High flux screening method for screening angiopoietin 2 receptor kinase inhibitor

An angiopoietin and receptor kinase technology, applied in the field of pharmacology, can solve problems such as time-consuming and labor-intensive, difficult to achieve high-throughput screening

Inactive Publication Date: 2015-03-25
CHINA PHARM UNIV
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Problems solved by technology

[0004] At present, there are many screening methods for Tie-2 kinase inhibitors, most of which use ELISA method to screen Tie-

Method used

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  • High flux screening method for screening angiopoietin 2 receptor kinase inhibitor
  • High flux screening method for screening angiopoietin 2 receptor kinase inhibitor
  • High flux screening method for screening angiopoietin 2 receptor kinase inhibitor

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specific Embodiment approach

[0019] 1. Establishment of screening method for Tie-2 kinase inhibitors

[0020] (1) Experimental materials

[0021] Tie-2 kinase detection kit (Cisbio, France), Tie-2 kinase (Invitrogen, USA), ATP (Shengxing, China), staurosporine (Beyond, China), 384 low-volume white plate (Corning, USA) , pipette tip (Axygen, USA).

[0022] (2) Experimental steps

[0023] 1) Carry out Tie-2 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 Tie-2 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 temperature for 1 ...

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Abstract

The invention discloses a high flux screening method for screening an angiopoietin 2 receptor kinase inhibitor, which comprises the following steps: 1)establishment and optimization of a screening model of the angiopoietin 2 receptor 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, utilizes homogeneous time-resolved fluorescence detection technology to construct a high-throughput screening model for angiopoietin 2 receptor tyrosine kinase (Tie-2) inhibitors, which is used for the detection of Tie-2 by samples to be tested. 2 High-throughput detection of kinase inhibitory activity. Background technique [0002] Tie-2, encoding 1124 amino acids, belongs to the tyrosine kinase receptor subclass. Its extracellular domain is composed of multiple functional domains, including an epidermal growth factor domain, 2 immunoglobulin-like domains, a Type plasmin repeat sequence, the intracellular part contains a tyrosine kinase domain, which contains a kinase insertion domain, and its core is at the carboxy terminus. Tie-2 is a tyrosine kinase receptor expressed in endothelial cells and some hematopoietic stem cells, and is uniformly expressed in the vascular endothelium of the embryo; in...

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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