AS-604850 is a specific and ATP-competitive PI3Kγ inhibitor (IC50: 250 nM), 18-fold more selective for PI3Kγ than PI3Kα, and over 80-fold selectivity for PI3Kγ than PI3Kδ/β.

PI3Kα:4.5 μM, PI3Kγ:250 nM

In vitro PI3Kγ Kinase Assay: Human PI3Kγ (100 ng) is incubated at RT with kinase buffer (10 mM MgCl2, 1 mM β-glycerophosphate, 1 mM DTT, 0.1 mM Na3VO4, 0.1% Na Cholate and 15 M ATP/100 nCi γ[33]ATP, final concentrations) and lipid vesicles containing 18 M PtdIns and 250 M of PtdSer (final concentrations), in the presence of AS-252424 or DMSO. Kinase reaction is stopped by adding 250 g of Neomycin-coated Scintillation Proximity Assay (SPA) beads and preceded.

Hepatocyte cultures are treated with diluent (DMSO), 25 μM TLC, 250 μM TCDC, 50 μM GCDC, or 50 ng/ml Fas for 2-4 hours HepG2-Ntcp and Huh7-Ntcp cells are treated with DMSO, 20 μM TLC, 75 μM TCDC or GCDC, 200 μM etoposide or 200 ng/ml TNFa plus 28 ng/ml actinomycin D for 2-4 hours. The number of apoptotic cells is determined morphologically using fluorescent staining and expressed as % of cells. Apoptosis is confirmed in human cell lines by determination of caspase-3/-7 activity and in rat hepatocytes by detection of the 17 kDa proteolytic cleavage fragment of caspase-3 by immunoblotting; equal protein loading is monitored by immunoblotting for actin.(Only for Reference)