CN104458672A

CN104458672A - High flux screening method for screening mitogen activated protein kinase inhibitor

Info

Publication number: CN104458672A
Application number: CN201310421160.9A
Authority: CN
Inventors: 严明; 张陆勇; 胡洁; 高鹏
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2013-09-12
Filing date: 2013-09-12
Publication date: 2015-03-25

Abstract

The invention discloses a high flux screening method for screening a mitogen activated protein kinase inhibitor, which comprises the following steps: 1)establishment and optimization of a screening model of the mitogen activated protein kinase inhibitor: performing kinases concentration, incubation time, substrate concentration and ATP concentration experiments; 2)model reliability verification with positive drug: selecting kinases with appropriate concentration, ATP Km and substrate Km, respectively adding 2muml kinases and the substrate in each pore, adding 4muml positive drug in each pore according to concentration gradient, adding 2muml ATP for reacting, incubating at room temperature according to optimization time, adding 10muml stopping solution in each pore for stopping the reaction, incubating for 1 hour at room temperature and then detecting, analyzing data to obtain the positive drug IC50; and 3)verification of the high flux screen model: operating according to the above steps, using a Biomek NXP automation sampling apparatus and a Multidrop automatic liquid separator for feeding samples, and then calculating Z factors. The method has the advantages of simpleness and rapidity, high sensitivity, stable and reliable result and good reappearance, and can be used for high flux screening.

Description

A kind of screening mitogen activated protein kinase inhibitor high-throughput screening method

Technical field

The invention belongs to area of pharmacology, utilize homogeneous phase time discrimination fluorescence detection technique, construct the high flux screening model of MAPK (MAPK) inhibitor, for testing sample, the high flux of mapk kinase inhibit activities is detected.

Background technology

MAPK (mitogen-activated protein kinase, MAPK) signal path is the bars transduction pathway that discovered in recent years is extensively present in various zooblast, has important regulating and controlling effect for the operation of cell cycle and gene expression.MAPK signal path is made up of the protein serine/threonine that a group activates in cascaded fashion successively, being amplified step by step by extracellular signal with this and be transmitted in cell and even nucleus, the effector molecule in born of the same parents' external stimulue that membrane receptor is combined and tenuigenin and nucleus couples together.MAPK is also referred to as born of the same parents and regulates kinases (extracellular regulating kinase outward, ERK), the core element of MAPK signal path, the signal transduction system of it and its upstream activat molecule and a downstream effect molecular composition precise and high efficiency.MAPK activates the phosphorylation needing Thr and Tyr two sites, and mapk kinase (MAPKkinase, MAPKK) is its upstream activat molecule, and the catalysis characteristics of its dual specificity phosphatase ensure that MAPK normal Activate, completes specific physiological activity.MAPKK is activated by phosphorylation, and MAPKKK (MAPK kinase kinase) participates in its activation, Raf and Mos is the important member of MAPKKK, and their expression is subject to the regulation and control of cell cycle and born of the same parents' external stimulus.The downstream molecules of MAPK comprises multiple protein kinases, phosphatidase, and transcription factor.MAPK makes their activation then activate the effector molecule in more downstream by these effector molecules of phosphorylation, thus is delivered in cell by extracellular signal and completes certain physiological activity.Therefore, study mapk kinase inhibitor to be 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.

At present, the screening technique of existing multiple mapk kinase inhibitor, utilize ELISA method to screen mapk kinase inhibitor, but the method wastes time and energy, and is difficult to accomplish high flux screening more.Therefore, set up convenient and swift detection method accurately, 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 mapk kinase inhibitor high flux screening model based on homogeneous phase time discrimination 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: adopt homogeneous phase time discrimination fluorescence method establishment external mapk kinase inhibitor high flux screening model, primary dcreening operation, sieves discovery one class again and has the candidate compound suppressing mapk kinase activity.Concrete steps are as follows:

The present invention utilizes a kind of mapk kinase inhibitor of the method establishment of homogeneous phase time discrimination fluorescence high flux screening model.

