A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis.

M Bruckner,S Scorzoni,A Stukalov,J Colinge,J W Bigenzahn,S Kubicek,S Krautwald,M Gridling,A Linkermann,G Superti-Furga,A Fauster,K Parapatics,K L Bennett,K V M Huber,M Rebsamen,C-H Lardeau

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.

Highlights

Given that many viruses have developed strategies to circumvent apoptotic cell death, necroptosis constitutes an important, pro-inflammatory back-up mechanism that limits viral spread in vivo.[7,8,9] In contrast, in the context of sterile inflammation, necroptotic cell death contributes to disease pathology, outlining potential benefits of therapeutic intervention.[10]
To identify novel necroptosis inhibitors for potential clinical use, we screened a panel of 268 Food and Drug Administration (FDA)-approved drugs with diverse mechanisms of action for their ability to block tumor necrosis factor (TNF)-αinduced necroptosis in Fas-associated protein with death domain (FADD) deficient human Jurkat Tcells[36,37] (Figure 1a)
Pazopanib is an oral receptor tyrosine kinase inhibitor approved for treatment of patients with advanced renal cell carcinoma and soft tissue sarcoma[40,41] targeting vascular endothelial growth factor receptor (VEGFR)-1, -2, -3, platelet-derived growth factor receptor β (PDGFRβ) and c-Kit.[42]

Summary

Introduction

Given that many viruses have developed strategies to circumvent apoptotic cell death, necroptosis constitutes an important, pro-inflammatory back-up mechanism that limits viral spread in vivo.[7,8,9] In contrast, in the context of sterile inflammation, necroptotic cell death contributes to disease pathology, outlining potential benefits of therapeutic intervention.[10]. Received 16.2.15; revised 31.3.15; accepted 06.4.15; Edited by M Agostini structurally distinct kinase inhibitors pazopanib and ponatinib as potent blockers of necroptosis targeting the key enzymes RIPK1/3

Methods

Results

Conclusion