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High flux screening method for screening tropomyosin-related kinase B inhibitor

A tropomyosin, high-throughput technology, applied in the field of pharmacology, can solve the problems of difficulty in high-throughput screening, time-consuming and labor-intensive

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

AI Technical Summary

Problems solved by technology

[0004] At present, there are many screening methods for TrkB kinase inhibitors, most of which use ELISA method to screen TrkB 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 tropomyosin-related kinase B inhibitor
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  • High flux screening method for screening tropomyosin-related kinase B inhibitor

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specific Embodiment approach

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

[0020] (1) Experimental materials

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

[0022] (2) Experimental steps

[0023] 1) Carry out TrkB 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 TrkB 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 hour. ...

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Abstract

The invention discloses a high flux screening method for screening a tropomyosin-related kinase B inhibitor, which comprises the following steps: 1)establishment and optimization of a screening model of the tropomyosin-related kinase B 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 tropomyosin-related kinase B (TrkB) inhibitors is constructed by using homogeneous time-resolved fluorescence detection technology, which is used for high-throughput screening of TrkB kinase inhibitory activity of test samples. volume detection. Background technique [0002] TrkB is the main receptor of BDNF and has the activity of receptor tyrosine kinase. Studies have shown that BDNF and TrkB are upregulated in a variety of human tumors, including bladder cancer and ovarian cancer. However, TrkB overexpression was found in pancreatic cancer cells with high metastatic potential, suggesting that TrkB may mediate the clinical features of aggressive growth and metastasis of pancreatic cancer. TrkB is activated by BDNF and induces the activation of a series of downstream signaling pathway molecules, such as protein kinase B (protein kinase B, PKB, also known as Akt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 严明张陆勇胡洁高鹏
Owner CHINA PHARM UNIV
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