Step one: the Establishment and optimization of mapk kinase inhibitor screening model.

Step 2: positive drug verification model reliability.

Step 3: high flux screening model is verified.

Accompanying drawing illustrates:

Fig. 1: mapk kinase concentration gradient Optimal Experimental result.

Fig. 2: mapk kinase temperature incubates time-optimized experimental result.

Fig. 3: mapk kinase concentration of substrate Optimal Experimental result.

Fig. 4: mapk kinase ATP concentration optimization experimental result.

Fig. 5: positive drug staurosporine is to the suppression curve map of mapk kinase.

Fig. 6: mapk kinase inhibitor 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.MAPK inhibitors of kinases screening technique is set up

(1) experiment material

Mapk kinase detection kit (Cisbio, France), mapk kinase (Invitrogen, the U.S.), ATP is (raw emerging, China), staurosporine (the green skies, China), 384 low volume blank (Corning, the U.S.), rifle head (Axygen, the U.S.).

(2) experimental procedure

1) carry out mapk kinase concentration gradient, temperature incubate the time, concentration of substrate, ATP concentration experiment, see Fig. 1-4.

2) 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.

3) in reaction vessel, every hole adds mapk kinase solution 2 μ l, substrate solution 2 μ l, damping fluid or treat sieve compound 4 μ l, ATP2 μ l.Room temperature reaction 1 hour.

4) every hole adds Estradiol-XL6655 μ l, Anti-Estradiol-cryptate5 μ l, incubated at room 1 hour.

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

6) draw positive drug staurosporine amount effect curve and measure its IC ₅₀value, is shown in Fig. 5.

7) acquisition testing signal drawing, by the reliability of signal window and Z ' value determination high flux screening model, is shown in 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 IC ₅₀.

Experimental result

Mapk kinase screening model optimum results: the mapk kinase needed for optimum response is 0.1ng/ μ l (see Fig. 1), the best temperature time of incubating is 60min (see Fig. 2), best concentration of substrate is 17.62nM (see Fig. 3), and best ATP concentration is 0.1359 μM (see Fig. 4).Positive drug half inhibiting rate IC ₅₀for 33760nM (see Fig. 5), and by signal to noise ratio (S/N ratio) and Z ' value (see Fig. 6,7) checking shows that the mapk kinase inhibitor 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 the high flux screening carrying out mapk kinase inhibitor.

Claims

1. a mitogen activated protein kinase inhibitor high flux screening model, is characterized in that, comprises step:

(1) Establishment and optimization of inhibitors of kinases screening model;

(2) positive drug verification model reliability;

(3) high flux screening model checking.

2. the method for claim 1, is characterized in that, described kinases is MAPK.

3. the method for claim 1, is characterized in that, step carries out MAPK concentration gradient in (1), temperature incubates the time, concentration of substrate, ATP concentration experiment.

4. method as claimed in claim 3, it is characterized in that, be 0.1ng/ μ l by step (1) the MAPK concentration that can obtain needed for optimum response, the best temperature time of incubating is 60min, best concentration of substrate is 17.62nM, and best ATP concentration is 0.1359 μM.

5. the method for claim 1, is characterized in that, the kinases of the suitable concn that step (2) optional step (1) arrives, ATP Km, substrate Km; Kinases and substrate are pressed 1:2 volume mixture, and every hole adds 4 μ l, then adds 4 μ l positive drug by the every hole of concentration gradient, and last every hole adds 2 μ l ATP and starts reaction, by optimization time incubated at room; Preparation SA-XL665 and TK-Ab, by SA-XL665 and TK Ab 1:1 mixing by volume, every hole adds 10 μ l cessation reactions, and incubated at room detected after 1 hour, analyzes data and obtains positive drug half inhibiting rate IC ₅₀for 33760nM.

6. either method described in claim 1-5 is in the application of screening mitogen activated protein kinase inhibitor.