GSK583 possesses comparable binding affinity for RIP3 kinase as demonstrated by an in-house FP binding assay configured similarly to the RIP2 FP assay (RIP2 FP IC50 = 5 nM; RIP3 FP IC50 = 16 nM). Despite this potent biochemical activity against RIP3 kinase, GSK583 shows little or no inhibition of RIP3-dependent necroptotic cell death in a cellular assay up to 10 μM concentration. GSK583 potently and dose dependently inhibits MDP-stimulated tumor necrosis factor-alpha (TNFα) production with an IC50 = 8 nM in primary human monocytes. Following treatment with GSK583 at 1 μM, little inhibition of pro-inflammatory signaling is observed upon activation of Toll-like receptors (TLR2, TLR4, TLR7) or cytokine receptors (IL-1R, TNFR) but complete inhibition is observed upon activation of both NOD1 and NOD2 receptors, which signal in a RIP2-dependent manner. Although GSK583 has excellent kinase selectivity, it does inhibit both the hERG channel and Cyp3A4, which precludes it from further progression as a drug candidate^[1].