CHEMBL5735154

Inhibitory Activity Assay: The in-vitro kinase assay was performed using the HTRF kinEASE TK kit available from Cisbio. The operation steps are indicated in the instructions of the kit. This method was used to detect the inhibitory effect of the compound to be tested on the activity of ALK enzymes in vitro, including wild-type ALK (Cat.PV3867, Invitrogen), ALK L1196M (Cat. PV6168, Life technologies), and ALK F1174L (Cat. PV6160, Life technologies). The specific operation steps were as follows.(1) First, a 2.5% DMSO solution was prepared with 1× kinase buffer previously formulated (where a too high concentration of DMSO had influence on the reaction, and the final concentration of DMSO was controlled to 1%), and then the compound to be tested was diluted with the 2.5% DMSO solution corresponding to the enzyme. The compound had a screening concentration of 100 nM, 10 nM and 1 nM. In addition to the control wells, 4 μL of the diluted compound solution to be tested was added to the reaction wells, and 4 μL of the 2.5% DMSO solution corresponding to the ALK enzyme previously formulated was added to the control wells.(2) 2 μL of a TK-biotin substrate solution previously formulated in a substrate concentration corresponding to the ALK enzyme was added to all reaction wells.(3) 2 μL of a previously prepared enzyme solution of corresponding concentration was added to all the reaction wells except the negative wells, and 2 μL of the 1× kinase buffer corresponding to the enzyme was added to the negative wells to make up the volume. The plate was sealed with a membrane, and incubated for 10 min at room temperature after mixing uniformly, such that the compounds were bond to the enzyme fully.(4) 2 μL of an ATP solution in a concentration corresponding to the ALK enzyme was added to all the reaction wells to initiate the kinase reaction, where the enzyme reaction time by ALK was 60 minutes.(5) An ALK test solution was prepared 5 minutes before the end of the kinase reaction. Streptavidin-XL665 and TK antibody europium cryptate (1:100) solutions for assay having a concentration corresponding to the enzyme were prepared using the detection buffer in the kit.(6) After the completion of the kinase reaction, 5 μl of diluted Streptavidin-XL665 was added to all the reaction wells respectively, and mixed uniformly, and then the diluted TK antibody europium cryptate solution for assay was added immediately.(7) The plate was sealed, mixed uniformly and allowed to react at room temperature for 1 h. The fluorescence signal (excitation at 320 nm, and emission at 665 nm and 615 nm) was detected using the ENVISION instrument (Perkinelmer). The inhibition rate for each well was calculated from the values of the fully active wells and the background signal wells. The values of replicated wells were averaged, and the half-maximal inhibitory activity (IC50) of each compound to be tested was fitted with a professional drawing analysis software PRISM 5.0.