Necroptosis – A Mechanism of Regulated Pathologic Necrosis
Cell death is conventionally categorized into either regulated apoptosis, characterized by uniform phenotypic and biochemical transitions such as cell shrinking, nuclear condensation and fragmentation, membrane blebbing, or necrosis, defined as a passive, unregulated, catastrophic cellular demise triggered by excessive insult. Necrosis is also characterized by certain phenotypic features, such as organelle swelling, early plasma membrane rupture and oxidative stress. However, these events are more reflective of the catastrophic cellular demise, rather than specific controlled execution events. While necrosis represents a primary component of pathologic tissue injury, it has not been perceived as a therapeutic target, due to the notion that it is an unregulated process. However, this notion has been recently challenged by the discoveries from our and other laboratories, suggesting that necrotic death can result from a specific, regulated cellular pathway, termed necroptosis. This process shares activating stimuli and regulated nature with apoptosis, yet displays morphology characteristics of an unregulated necrotic death. Therefore, the discovery that necrotic death can result from specific cellular regulation, rather than uncontrollable overwhelming stress, may provide a dramatic breakthrough in our ability to therapeutically target pathologic necrosis with selective small molecule inhibitors. Necroptosis can be triggered in cultured cells by “classic” apoptotic stimuli, such as Death domain Receptor (DR) engagement by cognate ligands (TNFα, Fas ligand (FasL) and TRAIL), under the conditions when apoptosis execution is blocked, for example by caspase inhibition. Recent evidence suggest important role of necroptosis in the tissue injury in the animal models of stroke, myocardial infarction, traumatic brain injury, retinal ischemia, acute pancreatitis and bacterial and viral infections in vivo. Our laboratory is interested in understanding pathologic stimuli, activating necroptosis in vivo in animal disease models, in the identification of the specific markers of necroptotic death in vivo and in the development of novel small molecule approaches to inhibition of pathological necroptosis.
Figure 1. Electron microscopy images of apoptosis-deficient human Jurkat T cells (FADD-deficient) undergoing necroptosis in response to TNFα stimulation (top row, arrows). Necroptosis is efficiently inhibited by a specific necroptosis inhibitor (necrostain-1, Nec-1). In contrast, wild type Jurkat cells undergo Nec-1-insensitive apoptosis in response to FasL and cycloheximide treatment (bottom row, arrows).
RIP1 Kinase is a Key Mediator of Necroptosis
The first insight into the mechanism of “programmed necrosis” or “necroptosis” came from the several studies, suggesting that kinase activity of TNF receptor-associated molecule, RIP1, is required for induction of necroptosis in a variety of cell types. Importantly, RIP1 kinase is dispensable for either apoptosis or NFκB activation, two other major pathways induced by DRs. These studies established RIP1 kinase as a first specific mediator of necroptosis. Subsequent experiments suggested that RIP1-related kinase, RIP3, is also involved in necroptosis. However, many questions remain and are the focus of our research. These include: a) What is the mechanistic basis for the activation of RIP1 kinase during necroptosis?; b) What are the critical necroptotic substrates of RIP1 kinase (none of which are currently known)?; c) What are the key signaling and execution pathways activated during necroptosis? We are combining cell based and biochemical analysis of signaling events downstream from RIP1 kinase with high resolution mass spectrometry to address these questions. We are also studying various downstream execution events, such as activation of oxidative stress, mitochondrial dysfunction and induction of autophagy to better understand mechanism of necroptotic demise in both primary cells and various cell lines.
Figure 2. Engagement of TNFα Receptor 1 leads to activation of multiple divergent signaling pathways, resulting in different alternative forms of cell death (apoptosis or necroptosis) or pro-survival/pro-inflammatory NFκB activation.
