Kinase assays IC50 determination

The potency and selectivity of CPL302-253 against different isotypes of class I PI3K were assessed using ADP-Glo Kinase Assay (Promega) according to manufacturer’s protocol. PI3Kα, PI3Kβ, PI3Kδ, PI3Kγ were purchased from Merck Millipore. The inhibition of examined kinases activity was evaluated by measuring their ability to converse ATP to ADP in the presence of decreasing doses of tested compound (from 400 μM to 0,00004 μM). Kinases were mixed with 1 mM PIP2:PS substrate (Phosphoinositol-4,5-bisphosphate (PIP2) lipid vesicles with phosphoserine (PS), Thermofisher Scientific) and 30 μM ATP in an assay buffer (50 mM HEPES pH 7.5,50 mM NaCl, 3 mM MgCl2, 25 μg/ml BSA). The mixture was incubated in the dark for 1 h at 25°C (PI3Kα and PI3Kδ) or at 30°C (PI3Kβ, PI3Kγ). Next ADP-Glo reagent 1 was added to stop the reaction and remove any remaining ATP. The samples were incubated for 40 min at 25°C. Subsequently the ADP-Glo reagent 2 was added and reactions were incubated for 40 min at 25°C. Finally, luminescence was assessed and the IC50 was calculated.