Method for in-vitro screening of CDKs family protein kinase inhibitor based on MALDI-TOF-MS

A technology for protein kinase inhibitors and in vitro screening, applied in biochemical equipment and methods, microbial measurement/testing, instruments, etc., can solve problems such as CDK-Cyclin pathway regulation disorders

Active Publication Date: 2021-05-25
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Dysregulation of the CDK-Cyclin pathway can be observed in various cancers, leading to uncontrolled proliferation 11 , but currently only anti-breast cancer drugs based on the inhibition of CDK4 / 6 have been approved for marketing, which not only provides hope and motivation for using CDKs as targets to treat various cancers, but also illustrates the search for a high-throughput screening method for CDK inhibitors of great significance

Method used

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  • Method for in-vitro screening of CDKs family protein kinase inhibitor based on MALDI-TOF-MS
  • Method for in-vitro screening of CDKs family protein kinase inhibitor based on MALDI-TOF-MS
  • Method for in-vitro screening of CDKs family protein kinase inhibitor based on MALDI-TOF-MS

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Take CDK9 as an example:

[0067] figure 1It is the diagram of the action mode of CDK9 phosphorylated substrate peptide PSYSPTSPSYSPT before and after drug addition. The results show that the core of the experiment is to use the protein kinase extracted from cells to phosphorylate the corresponding short peptide (2Rpeptide, PSYSPTSPSYSPT) in vitro with the help of ATP. Mass spectrometry can be used to Phosphorylated short peptides and unphosphorylated short peptides were detected, and the peak area ratio of the two reflected the activity of CDK9. Utilizing the characteristics of automatic and high-throughput detection of MALDI-TOF-MS, the activity of CDK9 in each group of experiments can be quickly obtained, so as to quickly determine the inhibitory effect of the drug to be screened on CDK9. In this experiment, the CDK9 inhibitor flavopiridol (FLP) was selected as the positive drug for research. The structure of FLP is as follows:

[0068]

[0069] The experiment i...

Embodiment 2

[0075] Using the CDK9 protein kinase obtained in Example 1, papaya saponin II (drug 1), evodiamine (drug 2), dehydroevodiamine (drug 3), dioscin (drug 4), evodiamine (drug 5) were detected , Aloperine (drug 6), Chelerythrine chloride (drug 7), Cytisine (drug 8) inhibit the activity of CDK9 protein kinase, and the specific experimental steps are similar to those in Implementation 1:

[0076] The chemical structures of Aloperine (Drug 6), Chelerythrine chloride (Drug 7) and Cytisine (Drug 8) are shown below:

[0077]

[0078] CDK9 protein kinase in vitro kinase assay:

[0079]

[0080] Incubate in a water bath at 30°C for 30 min, then add 1 μL of 10% TFA to terminate the reaction.

[0081] (Except 8 drug treatment groups also include the positive control group that adds 1 μ DDMSO and the positive control group that working concentration is 160nM FLP);

[0082] After processing the data, Figure 4 The results of screening these natural products using the method provided ...

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Abstract

The invention discloses a method for in-vitro screening of a CDKs family protein kinase inhibitor based on MALDI-TOF-MS. The method comprises the following steps: firstly, preparing mother liquor of a CDKs in-vitro phosphokinase experiment buffer solution (KRB); adding a to-be-detected medicine sample, and carrying out in-vitro phosphokinase reaction to prepare a mass spectrometry sample; and finally, comparing the peak areas of ion peaks of phosphorylated oligopeptide and non-phosphorylated oligopeptide in a sample through MALDI-TOF-MS detection to judge the in-vitro activity of the to-be-detected drug in inhibiting CDKs. According to the method provided by the invention, in-vitro phosphorylation is carried out on corresponding substrate oligopeptides by utilizing a drug sample and protein kinase extracted from cells under the help of ATP, then phosphorylated oligopeptides and non-phosphorylated oligopeptides are detected by utilizing mass spectrometry, and the peak area ratio of the phosphorylated oligopeptides and the non-phosphorylated oligopeptides reflects the activity of inhibiting CDKs. According to the method provided by the invention, in-vitro experiments and MALDI-TOF-MS are adopted to detect phosphorylation results, so that the method has the advantages of low sample demand, low drug dosage, no need of complex cell experiments and large-scale sample screening.

Description

technical field [0001] The invention belongs to the field of amino metabolite isomer identification, and specifically relates to a method for screening CDKs family protein kinase inhibitors in vitro based on MALDI-TOF-MS. Background technique [0002] Cyclin-dependent kinases (CDKs) are a group of serine / threonine protein kinases, named after being activated by cyclin. The complete sequence of the Homo sapiens genome shows that, among the 30,000 predicted genes, there are 13 CDKs and 25 Cyclins. Eleven CDKS and their associated cyclins have been identified in humans. [0003] Among numerous kinases, CDKs are involved in many important cellular processes due to the complexity of their actions. They regulate cell division, apoptosis, transcription and differentiation, which are involved in many diseases, such as various human cancers, AIDS and so on. Different subtypes of CDKs play different roles in the process of cell division, apoptosis, transcription, and differentiatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/48G01N33/68G01N27/626
CPCC12Q1/485G01N33/6851G01N27/626G01N2333/912
Inventor 高祥张展鸣陈春景卿萍
Owner XIAMEN UNIV
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