Necroptosis is an inflammatory form of programmed cell death that is controlled by a defined set of protein mediators, despite displaying the morphological characteristics of unregulated cell death (necrosis). Recently, there has been increasing interest in this type of cell death, ignited by studies demonstrating that necroptosis is involved in the pathophysiology of various human diseases – including inflammatory conditions, degenerative conditions, infectious diseases and cancers – and further kindled by the progression of small molecule necroptosis inhibitors into clinical trials. The best studied form of necroptosis is driven by the tumor necrosis factor (TNF) signalling pathway, which is initiated by TNF binding to its cell surface receptor TNFR1. Importantly, TNF-induced necroptosis is regulated by three key proteins: the kinases RIPK1 and RIPK3, and the pseudokinase MLKL, which acts as the cell death executioner.
In this study, we identified a potent small molecule inhibitor of necroptotic cell death. We investigated the mechanism of action and elucidated the cellular targets of this necroptosis inhibitor using a suite of biochemistry, chemical biology and cell biology approaches. We determined that this small molecule targets all three necroptotic effector proteins – MLKL, RIPK1 and RIPK3 – in vitro and in cells. We established that this inhibitor potently blocks necroptosis in human and murine cells at nanomolar concentrations and shows efficacy in vivo in a murine model of TNF-induced systemic inflammatory response syndrome (SIRS). This study demonstrates that necroptosis can be potently inhibited by targeting multiple effectors, suggesting that targeting multiple proteins in the pathway may be an ideal strategy for inhibiting necroptosis in a therapeutic context.