Alexei Degterev

Identification of RIP1 kinase as a specific cellular target of necrostatins.

Necroptosis is a cellular mechanism of necrotic cell death induced by apoptotic stimuli in the form of death domain receptor engagement by their respective ligands under conditions where apoptotic execution is prevented. Although it occurs under regulated conditions, necroptotic cell death is characterized by the same morphological features as unregulated necrotic death. Here we report that necrostatin-1, a previously identified small-molecule inhibitor of necroptosis, is a selective allosteric inhibitor of the death domain receptor-associated adaptor kinase RIP1 in vitro. We show that RIP1 is the primary cellular target responsible for the antinecroptosis activity of necrostatin-1. In addition, we show that two other necrostatins, necrostatin-3 and necrostatin-5, also target the RIP1 kinase step in the necroptosis pathway, but through mechanisms distinct from that of necrostatin-1. Overall, our data establish necrostatins as the first-in-class inhibitors of RIP1 kinase, the key upstream kinase involved in the activation of necroptosis.

A decade of caspases.

Caspases are a family of cysteine proteases that play important roles in regulating apoptosis. A decade of research has generated a wealth of information on the signal transduction pathways mediated by caspases, the distinct functions of individual caspases and the mechanisms by which caspases mediate apoptosis and a variety of physiological and pathological processes.

Identification of a Molecular Signaling Network that Regulates a Cellular Necrotic Cell Death Pathway

Stimulation of death receptors by agonists such as FasL and TNFalpha activates apoptotic cell death in apoptotic-competent conditions or a type of necrotic cell death dependent on RIP1 kinase, termed necroptosis, in apoptotic-deficient conditions. In a genome-wide siRNA screen for regulators of necroptosis, we identify a set of 432 genes that regulate necroptosis, a subset of 32 genes that act downstream and/or as regulators of RIP1 kinase, 32 genes required for death-receptor-mediated apoptosis, and 7 genes involved in both necroptosis and apoptosis. We show that the expression of subsets of the 432 genes is enriched in the immune and nervous systems, and cellular sensitivity to necroptosis is regulated by an extensive signaling network mediating innate immunity. Interestingly, Bmf, a BH3-only Bcl-2 family member, is required for death-receptor-induced necroptosis. Our study defines a cellular signaling network that regulates necroptosis and the molecular bifurcation that controls apoptosis and necroptosis.

Identification of small-molecule inhibitors of interaction between the BH3 domain and Bcl-xL

To study the role of the BH3 domain in mediating pro-apoptotic and anti-apoptotic activities of Bcl-2 family members, we identified a series of novel small molecules (BH3Is) that inhibit the binding of the Bak BH3 peptide to Bcl-xL. NMR analyses revealed that BH3Is target the BH3-binding pocket of Bcl-xL. Inhibitors specifically block the BH3-domain-mediated heterodimerization between Bcl-2 family members in vitro and in vivo and induce apoptosis. Our results indicate that BH3-dependent heterodimerization is the key function of anti-apoptotic Bcl-2 family members and is required for the maintenance of cellular homeostasis.

Diversity in the Mechanisms of Neuronal Cell Death

Neurons may die as a normal physiological process during development or as a pathological process in diseases. The best-understood mechanism of neuronal cell death is apoptosis, which is regulated by an evolutionarily conserved cellular pathway that consists of the caspase family, the Bcl-2 family, and the adaptor protein Apaf-1. Apoptosis, however, may not be the only cellular mechanism that regulates neuronal cell death. Neuronal cell death may exhibit morphological features of autophagy or necrosis, which differ from that of the canonical apoptosis. This review evaluates the evidence supporting the existence of alternative mechanisms of neuronal cell death and proposes the possible existence of an evolutionarily conserved pathway of necrosis.

Expansion and evolution of cell death programmes

Cell death has historically been subdivided into regulated and unregulated mechanisms. Apoptosis, a form of regulated cell death, reflects a cells decision to die in response to cues and is executed by intrinsic cellular machinery. Unregulated cell death (often called necrosis) is caused by overwhelming stress that is incompatible with cell survival. Emerging evidence, however, suggests that these two processes do not adequately explain the various cell death mechanisms. Recent data point to the existence of multiple non-apoptotic, regulated cell death mechanisms, some of which overlap or are mutually exclusive with apoptosis. Here we examine how and why these different cell death programmes have evolved, with an eye towards new cytoprotective therapeutic opportunities.

Small-Molecule Inhibition of the Interaction between the Translation Initiation Factors eIF4E and eIF4G

Assembly of the eIF4E/eIF4G complex has a central role in the regulation of gene expression at the level of translation initiation. This complex is regulated by the 4E-BPs, which compete with eIF4G for binding to eIF4E and which have tumor-suppressor activity. To pharmacologically mimic 4E-BP function we developed a high-throughput screening assay for identifying small-molecule inhibitors of the eIF4E/eIF4G interaction. The most potent compound identified, 4EGI-1, binds eIF4E, disrupts eIF4E/eIF4G association, and inhibits cap-dependent translation but not initiation factor-independent translation. While 4EGI-1 displaces eIF4G from eIF4E, it effectively enhances 4E-BP1 association both in vitro and in cells. 4EGI-1 inhibits cellular expression of oncogenic proteins encoded by weak mRNAs, exhibits activity against multiple cancer cell lines, and appears to have a preferential effect on transformed versus nontransformed cells. The identification of this compound provides a new tool for studying translational control and establishes a possible new strategy for cancer therapy.

