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
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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:
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[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:
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[0078] CDK9 protein kinase in vitro kinase assay:
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[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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