Alexei Degterev

Tufts University, Georgia, United States

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Publications (70)556.4 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: We previously reported encouraging in vitro and in vivo anti-cancer activity of N-((3-chloro-2-hydroxy-5-nitrophenyl)carbamothioyl)benzamide (termed PITENIN-1). In the current work, we describe the structure–activity relationship study of the PIT-1 series, based on the replacement of a central thiourea unit with 1,2,3-triazole, which leads to increased liver microsomal stability, drug likeness and toxicity towards cancer cells.
    Medicinal Chemistry Communication 07/2014; · 2.72 Impact Factor
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    ABSTRACT: Akt (protein kinase B) and mammalian target of rapamycin (mTOR) have been implicated in the pathogenesis of cell death and cognitive outcome after cerebral contusion in mice; however, a role for Akt/mTOR in concussive brain injury has not been well characterized. In a mouse closed head injury (CHI) concussion traumatic brain injury (TBI) model, phosphorylation of Akt (p-Akt), mTOR (p-mTOR), and S6RP (p-S6RP) was increased by 24 hours in cortical and hippocampal brain homogenates (P<0.05 versus sham for each), and p-S6RP was robustly induced in IBA-1+ microglia and glial fibrillary acidic protein-positive (GFAP+) astrocytes. Pretreatment with inhibitors of Akt or mTOR individually by the intracerebroventricular route reduced phosphorylation of their respective direct substrates FOXO1 (P<0.05) or S6RP (P<0.05) after CHI, confirming the activity of inhibitors. Rapamycin pretreatment significantly worsened hidden platform (P<0.01) and probe trial (P<0.05) performance in CHI mice. Intracerebroventricular administration of necrostatin-1 (Nec-1) before CHI increased hippocampal Akt and S6RP phosphorylation and improved place learning (probe trials, P<0.001 versus vehicle), whereas co-administration of rapamycin or Akt inhibitor with Nec-1 eliminated improved probe trial performance. These data suggest a beneficial role for Akt/mTOR signaling after concussion TBI independent of cell death that may contribute to improved outcome by Nec-1.Journal of Cerebral Blood Flow & Metabolism advance online publication, 18 June 2014; doi:10.1038/jcbfm.2014.113.
    06/2014;
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    ABSTRACT: Retinal ischemia-reperfusion (IR) injury remains a common cause of blindness and has a final pathway of retinal ganglion cell (RGC) death by apoptosis and necrosis. RGC apoptosis was intensively studied in IR injury, while RGC necrosis did not receive nearly enough consideration since it was viewed as an accidental and unregulated cellular event. However, there is evidence that necrosis, like apoptosis, can be implemented by a programmed mechanism. In this study, we tested the role of RGC programmed necrosis (necroptosis) in IR-induced retinal injury. We employed the mouse model of retinal IR injury for in vivo experiments. The oxygen and glucose deprivation (OGD) model was used as an IR model in vitro. Primary RGCs were isolated by an immunopanning technique. Necrostatin 1 (Nec1) was used to inhibit necroptosis in in vitro and in vivo experiments. The changes in gene expression were assessed by quantitative RT-PCR. The distribution of proteins in the retina and in RGC cultures was evaluated by immunohistochemistry and immunocytochemistry, respectively. Our data suggest that proteins (Ripk1 and Ripk3), which initiate necroptosis, were present in normal and ischemic RGCs. Treatment with Nec1 significantly reduced retinal damage after IR. Increased RGC survival and reduced RGC necrosis following OGD were observed in Nec1-treated cultures. We found significantly reduced expression of genes coding pro-inflammatory markers Il1b, Ccl5, Cxcl10, Nos2 and Cybb in Nec1-treated ischemic retinas. Thus, our findings suggest that RGC necroptosis contributes to retinal damage after IR through direct loss of cells and induction of associated inflammatory responses.