Regulation of intracellular accumulation of mutant Huntingtin by Beclin 1.

Intracellular accumulation of mutant Huntingtin with expanded polyglutamine provides a context-dependent cytotoxicity critical for the pathogenesis of Huntington disease (Everett, C. M., and Wood, N. W. (2004) Brain 127, 2385-2405). Here we demonstrate that the accumulation of mutant Huntingtin is highly sensitive to the expression of beclin 1, a gene essential for autophagy. Moreover, we show that the accumulated mutant Huntingtin recruits Beclin 1 and impairs the Beclin 1-mediated long lived protein turnover. Thus, sequestration of Beclin 1 in the vulnerable neuronal population of Huntington disease patients might further reduce Beclin 1 function and autophagic degradation of mutant Huntingtin. Finally, we demonstrate that the expression of beclin 1 decreases in an age-dependent fashion in human brains. Because beclin 1 gene is haploid insufficient in regulating autophagosome function (Qu, X., Yu, J., Bhagat, G., Furuya, N., Hibshoosh, H., Troxel, A., Rosen, J., Eskelinen, E. L., Mizushima, N., Ohsumi, Y., Cattoretti, G., and Levine, B. (2003) J. Clin. Invest. 112, 1809-1820; Yue, Z., Jin, S., Yang, C., Levine, A. J., and Heintz, N. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 15077-15082), we propose that the age-dependent decrease of beclin 1 expression may lead to a reduction of autophagic activity during aging, which in turn promotes the accumulation of mutant Htt and the progression of the disease.

Necrostatin-1 analogues: critical issues on the specificity, activity and in vivo use in experimental disease models

Necrostatin-1 (Nec-1) is widely used in disease models to examine the contribution of receptor-interacting protein kinase (RIPK) 1 in cell death and inflammation. We studied three Nec-1 analogs: Nec-1, the active inhibitor of RIPK1, Nec-1 inactive (Nec-1i), its inactive variant, and Nec-1 stable (Nec-1s), its more stable variant. We report that Nec-1 is identical to methyl-thiohydantoin-tryptophan, an inhibitor of the potent immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO). Both Nec-1 and Nec-1i inhibited human IDO, but Nec-1s did not, as predicted by molecular modeling. Therefore, Nec-1s is a more specific RIPK1 inhibitor lacking the IDO-targeting effect. Next, although Nec-1i was ∼100 × less effective than Nec-1 in inhibiting human RIPK1 kinase activity in vitro, it was only 10 times less potent than Nec-1 and Nec-1s in a mouse necroptosis assay and became even equipotent at high concentrations. Along the same line, in vivo, high doses of Nec-1, Nec-1i and Nec-1s prevented tumor necrosis factor (TNF)-induced mortality equally well, excluding the use of Nec-1i as an inactive control. Paradoxically, low doses of Nec-1 or Nec-1i, but not Nec -1s, even sensitized mice to TNF-induced mortality. Importantly, Nec-1s did not exhibit this low dose toxicity, stressing again the preferred use of Nec-1s in vivo. Our findings have important implications for the interpretation of Nec-1-based data in experimental disease models.

Necrostatin-1 Reduces Histopathology and Improves Functional Outcome after Controlled Cortical Impact in Mice:

Necroptosis is a newly identified type of programmed necrosis initiated by the activation of tumor necrosis factor alpha (TNFα)/Fas. Necrostatin-1 is a specific inhibitor of necroptosis that reduces ischemic tissue damage in experimental stroke models. We previously reported decreased tissue damage and improved functional outcome after controlled cortical impact (CCI) in mice deficient in TNFα and Fas. Hence, we hypothesized that necrostatin-1 would reduce histopathology and improve functional outcome after CCI in mice. Compared with vehicle-/inactive analog-treated controls, mice administered necrostatin-1 before CCI had decreased propidium iodide-positive cells in the injured cortex and dentate gyrus (6 h), decreased brain tissue damage (days 14, 35), improved motor (days 1 to 7), and Morris water maze performance (days 8 to 14) after CCI. Improved spatial memory was observed even when drug was administered 15 mins after CCI. Necrostatin-1 treatment did not reduce caspase-3-positive cells in the dentate gyrus or cortex, consistent with a known caspase-independent mechanism of necrostatin-1. However, necrostatin-1 reduced brain neutrophil influx and microglial activation at 48 h, suggesting a novel anti-inflammatory effect in traumatic brain injury (TBI). The data suggest that necroptosis plays a significant role in the pathogenesis of cell death and functional outcome after TBI and that necrostatin-1 may have therapeutic potential for patients with TBI.