Activity and specificity of necrostatin-1, small-molecule inhibitor of RIP1 kinase

Department of Biochemistry, Tufts University, Boston, MA, USA.
Cell death and differentiation (Impact Factor: 8.39). 11/2012; 20(2). DOI: 10.1038/cdd.2012.133
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    ABSTRACT: Cellular necrosis has long been regarded as an incidental and uncontrolled form of cell death. However, a regulated form of cell death termed necroptosis has been identified recently. Necroptosis can be induced by extracellular cytokines, pathogens and several pharmacological compounds, which share the property of triggering the formation of a RIPK3-containing molecular complex supporting cell death. Of interest, most ligands known to induce necroptosis (including notably TNF and FASL) can also promote apoptosis, and the mechanisms regulating the decision of cells to commit to one form of cell death or the other are still poorly defined. We demonstrate herein that intracellular nicotinamide adenine dinucleotide (NAD+) plays an important role in supporting cell progression to necroptosis. Using a panel of pharmacological and genetic approaches, we show that intracellular NAD+ promotes necroptosis of the L929 cell line in response to TNF. Use of a pan-sirtuin inhibitor and shRNA- mediated protein knockdown led us to uncover a role for the NAD+-dependent family of sirtuins, and in particular for SIRT2 and SIRT5, in the regulation of the necroptotic cell death program. Thus, and in contrast to a generally held view, intracellular NAD+ does not represent an universal pro-survival factor, but rather acts as a key metabolite regulating the choice of cell demise in response to both intrinsic and extrinsic factors.
    Cell Death and Differentiation 05/2015; AOP. DOI:10.1038/cdd.2015.60 · 8.39 Impact Factor
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    ABSTRACT: Intracerebral hemorrhage (ICH) is the most common form of hemorrhagic stroke, accounting for 15% of all strokes. ICH has the highest acute mortality and the worst long-term prognosis of all stroke subtypes. Unfortunately, the dearth of clinically effective treatment options makes ICH the least treatable form of stroke, emphasizing the need for novel therapeutic targets. Recent work by our laboratory identified a novel role for the necroptosis inhibitor, necrostatin-1, in limiting neurovascular injury in tissue culture models of hemorrhagic injury. In the present study, we tested the hypothesis that necrostatin-1 reduces neurovascular injury after collagenase-induced ICH in mice. Necrostatin-1 significantly reduced hematoma volume by 54% at 72 h after-ICH, as compared to either sham-injured mice or mice administered an inactive, structural analogue of necrostatin-1. Necrostatin-1 also limited cell death by 48%, reduced blood-brain barrier opening by 51%, attenuated edema development to sham levels, and improved neurobehavioral outcomes after ICH. These data suggest a potential clinical utility for necrostatin-1 and/or novel necroptosis inhibitors as an adjunct therapy to reduce neurological injury and improve patient outcomes after ICH.
    International Journal of Cell Biology 03/2014; 2014:495817. DOI:10.1155/2014/495817
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    ABSTRACT: Metastatic renal cell carcinoma (RCC) is an incurable disease in clear need of new therapeutic interventions. In early-phase clinical trials, the cytokine IFN-γ showed promise as a biotherapeutic for advanced RCC, but subsequent trials were less promising. These trials, however, focused on the indirect immunomodulatory properties of IFN-γ, and its direct anti-tumor effects, including its ability to kill tumor cells, remains mostly unexploited. We have previously shown that IFN-γ induces RIP1 kinase-dependent necrosis in cells lacking NF-κB survival signaling. RCC cells display basally-elevated NF-κB activity, and inhibiting NF-κB in these cells, for example by using the small-molecule proteasome blocker bortezomib, sensitizes them to RIP1-dependent necrotic death following exposure to IFN-γ. While these observations suggest that IFN-γ-mediated direct tumoricidal activity will have therapeutic benefit in RCC, they cannot be effectively exploited unless IFN-γ is targeted to tumor cells in vivo. Here, we describe the generation and characterization of two novel 'immunocytokine' chimeric proteins, in which either human or murine IFN-γ is fused to an antibody targeting the putative metastatic RCC biomarker CD70. These immunocytokines display high levels of species-specific IFN-γ activity and selective binding to CD70 on human RCC cells. Importantly, the IFN-γ immunocytokines function as well as native IFN-γ in inducing RIP1-dependent necrosis in RCC cells, when deployed in the presence of bortezomib. These results provide a foundation for the in vivo exploitation of IFN-γ-driven tumoricidal activity in RCC.
    PLoS ONE 04/2013; 8(4):e61446. DOI:10.1371/journal.pone.0061446 · 3.53 Impact Factor