    Experimental Eye Research 04/2014; · 3.03 Impact Factor
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    Nature 02/2014; 506(7489):E4-6. · 38.60 Impact Factor
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    ABSTRACT: Retinal ischemia–reperfusion (IR) injury remains a common cause of blindness and has a final pathway of retinal ganglion cell (RGC) death by apoptosis and necrosis. RGC apoptosis was intensively studied in IR injury, while RGC necrosis did not receive nearly enough consideration since it was viewed as an accidental and unregulated cellular event. However, there is evidence that necrosis, like apoptosis, can be implemented by a programmed mechanism. In this study, we tested the role of RGC programmed necrosis (necroptosis) in IR-induced retinal injury. We employed the mouse model of retinal IR injury for in vivo experiments. The oxygen and glucose deprivation (OGD) model was used as an IR model in vitro. Primary RGCs were isolated by an immunopanning technique. Necrostatin 1 (Nec1) was used to inhibit necroptosis in in vitro and in vivo experiments. The changes in gene expression were assessed by quantitative RT-PCR. The distribution of proteins in the retina and in RGC cultures was evaluated by immunohistochemistry and immunocytochemistry, respectively. Our data suggest that proteins (Ripk1 and Ripk3), which initiate necroptosis, were present in normal and ischemic RGCs. Treatment with Nec1 significantly reduced retinal damage after IR. Increased RGC survival and reduced RGC necrosis following OGD were observed in Nec1-treated cultures. We found significantly reduced expression of genes coding pro-inflammatory markers Il1b, Ccl5, Cxcl10, Nos2 and Cybb in Nec1-treated ischemic retinas. Thus, our findings suggest that RGC necroptosis contributes to retinal damage after IR through direct loss of cells and induction of associated inflammatory responses.
    Experimental Eye Research 01/2014; · 3.03 Impact Factor
  • Alexei Degterev, Wen Zhou, Jenny L Maki, Junying Yuan
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    ABSTRACT: 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.
    Methods in enzymology. 01/2014; 545:1-33.
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    ABSTRACT: Necroptosis is a newly described form of regulated necrosis that contributes to neuronal death in experimental models of stroke and brain trauma. Although much work has been done elucidating initiating mechanisms, signaling events governing necroptosis remain largely unexplored. Akt is known to inhibit apoptotic neuronal cell death. Mechanistic target of rapamycin (mTOR) is a downstream effector of Akt that controls protein synthesis. We previously reported that dual inhibition of Akt and mTOR reduced acute cell death and improved long term cognitive deficits after controlled-cortical impact in mice. These findings raised the possibility that Akt/mTOR might regulate necroptosis. To test this hypothesis, we induced necroptosis in the hippocampal neuronal cell line HT22 using concomitant treatment with tumor necrosis factor α (TNFα) and the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. TNFα/zVAD treatment induced cell death within 4 h. Cell death was preceded by RIPK1-RIPK3-pAkt assembly, and phosphorylation of Thr-308 and Thr473 of AKT and its direct substrate glycogen synthase kinase-3β, as well as mTOR and its direct substrate S6 ribosomal protein (S6), suggesting activation of Akt/mTOR pathways. Pretreatment with Akt inhibitor viii and rapamycin inhibited Akt and S6 phosphorylation events, mitochondrial reactive oxygen species production, and necroptosis by over 50% without affecting RIPK1-RIPK3 complex assembly. These data were confirmed using small inhibitory ribonucleic acid-mediated knockdown of AKT1/2 and mTOR. All of the aforementioned biochemical events were inhibited by necrostatin-1, including Akt and mTOR phosphorylation, generation of oxidative stress, and RIPK1-RIPK3-pAkt complex assembly. The data suggest a novel, heretofore unexpected role for Akt and mTOR downstream of RIPK1 activation in neuronal cell death.
    Cell Death & Disease 01/2014; 5:e1084. · 6.04 Impact Factor
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    ABSTRACT: 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.
    Proceedings of the National Academy of Sciences 07/2013; · 9.81 Impact Factor
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    ABSTRACT: 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, 1-3 mg of highly purified RIP1 kinase was typically obtained from a 1 L 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.
