Jenny L. Maki

Interferon-induced RIP1/RIP3-mediated necrosis requires PKR and is licensed by FADD and caspases

Significance The interferons are small secreted proteins with powerful antiviral and cytotoxic properties. Here, we outline a signaling pathway activated by interferons that results in the precipitous necrotic death of susceptible cells. Interferon-induced necrosis proceeds via a novel, progressive mechanism that requires RNA transcription, as well as the sequential activity of three serine-threonine kinases: PKR, RIP1, and RIP3. This pronecrotic kinase cascade is normally held in check by FADD and caspases. As FADD can be disabled by phosphorylation during mitosis, our findings suggest the existence of a putative cell cycle-dependent checkpoint that licenses interferon-induced necrosis. Interferons (IFNs) are cytokines with powerful immunomodulatory and antiviral properties, but less is known about how they induce cell death. Here, we show that both type I (α/β) and type II (γ) IFNs induce precipitous receptor-interacting protein (RIP)1/RIP3 kinase-mediated necrosis when the adaptor protein Fas-associated death domain (FADD) is lost or disabled by phosphorylation, or when caspases (e.g., caspase 8) are inactivated. IFN-induced necrosis proceeds via progressive assembly of a RIP1–RIP3 “necrosome” complex that requires Jak1/STAT1-dependent transcription, but does not need the kinase activity of RIP1. Instead, IFNs transcriptionally activate the RNA-responsive protein kinase PKR, which then interacts with RIP1 to initiate necrosome formation and trigger necrosis. Although IFNs are powerful activators of necrosis when FADD is absent, these cytokines are likely not the dominant inducers of RIP kinase-driven embryonic lethality in FADD-deficient mice. We also identify phosphorylation on serine 191 as a mechanism that disables FADD and collaborates with caspase inactivation to allow IFN-activated necrosis. Collectively, these findings outline a mechanism of IFN-induced RIP kinase-dependent necrotic cell death and identify FADD and caspases as negative regulators of this process.

Small molecule inhibition of phosphatidylinositol-3,4,5-triphosphate (PIP3) binding to pleckstrin homology domains

The PI3-kinase (PI3K) pathway regulates many cellular processes, especially cell metabolism, cell survival, and apoptosis. Phosphatidylinositol-3,4,5-trisphosphate (PIP3), the product of PI3K activity and a key signaling molecule, acts by recruiting pleckstrin-homology (PH) domain-containing proteins to cell membranes. Here, we describe a new structural class of nonphosphoinositide small molecule antagonists (PITenins, PITs) of PIP3–PH domain interactions (IC50 ranges from 13.4 to 31 μM in PIP3/Akt PH domain binding assay). PITs inhibit interactions of a number of PIP3-binding PH domains, including those of Akt and PDK1, without affecting several PIP2-selective PH domains. As a result, PITs suppress the PI3K-PDK1-Akt pathway and trigger metabolic stress and apoptosis. A PIT-1 analog displayed significant antitumor activity in vivo, including inhibition of tumor growth and induction of apoptosis. Overall, our studies demonstrate the feasibility of developing specific small molecule antagonists of PIP3 signaling.

The SCAN domain defines a large family of zinc finger transcription factors.

The SCAN domain is a highly conserved dimerization motif that is vertebrate-specific and found near the N-terminus of C(2)H(2) zinc finger proteins (SCAN-ZFP). Although the function of most SCAN-ZFPs is unknown, some have been implicated in the transcriptional regulation of growth factors, genes involved in lipid metabolism, as well as other genes involved in cell survival and differentiation. Here we utilize a bioinformatics approach to define the structures and gene locations of the 71 members of the human SCAN domain family, as well as to assess the conserved syntenic segments in the mouse genome and identify potential orthologs. The genes encoding SCAN domains are clustered, often in tandem arrays, in both the human and mouse genomes and are capable of generating isoforms that may affect the function of family members. Twenty-three members of the mouse SCAN family appear to be orthologous with human family members, and human-specific cluster expansions were observed. Remarkably, the SCAN domains in lower vertebrates are not associated with C(2)H(2) zinc finger genes, but are contained in large retrovirus-like polyproteins. Collectively, these studies define a large family of vertebrate-specific transcriptional regulators that may have rapidly expanded during recent evolution.

Inflammatory Signaling by NOD-RIPK2 Is Inhibited by Clinically Relevant Type II Kinase Inhibitors.

