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High flux screening method for screening anaplastic lymphoma kinase inhibitor

A lymphoma kinase and kinase inhibitor technology, applied in the field of pharmacology, can solve the problems of time-consuming, labor-intensive, and difficult to achieve high-throughput screening

Inactive Publication Date: 2015-03-25
CHINA PHARM UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are a variety of screening methods for ALK kinase inhibitors, most of which use ELISA to screen ALK kinase inhibitors, but this method is time-consuming and laborious, and it is difficult to achieve high-throughput screening

Method used

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  • High flux screening method for screening anaplastic lymphoma kinase inhibitor
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  • High flux screening method for screening anaplastic lymphoma kinase inhibitor

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specific Embodiment approach

[0019] 1. Establishment of screening method for ALK kinase inhibitors

[0020] (1) Experimental materials

[0021] ALK kinase detection kit (Cisbio, France), ALK kinase (Invitrogen, the United States), ATP (Shengxing, China), staurosporine (Beyond, China), 384 low-volume white plate (Coming, the United States), pipette tip ( Axygen, USA).

[0022] (2) Experimental steps

[0023] 1) Carry out ALK kinase concentration gradient, incubation time, substrate concentration, ATP concentration experiments, see Figure 1-4 .

[0024] 2) Accurately weigh the compound to be tested, add DMSO solvent to form a mother solution, and then use the detection buffer to prepare the solution of the compound to be tested to the required concentration. The initial screening concentration is about 1×10 -3 mol / L.

[0025] 3) Add 2 μl of ALK kinase solution, 2 μl of substrate solution, 4 μl of buffer or compound to be screened, and 2 μl of ATP into each well of the reaction vessel. React at room t...

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PUM

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Abstract

The invention discloses a high flux screening method for screening an anaplastic lymphoma kinase inhibitor, which comprises the following steps: 1)establishment and optimization of a screening model of the anaplastic lymphoma 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

technical field [0001] The invention belongs to the field of pharmacology. A high-throughput screening model for anaplastic lymphoma kinase (ALK) kinase inhibitors is constructed by using a homogeneous time-resolved fluorescence detection technology, which is used for high-throughput screening of test samples for ALK kinase inhibitory activity. detection. Background technique [0002] ALK is a receptor-type protein tyrosine phosphokinase, which can receive extracellular signals and regulate cell growth, differentiation, survival and transformation. Structurally, ALK includes an extracellular ligand-binding domain, a transmembrane domain and an intracellular domain. The full-length cDNA of ALK is 6226bp, encoding a protein of 177kDa, and the molecular weight of the modified ALK protein after compilation is close to 200-220kDa. ALK is a single-chain transmembrane protein with 1620 amino acid residues. The extracellular portion of ALK consists of 1030 amino acid residues, fo...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 严明张陆勇胡洁高鹏
Owner CHINA PHARM UNIV
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