Abstract SY27-03: Development of necrostatins, small molecule inhibitors of RIP1-kinase signaling and necroptosis
Authors: Maki Jenny L. , Teng Xin , Smith Elizabeth E. , Ray Soumya , Yuan Junying , Cuny Gregory D. , Degterev Alexei 
Published: August 2012

Abstract
Receptor Interacting Protein 1 (RIP1) kinase, a component of the Tumor Necrosis Factor alpha (TNFα) receptor signaling complexes, has recently emerged as a new important mediator of cell death signaling. Firstly, RIP1, in complex with a homologous RIP3 kinase, mediates activation of regulated necrotic death, termed necroptosis. Necroptosis is activated by TNFα family members as well as other signals under apoptosis-deficient conditions. Secondly, recent evidence suggests that in some cases RIP1 kinase can also contribute to the induction of apoptosis through the formation of the “ripoptosome” complex, leading to the activation of caspase-8. Ser/Thr kinase activity of RIP1 plays a critical role in the activation of both forms of cell death.
Necrosis is a major form of cell death contributing to most acute pathologic injuries. Necroptosis displays all the major hallmarks of pathologic necrosis. This raises an intriguing possibility that RIP1-dependent regulated necroptosis, rather than the unregulated passive necrosis, may be responsible for the pathologic injury in vivo in various necrotizing diseases and, furthermore, that these injuries may be targeted therapeutically by inhibitors of necroptotic signaling. Indeed, emerging evidence suggests that inhibition of RIP1 kinase activity or genetic deletion of RIP3 kinase provides significant tissue and functional protection in a variety of mouse models of human disease, including those of stroke, myocardial infarction, retinal injury, septic shock and acute pancreatitis. On the other hand, recent evidence also suggest that under physiologic conditions necroptosis is activated as a secondary response to the genetic loss of several apical apoptosis regulators, such as caspase-8 and FADD, indicating that normally necroptosis may act as a surveillance mechanism ensuring the integrity of the apoptotic signaling machinery.
In order to explore therapeutic potential of necroptosis inhibition, we identified several structurally diverse small molecule inhibitors of necroptosis, termed necrostatins, in a high throughput screen for the suppressors of TNFα-induced necroptotic death. These molecules are specific and efficient inhibitors of necrotic death in a variety of cellular models of necroptosis. Medicinal chemistry optimization of necrostatins led to significantly improved biologic activity, resulting in a panel of four diverse sub-micromolar, metabolically stable small molecule inhibitors of cell death. Our further studies indicated that even though necrostatins were selected in a random cell-based screen, all four molecules specifically target RIP1 kinase in necroptotic cells. Importantly, we found that all necrostatins lack activity in RIP1-deficient cells. This finding highlights the role of RIP1 kinase as a critical druggable target in necroptosis pathway. Furthermore, selectivity profiling against a panel of human kinases, revealed that necrostatin-1 (Nec-1), displays exquisite selectivity for RIP1.
The critical role of RIP1 kinase in necroptosis raises the need to develop methods to study RIP1 kinase activity in vitro to understand the molecular mechanism of RIP1 kinase inhibition by the different necrostatins. For that, we optimized the expression and purification of recombinant RIP1 kinase in Sf9 cells. Using recombinant kinase, we have developed several new assays to measure both catalytic activity and RIP1/necrostatin binding interactions in vitro, including methods utilizing scintillation proximity and homogeneous time resolved fluorescence. In addition, fluorescent and photo-crosslinkable necrostatins analogs were developed to directly access binding of the inhibitors to recombinant RIP1 kinase. These assays provide useful tools to study RIP1 kinase activity and can be adapted to high throughput screening for use in the discovery of novel RIP1 kinase inhibitors.
We further discovered that all necrostatins likely target overlapping, but not identical sites in the active center of RIP1 kinase and these differences in binding sites result in significantly different modes of RIP1 inhibition. Strikingly, this translates into dramatic differences in the potency of inhibition of necroptosis in different cell types by the distinct structural classes of necrostatins. Notably, some of these molecules differentiate between human and mouse RIP1 kinases, despite ∼85% identity in protein sequence. Using bioinformatics, molecular modeling and site-directed mutagenesis approaches, we begun to look into critical structural determinants that may be responsible for the activity of necrostatins and their selectivity towards particular species of RIP1 kinase.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY27-03. doi:1538-7445.AM2012-SY27-03