Necrostatins – Specific Small Molecule Inhibitors of RIP1 Kinase
We are focusing on the characterization of the mechanism of action of small molecule inhibitors of necroptosis, necrostatins. These molecules were originally discovered in a cell based screen of ~50,000 compounds for the inhibitors of cellular necroptosis in human monocytic U-937 cells. Subsequently, these diverse molecules were all found to represent first-in-class inhibitors of RIP1 kinase, highlighting critical role of RIP1 kinase in necrotic cell death. Following initial discovery and optimization, these molecules were found to display unique selectivity towards RIP1 kinase, lacking activity against other targets in human kinome. Necrostatins efficiently (at sub-micromolar concentrations) block necroptotic cell death in various cellular models and provide therapeutic benefit in animal models of stroke, myocardial infarction, retinal ischemia and brain trauma. We are pursuing multiple lines of investigation, including further medicinal chemistry optimization of the necrostatins, development of the necrostatin-based affinity probes, such as fluorescent and crosslinkable necrostatin analogs, biochemical analysis of the mechanism of RIP1 kinase inhibition by various necrostatins, development of novel RIP1 kinase activity assays, and structural analysis of RIP1 complexes with inhibitors.
Figure 3. A) Structures of necrostatins identified in the original cell-based screen. B) Inhibition of necroptosis, induced by TNFa in mouse fibrosarcoma L929 cells, by Nec-1. C) Molecular model of Nec-1 binding to the active site of RIP1 kinase.
Small Molecule Inhibitors of the PI3-kinase Signaling Network
Activation of the phosphoinositide 3-kinase (PI3K) represents one of the key mechanisms controlling regulation of cell viability, growth, metabolism and migration in response to growth factor, neurotrophic and antigen receptor stimulation. Class I PI3Ks (α, β, and γ) catalyze phosphorylation of the lipid phosphatidylinositol-4,5-bisphosphate (PIP2) at the D3 position to generate the second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3) (Figure 4). PIP3 controls all of the abovementioned PI3K-dependent cellular functions through binding to Pleckstrin Homology (PH) domains of a network of effector proteins. PIP3 target proteins are located in the cytosol of unstimulated cells, but in response to the PIP3 synthesis translocate to plasma and other membranes. Membrane translocation and, in some cases, PIP3-induced conformational changes in the target factors initiate a variety of local responses, including polymerization of actin, assembly of signaling complexes, and priming of protein kinase cascades.
Figure 4. Organization of the PI3K/PIP3 signaling network. Steps inhibited by different classes of inhibitors are shown as yellow boxes: 1 – PI3K inhibitors, 2 – PITs, antagonists of PIP3/protein.
Upregulation of PI3K pathway, which occurs through changes in the upstream steps controlling PI3K activity, activating mutations of PI3K isoforms, loss of PIP3 phosphatase PTEN, or activation of downstream PIP3 targets such as Akt kinase, is one of the most frequent alterations in human cancers. Dysregulation of PI3K signaling contributes to tumor formation and growth on multiple levels, including promoting resistance of tumor cells to chemotherapy, increasing their growth rate, causing profound changes in cancer cell metabolism towards anaerobic glycolysis, promoting increased cancer cell invasiveness and, ultimately, metastasis and helping increased formation of blood vessels (“angiogenesis”), feeding a growing tumor mass. Not surprizingly, development of inhibitors of the kinase activity of PI3Ks as anti-cancer agents has attracted significant interest. Several candidate molecules have been developed and are entering early stages of clinical testing. In addition, several downstream effector kinases, e.g. PDK1 and Akt kinases, have also been targeted for inhibition.
We are pursuing a different approach, focusing on the development of small molecule antagonists of PIP3 binding to PH domains (Figure 4, box 2), termed PITENINs. This provides, in principle, a universal method for targeting all aspects of PIP3 signaling either globally (pan-PIP3 antagonists) or, specifically, though developing small molecules antagonizing particular subsets of PIP3/PH domain interactions. These molecules may represent unique and valuable tools for dissecting endogenous signaling pathway activated by PI3K in various cellular environments as well as in vivo. We are pursuing medicinal chemistry optimization of PITENINs, characterization of their effects on PIP3-dependent signaling in various cell types and analysis of the anti-cancer properties of these molecules in the in vivo xenograft tumor models.
Figure 5. Inhibition of growth factor (PDGF) – induced membrane translocation of PH domains (GFP-fused) of Akt and Btk kinases by PITENIN-1 and PITENIN-2, but not by closely related inactive analogs (PITis).