distribution value.
Embodiment
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described:
1. activity of aromatizing enzyme detects
1) experiment material
Estradiol detection kit (Cisbio, France), testosterone (Sigma-aldrich, the U.S.), NADPH (Roche, 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 incubates the time, concentration of substrate, NADPH concentration experiment, see Fig. 1-4.
● testing compound accurate weighing, adds DMSO solvent and becomes mother liquor, and then use and detect buffer testing compound solution to desired concn, primary dcreening operation concentration is about 1 × 10
-3mol/L.
● in reaction vessel, every hole adds aromatizing enzyme solution 2 μ l, substrate solution 2 μ l, damping fluid or treat sieve compound 4 μ l, NADPH2 μ l.37 DEG C are 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) company detection platform HTRF module to detect the fluorescence intensity at 665nm and 610nm place respectively.
● draw positive drug Letrozole amount effect curve and measure its IC
50value, is shown in Fig. 5.
● acquisition testing signal is also drawn, and by the reliability of signal window and Z ' value determination high flux screening model, sees Fig. 6,7.
2. data processing
1) according to the ratio (Ratio665/610) of each hole 665nm of formulae discovery and 610nm place fluorescence intensity;
2) according to the relative inhibition in each hole of formulae discovery
3) the relative inhibition value that detects after carrying out concentration dilution of active sample, making to be used as the mapping of Graphpad software and asking and calculate half inhibiting rate IC50.
Experimental result
Aromatizing enzyme screening model optimum results: the aromatizing enzyme needed for optimum response is 0.15pmol (see Fig. 1), the best temperature time of incubating is 1 hour (see Fig. 2), best concentration of substrate is 30nM (see Fig. 3), and optimum N ADPH concentration is 6 μMs (see Fig. 4).With positive drug Letrozole half inhibiting rate IC
50for 40nM (see Fig. 5), and by signal to noise ratio (S/N ratio) and Z ' value (see Fig. 6,7) checking shows that the arimedex in-vitro screening model adopting this method to set up reaches the requirement of high flux screening, experimental result is reliable and stable, may be used for substituting the aromatizing enzyme high flux screening model based on isotope detection technology.
Claims (1)
1. an arimedex high flux screening model, select testosterone as substrate, carry out quantitatively with the Estradiol antibody of cryptate mark to reaction product, and arimedex high flux screening model is set up in the optimization carrying out reaction conditions on this basis, it is characterized in that the aromatizing enzyme needed for optimum response is 0.15pmol, the best temperature time of incubating is 1 hour, best concentration of substrate is 30nM, optimum N ADPH concentration is 6 μMs, Z ' close to and be greater than 0.8, the requirement of high flux screening can be met.
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| CN104280366B (en) * | 2013-07-03 | 2017-10-10 | 复旦大学 | A kind of method of the target proteinses of high flux detection compound |
| CN104458709A (en) * | 2013-09-12 | 2015-03-25 | 中国药科大学 | High flux screening method for screening calcium activated neutral protease-1 inhibitor |
| CN106872433B (en) * | 2017-03-31 | 2019-03-08 | 中国农业大学 | Beta-cyclodextrin functionalization carbon dots material and its applied to detection testosterone method |
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Inventor after: Yan Ming Inventor after: Ji Jinzi Inventor after: An Xiaofei Inventor after: Miao Jingpan Inventor after: Xiang Hua Inventor after: Zhang Luyong Inventor before: Yan Ming Inventor before: Ji Jinzi Inventor before: Miao Jingpan Inventor before: Xiang Hua Inventor before: Zhang Luyong |
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