Necrosis, a catastrophic cell death caused by overwhelming stress, is a major contributor to human disease. However, very little effort has been made to develop therapies targeting pathologic necrosis due to its perceived uncontrollable nature. This notion has been recently challenged through the discovery that necrosis can result from the activation of intrinsic cellular regulatory pathways, suggesting that necrosis can be specifically targeted for inhibition. As a direct demonstration of the feasibility of this approach, we have developed potent and selective small molecule inhibitors of such regulated necrosis or "necroptosis". Furthermore, we have used one of these molecules, Necrostatin-1, to demonstrate that necroptosis is an important component of acute pathologic injury in vivo in the case of ischemic brain and heart damage and endotoxic liver destruction. Discovery of necrostatins provides an unprecedented opportunity to develop novel necrosis-specific therapies for human disease. In Preliminary studies, we identified five structurally distinct necrostatins in a cell based high-throughput screen of 100,000+ compounds. Surprisingly, all necrostatins inhibit necroptosis at the level of RIP kinase complex, an established mediator of necroptosis induction, suggesting that it is a key step of this pathway susceptible to inhibition. Interestingly, while all necrostatins act through a similar target, they display distinct activities in different cells, suggesting the possibility of developing cell type-specific inhibitors. In this proposal, we will further investigate the mechanism of necrostatins'activity in vitro and in vivo.
In Aim 1, we will investigate in vitro interactions of necrostatins with RIP and associated factors using combination of biochemical, mass spectrometry, medicinal chemistry and structural biology methods.
In Aim 2, we will apply the insights from Aim 1 to identify key parameters defining differential cellular sensitivity to necrostatins in order to develop specific markers for predicting optimal strategy for necroptosis suppression in various paradigms of pathologic necrosis.
In Aim 3, we propose to study how in vitro activities of necrostatins translate into therapeutic benefit in the animal model of acute liver injury. Overall, our project should further define necroptosis as the key component of pathologic injury and provide mechanistic basis for development of novel necroptosis-specific therapies.

Public Health Relevance

Necrosis is a key component of acute tissue injury in many human diseases from stroke and myocardial infarction to septic shock. The goal of our proposal is to characterize the mechanism of action of novel chemical inhibitors of cellular necrosis, identified in our preliminary studies, and establish their potential as therapeutic agents in animal models of human disease. These molecules may present a principally novel direction for drug development for many devastating human disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM084205-04
Application #
8206661
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Maas, Stefan
Project Start
2009-01-01
Project End
2013-06-30
Budget Start
2012-01-01
Budget End
2013-06-30
Support Year
4
Fiscal Year
2012
Total Cost
$371,857
Indirect Cost
$123,106
Name
Tufts University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
039318308
City
Boston
State
MA
Country
United States
Zip Code
02111
Degterev, Alexei; Zhou, Wen; Maki, Jenny L et al. (2014) Assays for necroptosis and activity of RIP kinases. Methods Enzymol 545:1-33
Dvoriantchikova, Galina; Degterev, Alexei; Ivanov, Dmitry (2014) Retinal ganglion cell (RGC) programmed necrosis contributes to ischemia-reperfusion-induced retinal damage. Exp Eye Res 123:1-7
McNamara, Colleen R; Ahuja, Ruchita; Osafo-Addo, Awo D et al. (2013) Akt Regulates TNF? synthesis downstream of RIP1 kinase activation during necroptosis. PLoS One 8:e56576
Maki, Jenny L; Degterev, Alexei (2013) Activity assays for receptor-interacting protein kinase 1:a key regulator of necroptosis. Methods Mol Biol 1004:31-42
Maki, Jenny L; Tres Brazell, J; Teng, Xin et al. (2013) Expression and purification of active receptor interacting protein 1 kinase using a baculovirus system. Protein Expr Purif 89:156-61
Thapa, Roshan J; Nogusa, Shoko; Chen, Peirong et al. (2013) Interferon-induced RIP1/RIP3-mediated necrosis requires PKR and is licensed by FADD and caspases. Proc Natl Acad Sci U S A 110:E3109-18
McNamara, Colleen R; Degterev, Alexei (2011) Small-molecule inhibitors of the PI3K signaling network. Future Med Chem 3:549-65