Potent Inhibition of Necroptosis by Simultaneously Targeting Multiple Effectors of the Pathway

Abstract

Necroptosis is an inflammatory form of programmed cell death that has been implicated in various human diseases. Compound <b>2</b> is a more potent analogue of the published compound <b>1</b> and inhibits necroptosis in human and murine cells at nanomolar concentrations. Several target engagement strategies were employed, including cellular thermal shift assays (CETSA) and diazirine-mediated photoaffinity labeling via a bifunctional photoaffinity probe derived from compound <b>2</b>. These target engagement studies demonstrate that compound <b>2</b> binds to all three necroptotic effector proteins (mixed lineage kinase domain-like protein (MLKL), receptor-interacting serine/threonine protein kinase 1 (RIPK1) and receptor-interacting serine/threonine protein kinase 3 (RIPK3)) at different levels <i>in vitro</i> and in cells. Compound <b>2</b> also shows efficacy <i>in vivo</i> in a murine model of systemic inflammatory response syndrome (SIRS).