PI3K enzyme inhibition assays

The assay was performed using homogenous time resolved fluorescence (HTRF) based assay kit as per the manufacturer instructions. Briefly, phospotidyl inositol bis phosphate (PIP2) was added to the working reaction buffer that contained desired isoform of PI3K enzyme (delta/gamma) along with appropriate drug and enzyme controls. After adding the desired concentrations of CB-006-3 along with suitable blanks, the reaction mixture was pre incubated for ten minutes at room temperature followed by optimal concentration of ATP. After 30 min incubation at room temperature, the reaction was terminated using stop solution. Detection mixture from the kit was added to all wells, followed by six-hour incubation in dark. HTRF ratio was measured at excitation 337 nM, emission 665 nm, with a delay of 50 micro seconds delay and counting window of 400 micro seconds using FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany). The percentage of inhibition was normalized to control and IC₅₀ determined using GraphPad prism software.