GSK′963A is a potent and selective inhibitor of RIP1 kinase. (a) Chemical structures of GSK′963A (active analog), GSK′962A (inactive analog) and Necrostatin-1. (b) Dose–response curves for GSK′963, GSK′962 and Nec-1 in the FP binding assay evaluating the affinity of compounds for RIP1 (ATP-binding pocket). Graphs represents n=52 for Nec-1, n=4 for GSK′962A and n=7 for GSK′963A. Error bars indicate S.D. (c) Dose–response curves for GSK′963, GSK′962 and Nec-1 in ADP-Glo kinase assay measuring autophosphorylation of RIP1 kinase domain in vitro. Graph represents n=20 for Nec-1, n=2 for GSK′962A and n=2 for GSK′963A. Error bars indicate S.D. (d) GSK′963 does not block the activity of any of the tested 339 human kinases at 10μM concentration, as evaluated at the Reaction Biology Corporation. Each dot represents an individual kinase with the color indicating the level of inhibition (red>90%, yellow 70–90%, orange 50–70% and green <50%) (e) Effect of GSK′963, GSK′962 and Nec-1 on indoleamine 2,3-dioxygenase (IDO) activity evaluated by an in vitro enzymatic assay. Menadione was used as a positive control for IDO inhibition. The results are representative of three independent experiments. =Nec-1, =GSK′962, =GSK′963 and =Menadione.