In vitro PI3K gamma kinase assay

The in vitro PI3K gamma kinase assay was carried out using a PI3-Kinase (human) HTRF^TM Assay kit (Millipore, USA) according to the manufacturer’s protocol. Briefly, the PI3K gamma kinase was incubated with 10 μM sample (tested compounds) in the reaction buffer containing 10 μM PIP2 in the wells of a 384-well opaque black plate (PerkinElmer, USA) at room temperature. Kinase reactions were initiated by adding 10 μM ATP and incubating the plate at room temperature for 1 h. The reactions were quenched by adding 5 μL of Stop Solution (containing EDTA and biotin-PIP3), and then the detection buffer (containing the Europium-labeled anti-GST antibody, the GST-tagged receptor of the phosphoinositide (GRP1) PH domain, and streptavidin–APC) was added to each well. Following 2 h incubation in the dark, the HTRF signal was measured with a 2104 EnVision® Multilabel Reader (PerkinElmer, USA) in the time-resolved fluorescence mode, set for excitation at 320 nm and dual emission detection at 615 nm (Eu) and 665 nm (APC). The emission ratio (ER) of 665 nm emission signal to 615 nm emission signal was then calculated and the inhibition ratio of a certain sample on PI3K was calculated according to the following formula: PI3K inhibition (%) = (ER_sample − ER_0%)/(ER_100% − ER_0%) × 100%. ER_0% means 0% inhibition, which was calculated from the reaction of PI3K, PIP2, and ATP in the absence of sample. ER_100% means 100% inhibition, which was calculated from the reaction of PIP2 and ATP without PI3K. BEZ235 was used as a positive control. For active inhibitors (relative inhibitory activity ≥50%), the IC₅₀ values were estimated using the “Sigmoidal dose-response (variable slope)” equation in the GraphPad Prism® Version 5.0 software, with the maximum and minimum of the curve constrained to 100 and 0, respectively.