A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis

Cell Death Dis. 2015 May 21;6(5):e1767. doi: 10.1038/cddis.2015.130.

Abstract

Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Necroptotic cell death contributes to the pathophysiology of several disorders involving tissue damage, including myocardial infarction, stroke and ischemia-reperfusion injury. However, no inhibitors of necroptosis are currently in clinical use. Here we performed a phenotypic screen for small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α)-induced necroptosis in Fas-associated protein with death domain (FADD)-deficient Jurkat cells using a representative panel of Food and Drug Administration (FDA)-approved drugs. We identified two anti-cancer agents, ponatinib and pazopanib, as submicromolar inhibitors of necroptosis. Both compounds inhibited necroptotic cell death induced by various cell death receptor ligands in human cells, while not protecting from apoptosis. Ponatinib and pazopanib abrogated phosphorylation of mixed lineage kinase domain-like protein (MLKL) upon TNF-α-induced necroptosis, indicating that both agents target a component upstream of MLKL. An unbiased chemical proteomic approach determined the cellular target spectrum of ponatinib, revealing key members of the necroptosis signaling pathway. We validated RIPK1, RIPK3 and transforming growth factor-β-activated kinase 1 (TAK1) as novel, direct targets of ponatinib by using competitive binding, cellular thermal shift and recombinant kinase assays. Ponatinib inhibited both RIPK1 and RIPK3, while pazopanib preferentially targeted RIPK1. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis highlights them as potentially interesting for the treatment of pathologies caused or aggravated by necroptotic cell death.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • Animals
  • Apoptosis / drug effects
  • Cell Line, Tumor
  • Fas-Associated Death Domain Protein / genetics
  • HEK293 Cells
  • HT29 Cells
  • Humans
  • Imidazoles / pharmacology*
  • Jurkat Cells
  • L Cells
  • MAP Kinase Kinase Kinases / antagonists & inhibitors
  • MAP Kinase Kinase Kinases / metabolism
  • Mice
  • Necrosis / drug therapy*
  • Phosphorylation / drug effects
  • Protein Binding / drug effects
  • Protein Kinase Inhibitors / pharmacology
  • Protein Kinases / metabolism
  • Pyridazines / pharmacology*
  • Pyrimidines / pharmacology*
  • Receptor-Interacting Protein Serine-Threonine Kinases / antagonists & inhibitors*
  • Receptor-Interacting Protein Serine-Threonine Kinases / metabolism
  • Sulfonamides / pharmacology*
  • Tumor Necrosis Factor-alpha / metabolism

Substances

  • FADD protein, human
  • Fas-Associated Death Domain Protein
  • Imidazoles
  • Protein Kinase Inhibitors
  • Pyridazines
  • Pyrimidines
  • Sulfonamides
  • Tumor Necrosis Factor-alpha
  • ponatinib
  • pazopanib
  • MLKL protein, human
  • Protein Kinases
  • RIPK1 protein, human
  • RIPK3 protein, human
  • Receptor-Interacting Protein Serine-Threonine Kinases
  • MAP Kinase Kinase Kinases
  • MAP kinase kinase kinase 7