The Comparison Of Three Non-radioactivity Homogeneous Kinase Activity Detection Assays

Kinase over expression can induce cancer,Anaplastic Lymphoma kinase?ALK?always regarded as driver genes in the generation of tumors.Hitherto,there is no study focus on ALK biochemical test.This article aims to introduce three different principle non-radioactivity homogeneous kinase activity detection assays,ADP-Glo,HTRF and Z-lyte separately.We chose ALK as the target,setup and optimize three methods.Then we determined the enzyme concentration?apparent ATP Km?substrate peptide Km?DMSO tolerance and plate uniformity etc.It is very important and meaningful to screen and obtain efficient ALK-targeting inhibitors,which can developed into excellent anti-cancer drugs.So far,there are many ALK-targeting inhibitors have been approved by Food and Drug Administration?FDA?,for example,Crizotinib,Ceritinib,and Alectinib and so on.Previous research showed that,the first-generation ALK inhibitor,Crizotinib showed a good efficiency in clinic,however,most patients develop acquired resistance to Crizotinib.In order to address the problem,screen and obtain more ALK inhibitors need to be solved.In this research,we detected 41 compounds IC₅₀ values,including 5 kinase inhibitors with known IC₅₀ values.As the results indicated,the assays showed reproducibility and stability,all the three technologies can applied to the high-throughput screening.According to the results of the comparison among three methods,we presented the advantages and disadvantages among three assay technologies:ADP-Glo assay is no claim for substrate and antibody;HTRF assay can screen potent compounds with less kinase concentration;Z-lyte assay is stable and fast.We should select the above methods according to different research stages and research contents,and provided meaningful reference value for searching novel kinase inhibitors.