In vitro activity: GSK-963 (also known as GSK'963 or GSK963), is a potent and selective RIP1 kinase inhibitor. GSK'963 is significantly more potent than Nec-1 (a known RIP1 kinase inhibitor ) in both biochemical and cellular assays, inhibiting RIP1-dependent cell death with an IC50 of between 1 and 4 nM in human and murine cells. GSK'963 is>10 000-fold selective for RIP1 over 339 other kinases, lacks measurable activity against IDO and has an inactive enantiomer, GSK'962, which can be used to confirm on-target effects. The increased in vitro potency of GSK'963 also translates in vivo, where GSK'963 provides much greater protection from hypothermia at matched doses to Nec-1, in a model of TNF-induced sterile shock. Together, we believe GSK'963 represents a next-generation tool for examining the function of RIP1 in vitro and in vivo, and should help to clarify our current understanding of the role of RIP1 in contributing to disease pathogenesis.

Kinase Assay: Compound potency against RIP1 kinase activity was determined using an ADP-Glo luminescence assay, which measures the conversion of ATP to ADP as previously described. In brief, the primary reaction consisted of 10 nM GST-RIPK1 (1–375) and 50 μM ATP in 50 mM HEPES pH 7.5, 50 mM NaCl, 30 mM MgCl2, 1 mM DTT, 0.5 mg/ml BSA, and 0.02% CHAPS. Five microliter of enzyme and 5 μl of ATP were added to the plate at twice the final assay concentration and incubated at room temperature for 4 h. The luminescence was measured on a plate reader. Test compound inhibition was expressed as percent inhibition of internal assay controls.

Cell Assay: Necroptotic cell death was induced in BMDM, L929 and U937 cells with TNF in the presence of caspase inhibitor zVAD-FMK or QVD-Oph (BMDM: 50 ng/ml TNF+50 μM zVAD; L929: 100 ng/ml TNF+50 μM QVD; U937: 100 ng/ml TNF+25 μM QVD). To evaluate the effect of RIP1 inhibitors, cells were pretreated with compound (dose–response) for 30 min. Induced cell death was evaluated 19–21 h later by measuring cellular ATP levels using CellTiter-Glo Luminescent Cell Viability assay (Promega, Fitchburg, WI, USA). To induce necroptosis in neutrophils, freshly isolated human neutrophils were stimulated with TNF (10 ng/ml), QVD-Oph (50 μM) and SMAC mimetic (100 nM). Induced cell death was evaluated as above.