Method for rapidly detecting activity of CDK9/CyclinT1 enzyme and application of method

A detection method and enzyme activity technology, applied in the field of biochemistry, can solve the problems of high cost of reagent waste liquid treatment, expensive isotope reagents, hardware and qualification requirements, etc., to shorten the detection time, easy to operate, improve data volume effect

Inactive Publication Date: 2019-09-10
武汉合研生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The use of isotope methods to evaluate the activity of CDK9 / CyclinT1 compounds requires laboratory hardware and qualifications, which limits the development of experiments
Moreover, isotope reagents are expensive, and the cost of reagent waste treatment is high, so it is not suitable for large-scale screening experiments to evaluate the activity of compound libraries.

Method used

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  • Method for rapidly detecting activity of CDK9/CyclinT1 enzyme and application of method
  • Method for rapidly detecting activity of CDK9/CyclinT1 enzyme and application of method
  • Method for rapidly detecting activity of CDK9/CyclinT1 enzyme and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: Rapid detection method of CDK9 / CyclinT1 enzymatic activity

[0039] In this example, CDK9 / CyclinT1 enzyme was purchased from Carna Biosciences, Inc., PKDTide polypeptide substrate, 5X polypeptide buffer and DTT were purchased from Signalchem, ADP-Glo TM Kinase reagents were purchased from Promega.

[0040] 1. A rapid detection method for CDK9 / CyclinT1 kinase activity, comprising the following steps:

[0041] (1) Take the sample to be tested, the blank control substance, and the positive control substance and incubate with the CDK9 / CyclinT1 enzyme reagent and PDKTide / ATP mixture respectively to generate ADP by kinase reaction; the sample to be tested, the positive control substance, the CDK9 / CyclinT1 enzyme reagent, The PDKTide / ATP mixture uses buffer as the solvent, and the positive control substance is a solution formed by dissolving staurosporine, a universal kinase inhibitor, in buffer, with a concentration of 50μM-0.64nM; the concentration of CDK9 / Cyc...

Embodiment 2

[0083] Example 2: The detection method of the present invention screens two compounds, CDK9-1N-1 and Atuveciclib S-Enantiomer, for CDK9 / CyclinT1 target inhibitors

[0084] Take CDK9-1N-1 and Atuveciclib S-Enantiomer to experiment according to the method of Example 1, and calculate the enzymatic activity and IC50 value of the two compounds against CDK9 / CyclinT1, as image 3 shown. The IC50 of CDK9-1N-1 and Atuveciclib S-Enantiomer are 33.95nM and 11.15nM, respectively.

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Abstract

The invention relates to a method for rapidly detecting the activity of a CDK9/CyclinT1 enzyme and application of the method. According to the detecting method, the principle that the CDK9/CyclinT1 enzyme can phosphorylate a substrate at the molecular level is utilized in combination with an ADP-Glo technology which has very high sensitivity, and then the amount of ATP consumed in a reaction is directly determined to quantify the process of the reaction. The whole reaction can be operated under a common laboratory condition, isotopic laboratory conditions are not needed, radiation hazards possibly caused to operating personnel and environments are reduced, and the safety is high. In the meanwhile, the method is simple and feasible, all experiments of the whole reaction can be completed byoperation within one day, the detection time is greatly shortened, the data volume of single-time detection can also be increased, and the method is more suitable for vast compound activity evaluationexperiments. Therefore, the method can be applied to screening of CDK9/CyclinT1 enzyme receptor inhibitors and screening of anti-tumor drugs relevant to target spots.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and in particular relates to a rapid detection method of CDK9 / CyclinT1 enzyme activity and its application. Background technique [0002] A member of the cyclin-dependent kinase (CDK9 / CyclinT1) complex, also known as positive transcriptional elongation factor B (P-TEFb), promotes Transition from terminal extension to active elongation. This complex is inactive in the presence of the 7SK SNNP complex. After phosphorylation of EP300, MYOD1, RPB1 / POLR2a, and AR, as well as the negative elongation factors DSIF and NELF, regulation of Cytokines induce transcriptional networks that promote RNA synthesis in genetic programs that promote cell growth and differentiation. [0003] P-TEFb is also involved in co-transcriptional histone modification, mRNA processing and mRNA efflux. Regulates a complex network of chromatin modifications including H2Bub1, H3K4me3, and H3K36me3; couples phosphorylation...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/48
CPCC12Q1/485G01N2333/912
Inventor 石榴徐燕华
Owner 武汉合研生物医药科技有限公司
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