CN104458675A - High flux screening method for screening stem cell factor acceptor kinase inhibitor - Google Patents

Abstract

The invention discloses a high flux screening method for screening a stem cell factor acceptor kinase inhibitor, which comprises the following steps: 1)establishment and optimization of a screening model of the stem cell factor acceptor 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 stem cell factor receptor 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 stem cell factor receptor tyrosine kinase (Kit) inhibitor, for testing sample, the high flux of Kit kinase inhibiting activity is detected.

Background technology

Kit is a kind of proto-oncogene, and be the endochylema retrovirus homologue of HZ4 feline sarcoma virus, be positioned at No. 4 chromosome 4q11-12, a kind of relative molecular mass of encoding is the transmembrane glycoprotein of 145kD, and this albumen belongs to receptor tyrosine kinase family member.Kit acceptor is divided into film outskirt, cross-film district and cytoplasmic region three part.Kit/SCF system is an important step in protein kinase/phosphatase signal transductive process.The activation of Kit acceptor needs SCF to participate under normal circumstances, after the specific binding of Kit acceptor and SCF, triggers the phosphorylation of tyrosine residue in the homodimer of Kit acceptor and cell membrane, thus by extracellular signal transduction in cell.Kit gene mutation result in the spontaneous receptor dimer that Kit does not rely on part, causes the sustained activation of Kit acceptor, irritation cell hyper-proliferative and anti-apoptotic signal out of control.Oneself finds the high expressed all having Kit in many malignant tumours, comprises gastrointestinal stromal tumor, prostate cancer, gonioma, breast cancer and small-cell carcinoma of the lung etc.The signal transduction pathway of Kit mediation is suppressed to become the focus of oncotherapy in recent years with the object reaching treatment tumour.

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 Kit inhibitors of kinases, utilize ELISA method to screen Kit 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 Kit 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 Kit inhibitors of kinases high flux screening model, primary dcreening operation, sieves discovery one class again and has the candidate compound suppressing Kit kinase activity.Concrete steps are as follows:

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

Step one: the Establishment and optimization of Kit 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: Kit kinase concentration gradient optimizing experimental result.(n＝3， )

Fig. 2: Kit kinases temperature incubates time-optimized experimental result.(n＝3， )

Fig. 3: Kit kinase substrate concentration optimization experimental result.(n＝3， )

Fig. 4: Kit kinases ATP concentration optimization experimental result.(n＝3， )

Fig. 5: positive drug staurosporine is to the kinase whose suppression curve map of Kit.(n＝3， )

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

(1) experiment material

Kit kinase assay kit (Cisbio, France), Kit kinases (Invitrogen, the U.S.), ATP is (raw emerging, China), staurosporine (the green skies, China), 384 low volume blank (Coming, the U.S.), rifle head (Axygen, the U.S.).

(2) experimental procedure

1) carry out Kit 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 Kit 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

Kit kinases screening model optimum results: the Kit kinases 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 0.38 μM (see Fig. 3), and best ATP concentration is 21.31 μMs (see Fig. 4).Positive drug half inhibiting rate IC ₅₀for 1.599nM (see Fig. 5), and by signal to noise ratio (S/N ratio) and Z ' value (see Fig. 6,7) checking shows that the Kit 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 Kit inhibitors of kinases.

Claims (6)

1. a stem cell factor receptor inhibitors of kinases 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 stem cell factor receptor kinases.

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

4. method as claimed in claim 3, it is characterized in that, be 0.1ng/ μ l by step (1) the stem cell factor receptor kinase concentration that can obtain needed for optimum response, the best temperature time of incubating is 60min, best concentration of substrate is 0.38 μM, and best ATP concentration is 21.31 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 1.599nM.

6. either method described in claim 1-5 is in the application of screening stem cell factor receptor inhibitors of kinases.