Summary RIPK2 mediates pro-inflammatory signaling from the bacterial sensors NOD1 and NOD2, and is an emerging therapeutic target in autoimmune and inflammatory diseases. We observed that cellular RIPK2 can be potently inhibited by type II inhibitors that displace the kinase activation segment, whereas ATP-competitive type I inhibition was only poorly effective. The most potent RIPK2 inhibitors were the US Food and Drug Administration-approved drugs ponatinib and regorafenib. Their mechanism of action was independent of NOD2 interaction and involved loss of downstream kinase activation as evidenced by lack of RIPK2 autophosphorylation. Notably, these molecules also blocked RIPK2 ubiquitination and, consequently, inflammatory nuclear factor κB signaling. In monocytes, the inhibitors selectively blocked NOD-dependent tumor necrosis factor production without affecting lipopolysaccharide-dependent pathways. We also determined the first crystal structure of RIPK2 bound to ponatinib, and identified an allosteric site for inhibitor development. These results highlight the potential for type II inhibitors to treat indications of RIPK2 activation as well as inflammation-associated cancers.

Assays for necroptosis and activity of RIP kinases.

Necrosis is a primary form of cell death in a variety of human pathologies. The deleterious nature of necrosis, including its propensity to promote inflammation, and the relative lack of the cells displaying necrotic morphology under physiologic settings, such as during development, have contributed to the notion that necrosis represents a form of pathologic stress-induced nonspecific cell lysis. However, this notion has been challenged in recent years by the discovery of a highly regulated form of necrosis, termed regulated necrosis or necroptosis. Necroptosis is now recognized by the work of multiple labs, as an important, drug-targetable contributor to necrotic injury in many pathologies, including ischemia-reperfusion injuries (heart, brain, kidney, liver), brain trauma, eye diseases, and acute inflammatory conditions. In this review, we describe the methods to analyze cellular necroptosis and activity of its key mediator, RIP1 kinase.

Use of synthetic signal sequences to explore the protein export machinery

The information for correct localization of newly synthesized proteins in both prokaryotes and eukaryotes resides in self‐contained, often transportable targeting sequences. Of these, signal sequences specify that a protein should be secreted from a cell or incorporated into the cytoplasmic membrane. A central puzzle is presented by the lack of primary structural homology among signal sequences, although they share common features in their sequences. Synthetic signal peptides have enabled a wide range of studies of how these “zipcodes” for protein secretion are decoded and used to target proteins to the protein machinery that facilitates their translocation across and integration into membranes. We review research on how the information in signal sequences enables their passenger proteins to be correctly and efficiently localized. Synthetic signal peptides have made possible binding and crosslinking studies to explore how selectivity is achieved in recognition by the signal sequence‐binding receptors, signal recognition particle, or SRP, which functions in all organisms, and SecA, which functions in prokaryotes and some organelles of prokaryotic origins. While progress has been made, the absence of atomic resolution structures for complexes of signal peptides and their receptors has definitely left many questions to be answered in the future.

Expression and purification of active receptor interacting protein 1 kinase using a baculovirus system

Receptor Interacting Protein 1 (RIP1) kinase is one of the key mediators of tumor necrosis factor alpha (TNF-α) signaling and is critical for activation of necroptotic cell death. We developed a method for expression of recombinant kinase, utilizing baculovirus co-infection of Cdc37, an Hsp90 co-chaperone, and RIP1-His, followed by a two-step purification scheme. After optimization, 1-3mg of highly purified RIP1 kinase was typically obtained from a 1L of Sf9 cells. The recombinant protein displayed kinase activity that was blocked by RIP1 inhibitors, necrostatins. The purified protein was used to develop a simple and robust thermal shift assay for further assessment of RIP1 inhibitors.

Activity assays for receptor-interacting protein kinase 1:a key regulator of necroptosis.

Necroptosis is a novel form of regulated non-apoptotic cell death, which displays morphological features of necrosis. The kinase activity of receptor-interacting protein kinase 1 (RIP1) is a critical component in signaling for necroptosis. The development of assays to evaluate RIP1 kinase activity is important in the further development of existing and novel inhibitors of necroptosis. Here, we describe RIP1 protein expression and purification from mammalian and insect cells as well as two in vitro kinase assays to detect RIP1 kinase activity and inhibition.

Abstract SY27-03: Development of necrostatins, small molecule inhibitors of RIP1-kinase signaling and necroptosis

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