title: A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis.
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.
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
authors.forename: A
authors.initials: A
authors.lastname: Fauster
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
authors.forename: M
authors.identifier: 0000000161724211
authors.initials: M
authors.lastname: Rebsamen
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
authors.forename: K V M
authors.initials: KV
authors.lastname: Huber
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
authors.forename: J W
authors.initials: JW
authors.lastname: Bigenzahn
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
authors.forename: A
authors.initials: A
authors.lastname: Stukalov
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
authors.forename: C-H
authors.initials: CH
authors.lastname: Lardeau
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
authors.forename: S
authors.initials: S
authors.lastname: Scorzoni
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
authors.forename: M
authors.initials: M
authors.lastname: Bruckner
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
authors.forename: M
authors.initials: M
authors.lastname: Gridling
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
authors.forename: K
authors.initials: K
authors.lastname: Parapatics
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
authors.forename: J
authors.initials: J
authors.lastname: Colinge
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
authors.forename: K L
authors.initials: KL
authors.lastname: Bennett
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
authors.forename: S
authors.initials: S
authors.lastname: Kubicek
authors.affiliation: Division of Nephrology and Hypertension, Christian-Albrechts-University, Kiel 24105 Germany.
authors.forename: S
authors.initials: S
authors.lastname: Krautwald
authors.affiliation: Division of Nephrology and Hypertension, Christian-Albrechts-University, Kiel 24105 Germany.
authors.forename: A
authors.identifier: 0000000162879725
authors.initials: A
authors.lastname: Linkermann
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
authors.forename: G
authors.identifier: 0000000205701768
authors.initials: G
authors.lastname: Superti-Furga
mesh_terms: D016475:3T3 Cells; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D053200:Fas-Associated Death Domain Protein / Q000235:genetics; D057809:HEK293 Cells; D019073:HT29 Cells; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D007191:Indazoles; D019169:Jurkat Cells; D007739:L Cells; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051379:Mice; D009336:Necrosis / Q000188:drug therapy*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D011724:Pyridazines / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D000099151:MAP Kinase Kinase Kinase 7
chemical_list: D053200:Fas-Associated Death Domain Protein; D007093:Imidazoles; D007191:Indazoles; D020930:MAP Kinase Kinase Kinases; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D011724:Pyridazines; D011743:Pyrimidines; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D013449:Sulfonamides; D014409:Tumor Necrosis Factor-alpha; D000099151:MAP Kinase Kinase Kinase 7; C505533:FADD protein, human; C545373:ponatinib; C516667:pazopanib; C570693:MLKL protein, human; C506249:RIPK1 protein, human; C119705:RIPK3 protein, human