CN104458674A - High flux screening method for screening vascular endothelial growth factor 1 kinases inhibitor - Google Patents

Abstract

The invention discloses a high flux screening method for screening a vascular endothelial growth factor 1 kinases inhibitor, which comprises the following steps: 1)establishment and optimization of a screening model of the vascular endothelial growth factor 1 kinases 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 vascular endothelial growth factor 1 kinase inhibition agent high flux 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 vascular endothelial growth factor receptor tyrosine kinase (VEGFR) inhibitor, for testing sample, the high flux of VEGFR kinase inhibiting activity is detected.

Background technology

VEGFR family mainly comprises VEGFR-1 (Flt-1), VEGFR-2 (Flk-1/KDR), VEGFR-3 (Flt-4) three members, belong to receptor type tyrosine kinase (receptor tyrosine kinases, RTKs), be made up of 3 parts, comprise the tyrosine kinase activity district in 7 immunoglobulin like domain outside born of the same parents, cross-film district and born of the same parents.VEGFR-1 is the high-affinity receptor of VEGF-A, VEGF-B and PIGF, is present in vascular endothelial cell, candidate stem cell, macrophage and onthe surface of monocytes, relevant with the migration of these cells.The overexpression of acceptor or respective ligand, can by signal transduction in mulitpath, number of mechanisms active cell, signal protein enters nucleus activating transcription factor, cause cell proliferative disorder, Apoptosis inhibitor, Angiogenesis, cell invasion and transfer etc., and then cause the generation of tumour and other relevant diseases.VEGFR tyrosine kinase inhibitor acts on the most upstream of VEGFR signal transduction process, can block many paths, has that therapeutic domain is wide, curative effect is high and the not easily advantage such as resistance.Therefore, study VEGFR inhibitors of kinases 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 VEGFR1 inhibitors of kinases, utilize ELISA method to screen VEGFR1 inhibitors of kinases, 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 VEGFR1 inhibitors of kinases 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 VEGFR1 inhibitors of kinases high flux screening model, primary dcreening operation, sieves discovery one class again and has the candidate compound suppressing VEGFR1 kinase activity.Concrete steps are as follows:

The present invention utilizes a kind of VEGFR1 inhibitors of kinases of the method establishment of homogeneous phase time discrimination fluorescence high flux screening model.

Step one: the Establishment and optimization of VEGFR1 inhibitors of kinases screening model.

Step 2: positive drug verification model reliability.

Step 3: high flux screening model is verified.

Accompanying drawing illustrates:

Fig. 1: VEGFR1 kinase concentration gradient optimizing experimental result.

Fig. 2: VEGFR1 kinases temperature incubates time-optimized experimental result.

Fig. 3: VEGFR1 kinase substrate concentration optimization experimental result.

Fig. 4: VEGFR1 kinases ATP concentration optimization experimental result.

Fig. 5: positive drug staurosporine is to the kinase whose suppression curve map of VEGFR1.

Fig. 6: VEGFR1 inhibitors of kinases 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.VEGFR1 inhibitors of kinases screening technique is set up

(1) experiment material

VEGFR1 kinase assay kit (Cisbio, France), VEGFR1 kinases (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 VEGFR1 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 VEGFR1 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-XL665 5 μ l, Anti-Estradiol-cryptate 5 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

VEGFR1 kinases screening model optimum results: the VEGFR1 kinases needed for optimum response is 0.2ng/ μ l (see Fig. 1), the best temperature time of incubating is 60min (see Fig. 2), best concentration of substrate is 148.8nM (see Fig. 3), and best ATP concentration is 1.1 μMs (see Fig. 4).Positive drug half inhibiting rate IC ₅₀for 14.18nM (see Fig. 5), and by signal to noise ratio (S/N ratio) and Z ' value (see Fig. 6,7) checking shows that the VEGFR1 inhibitors of kinases 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 VEGFR1 inhibitors of kinases.

Claims (6)

1. a vascular endothelial growth factor 1 inhibitors of kinases high flux screening model, is characterized in that, comprise 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 vascular endothelial growth factor 1 kinases.

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

4. method as claimed in claim 3, it is characterized in that, be 0.2ng/ μ l by step (1) vascular endothelial growth factor 1 kinase concentration that can obtain needed for optimum response, the best temperature time of incubating is 60min, best concentration of substrate is 148.8nM, and best ATP concentration is 1.1 μMs.

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 14.18nM.

6. either method described in claim 1-5 is in the application of screening vascular endothelial growth factor 1 inhibitors of kinases.