    Protein Expression and Purification 03/2013; · 1.43 Impact Factor
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    ABSTRACT: Abstract We have developed and characterized micellar formulations of analogs to the recently developed inhibitor of the phosphatidylinositol-3-kinase (PI3K) pathway (N-[(2-hydroxy-5-nitrophenyl)amino]carbonothioyl-3,5-dimethylbenzamide (DM-PIT-1)) for their physicochemical, loading and cytotoxic properties. The first generation inhibitor DM-PIT-1 is a non-lipid, small molecule inhibitor of phosphatidylinositol-3,4,5-triphosphate/Pleckstrin homology (PIP3/PH) binding capable of inhibiting the growth of tumor cells both in vitro and in vivo. A second generation of improved and druggable analogs has been developed. All compounds were successfully loaded (>70%) in PEG2000-PE micelles of 16-20 nm in size with several analogs demonstrating favorable cytotoxic activity against A2780 ovarian carcinoma. These compounds were also successfully incorporated into polyethylene glycol-phosphatidylethanolamine (PEG-PE) micelles combined with surface-bound tumor necrosis factor related apoptosis inducing ligand (TRAIL). The resulting multifunctional combination micelles were able to significantly enhance cytotoxic activity in the TRAIL-resistant A2780 cell line. Additionally, analogs NCL-176 and NCL-240 were effective in inhibiting tumor growth in an in vivo subcutaneous tumor model of A2780. These results indicate the utility of delivering TRAIL and PI3K pathway inhibitors in a combined micellar preparation.
    Drug Delivery 03/2013; · 2.02 Impact Factor
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    ABSTRACT: Necroptosis is a cellular mechanism that mediates necrotic cell death. The receptor-interacting serine/threonine protein kinase 1 (RIP1) is an essential upstream signaling molecule in tumor-necrosis-factor-α-induced necroptosis. Necrostatins, a series of small-molecule inhibitors, suppress necroptosis by specifically inhibiting RIP1 kinase activity. Both RIP1 structure and the mechanisms by which necrostatins inhibit RIP1 remain unknown. Here, we report the crystal structures of the RIP1 kinase domain individually bound to necrostatin-1 analog, necrostatin-3 analog, and necrostatin-4. Necrostatin, caged in a hydrophobic pocket between the N- and C-lobes of the kinase domain, stabilizes RIP1 in an inactive conformation through interactions with highly conserved amino acids in the activation loop and the surrounding structural elements. Structural comparison of RIP1 with the inhibitor-bound oncogenic kinase B-RAF reveals partially overlapping binding sites for necrostatin and for the anticancer compound PLX4032. Our study provides a structural basis for RIP1 inhibition by necrostatins and offers insights into potential structure-based drug design.
    Structure 03/2013; 21(3):493-9. · 5.99 Impact Factor
  • Jenny L Maki, Alexei Degterev
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    ABSTRACT: 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.
    Methods in molecular biology (Clifton, N.J.) 01/2013; 1004:31-42. · 1.29 Impact Factor
  • Robert D Riehle, Sinziana Cornea, Alexei Degterev
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    ABSTRACT: Many lipids present in cellular membranes are phosphorylated as part of signaling cascades and participate in the recruitment, localization, and activation of downstream protein effectors. Phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) is one of the most important second messengers and is capable of interacting with a variety of proteins through specific PtdIns(3,4,5)P3 binding domains. Localization and activation of these effector proteins controls a myriad of cellular functions including cell survival, proliferation, cytoskeletal rearrangement, and gene expression. Aberrations in the production and metabolism of PtdIns(3,4,5)P3 have been implicated in many human diseases including cancer, diabetes, inflammation, and heart disease. This chapter provides an overview of the role of PtdIns(3,4,5)P3 in cellular regulation and the implications of PtdIns(3,4,5)P3 dysregulation in human diseases. Additionally, recent attempts at targeting PtdIns(3,4,5)P3 signaling via small molecule inhibitors are summarized.
    Advances in experimental medicine and biology 01/2013; 991:105-39. · 1.83 Impact Factor
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    ABSTRACT: Necroptosis is a regulated form of necrotic cell death that has been implicated in the pathogenesis of various diseases including intestinal inflammation and systemic inflammatory response syndrome (SIRS). In this work, we investigated the signaling mechanisms controlled by the necroptosis mediator receptor interacting protein-1 (RIP1) kinase. We show that Akt kinase activity is critical for necroptosis in L929 cells and plays a key role in TNFα production. During necroptosis, Akt is activated in a RIP1 dependent fashion through its phosphorylation on Thr308. In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1. Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1). Akt activity, mediated in part through mTORC1, links RIP1 to JNK activation and autocrine production of TNFα. In other cell types, such as mouse lung fibroblasts and macrophages, Akt exhibited control over necroptosis-associated TNFα production without contributing to cell death. Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation.
