Method for screening kinase inhibitor by utilizing HTRF one-step method

A kinase inhibitor and one-step technology, which is applied in the field of one-step screening of kinase inhibitors using HTRF, can solve the problems of high-throughput or ultra-high-throughput screening of difficult compounds, experimental errors, and time-consuming, and achieve economical and reliable screening results. , The effect of saving experimental workload and saving experimental time

Inactive Publication Date: 2019-05-28
浠思(上海)生物技术有限公司
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  • Abstract
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  • Claims
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Problems solved by technology

[0005] The radioisotope method has the following disadvantages: 1. It produces a large amount of radioactive waste
It has certain damage to the human body; 2. The half-life of radioactive ATP is very short, only 14 days (32P) or 25 days (33P), so new isotopes need to be ordered frequently; 3. The energy of radioactive isotopes is very high, and there are obvious Cross-interference phenomenon, difficult to apply to high-throughput or ultra-high-throughput screening of compounds
[0006] As a traditional meth

Method used

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  • Method for screening kinase inhibitor by utilizing HTRF one-step method
  • Method for screening kinase inhibitor by utilizing HTRF one-step method
  • Method for screening kinase inhibitor by utilizing HTRF one-step method

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Experimental program
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Effect test

Embodiment 1

[0040] 1) To prepare EGFR kinase, start with 3-fold serial dilution from 1ng / ul. The substrate concentration is fixed at 1uM, the ATP concentration is 100uM, and the enzyme reaction time is 10, 20, 30, 40, 50, 60 minutes respectively;

[0041] 2) Add the prepared kinase, substrate, and ATP into the 384-well detection plate, incubate at room temperature for a corresponding period of time, add the detection reagent coupled with Donor and Acceptor (or add 10ul after equal volume mixing), incubate for 1 hour and read ;

[0042] According to the principle of HTRF method, the fluorescence ratio of 665nm / 620nm is detected. According to this ratio, the phosphorylation of the substrate is judged. The higher the signal, the higher the phosphorylation degree of the substrate, and the higher the kinase activity. The results are shown in figure 2 . figure 2 The Kd value was determined by MCL1 and BAK binding test.

Embodiment 2

[0044] 1) Configure a positive compound, and perform a gradient dilution of the compound staurosporine, starting from 100000nM, and diluting it 10 times;

[0045] 2) Prepare EGFR, and perform follow-up inhibitor screening according to the appropriate kinase concentration of Example 1, EGFR is 0.0041ng / ul, and the enzyme reaction time is 40min;

[0046] 3) Add the prepared inhibitor, EGFR, substrate, and ATP to the 384-well detection plate in sequence, and after incubating at room temperature for 40 minutes, add the detection reagent coupled with Donor and Acceptor (or add 10ul after mixing in equal amounts), and incubate for 1 hour reading;

[0047] According to the principle of HTRF method, the fluorescence ratio of 665nm / 620nm is detected, and according to the ratio, the inhibition of the kinase is judged. The weaker the signal, the lower the kinase activity and the stronger the inhibitory effect. see results image 3 . image 3 The IC50 of compound Selleck A-1210477, AP...

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Abstract

The invention discloses a method for screening a kinase inhibitor by utilizing an HTRF one-step method, and belongs to the field of inhibitor screening methods. The method for screening the kinase inhibitor by utilizing the HTRF one-step method comprises the following steps: sequentially adding a compound to be detected, a kinase, a substrate and ATP into a microporous plate for co-incubation; sequentially adding an antibody coupled with a recognition substrate phosphorylation site of Donor and streptavidin coupled with biotin on a recognition substrate of Acceptor into the microporous plate;incubating at room temperature, and detecting fluorescence by using an enzyme marker reading, wherein the ratio of 665nm/ 620nm is original data; according to the method, screening the kinase inhibitor at the biochemical level can be achieved, and constructing cells is not needed, and the method is more direct and quicker.

Description

technical field [0001] The invention belongs to the field of inhibitor screening methods, in particular to a method for screening kinase inhibitors using HTRF in one step. Background technique [0002] In biochemistry, protein kinases are a class of enzymes that catalyze the phosphorylation of proteins. It can catalyze the transfer of a phosphate group from adenosine triphosphate (ATP) and covalently bind to a specific substrate, thereby changing the conformation and activity of the substrate. Protein kinases can be divided into two categories: tyrosine kinases and serine / threonine kinases according to their selectivity for substrate phosphorylation sites. Protein kinases play key regulatory roles in nearly every aspect of cell biology. They are involved in the regulation of apoptosis, cell cycle progression, cytoskeletal rearrangement, differentiation, development, immune response, nervous system function and signal transduction. Once the kinase is out of balance, it wil...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/573G01N21/64
Inventor 王心媛
Owner 浠思(上海)生物技术有限公司
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