    PLoS ONE 01/2013; 8(3):e56576. · 3.53 Impact Factor
  • A Degterev, J L Maki, J Yuan
    Cell death and differentiation 11/2012; · 8.24 Impact Factor
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    ABSTRACT: Necroptosis is a regulated caspase-independent cell death pathway with morphological features resembling passive non-regulated necrosis. Several diverse structure classes of necroptosis inhibitors have been reported to date, including a series of 3,3a,4,5-tetrahydro-2H-benz[g]indazoles (referred to as the Nec-3 series) displaying potent activity in cellular assays. However, evaluation of the tricyclic necroptosis inhibitor's stability in mouse liver microsomes indicated that they were rapidly degraded. A structure-activity relationship (SAR) study of this compound series revealed that increased liver microsomal stability could be accomplished by modification of the pendent phenyl ring and by introduction of a hydrophilic substituent (i.e., α-hydroxyl) to the acetamide at the 2-position of the tricyclic ring without significantly compromising necroptosis inhibitory activity. Further increases in microsomal stability could be achieved by utilizing the 5,5-dioxo-3-phenyl-2,3,3a,4-tetrahydro-[1]benzothiopyrano[4,3-c]pyrazoles. However, in this case necroptosis inhibitory activity was not maintained. Overall, these results provide a strategy for generating potent and metabolically stable tricyclic necrostatin analogs (e.g., 33, LDN-193191) potentially suitable for in vivo studies.
    Bioorganic & medicinal chemistry letters 07/2012; 22(17):5685-8. · 2.65 Impact Factor
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    ABSTRACT: Necrotic cell death is prevalent in many different pathological disease states and in traumatic injury. Necroptosis is a form of necrosis that stems from specific signaling pathways, with the key regulator being receptor interacting protein 1 (RIP1), a serine/threonine kinase. Specific inhibitors of RIP1, termed necrostatins, are potent inhibitors of necroptosis. Necrostatins are structurally distinct from one another yet still possess the ability to inhibit RIP1 kinase activity. To further understand the differences in the binding of the various necrostatins to RIP1 and to develop a robust high-throughput screening (HTS) assay, which can be used to identify new classes of RIP1 inhibitors, we synthesized fluorescein derivatives of Necrostatin-1 (Nec-1) and Nec-3. These compounds were used to establish a fluorescence polarization (FP) assay to directly measure the binding of necrostatins to RIP1 kinase. The fluorescein-labeled compounds are well suited for HTS because the assays have a dimethyl sulfoxide (DMSO) tolerance up to 5% and Z' scores of 0.62 (fluorescein-Nec-1) and 0.57 (fluorescein-Nec-3). In addition, results obtained from the FP assays and ligand docking studies provide insights into the putative binding sites of Nec-1, Nec-3, and Nec-4.
    Analytical Biochemistry 05/2012; 427(2):164-74. · 2.58 Impact Factor
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    ABSTRACT: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
    Autophagy 04/2012; 8(4):445. · 12.04 Impact Factor
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    Autophagy 04/2012; 8(4):1-100. · 12.04 Impact Factor

Publication Stats

4k Citations
556.40 Total Impact Points

Institutions

  • 2007–2014
    • Tufts University
      • • Department of Biochemistry
      • • Department of Medicine
      Georgia, United States
  • 2009–2013
    • Northeastern University
      • Department of Pharmaceutical Sciences
      Boston, MA, United States
    • Universidad Autónoma de San Luis Potosí
      • Instituto de Física
      San Luis Potosí, San Luis Potosi, Mexico
    • Weill Cornell Medical College
      • Department of Radiology
      New York City, NY, United States
  • 2012
    • University of Michigan
      • Life Sciences Institute
      Ann Arbor, MI, United States
  • 2005–2012
    • Brigham and Women's Hospital
      • Department of Medicine
      Cambridge, MA, United States
  • 1999–2012
    • Harvard Medical School
      • Department of Cell Biology
      Boston, MA, United States
  • 2010
    • Houston Methodist Hospital
      Houston, Texas, United States
  • 2006
    • Tsinghua University
      Peping, Beijing, China
  • 2002
    • Harvard University
      Cambridge, Massachusetts, United States