Browsing by Author "Degterev, Alexei"
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Item A Potential New Treatment Paradigm for Multiple Sclerosis(2017) Nikhar, Sameer; Degterev, Alexei; Cuny, Gregory D.This project was completed with contributions from Alexei Degterev from the Department of Developmental, Molecular and Chemical Biology, School of Medicine, Tufts University, Boston.Item Akt Regulates TNF? Synthesis Downstream of RIP1 Kinase Activation during Necroptosis(PLoS One, 2013-03) McNamara, Colleen R.; Ahuja, Ruchita; Osafo-Addo, Awo D.; Barrows, Douglas; Kettenbach, Arminja; Skidan, Igor; Teng, Xin; Cuny, Gregory D.; Gerber, Scott A.; Degterev, AlexeiNecroptosis 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 TNF? 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 TNF?. In other cell types, such as mouse lung fibroblasts and macrophages, Akt exhibited control over necroptosis-associated TNF? 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.Item Expression and purification of active human receptor interacting protein 1 kinase using a baculovirus system(Protein Expression and Purification, 2014-06) Maki, Jenny L.; Brazell, Tres; Cuny, Gregory D.; Degterev, AlexeiReceptor Interacting Protein 1 (RIP1) kinase is one of the key mediators of tumor necrosis factor alpha (TNF-?) 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.Item Fluorescence polarization assay for inhibitors of the kinase domain of receptor interacting protein 1(Analytical Biochemistry, 2013-05) Maki, Jenny L.; Smith, Elizabeth E.; Teng, Xin; Ray, Soumya S.; Cuny, Gregory D.; Degterev, AlexeiNecrotic 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.Item Identification of RIP1 kinase as a specific cellular target of necrostatins(Nature Chemical Biology, 2017-05) Degterev, Alexei; Hitomi, Junichi; Germscheid, Megan; Ch'en, Irene L.; Korkina, Olga; Teng, Xin; Abbott, Derek; Cuny, Gregory D.; Yuan, Chengye; Wagner, Gerhard; Hedrick, Stephen M.; Gerber, Scott A.; Lugovskoy, Alexey; Yuan, JunyingNecroptosis 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.Item Necrostatin-1 Reduces Histopathology and Improves Functional Outcome after Controlled Cortical Impact in Mice(Journal of Cerebral Blood Flow and Metabolism, 2010-03) You, Zerong; Savitz, Sean I.; Yang, Jinsheng; Degterev, Alexei; Yuan, Junying; Cuny, Gregory D.; Moskowitz, Michael A.; Whalen, Michael J.Necroptosis is a newly identified type of programmed necrosis initiated by the activation of tumor necrosis factor alpha (TNF?)/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 TNF? 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.Item Optimization of tricyclic Nec-3 necroptosis inhibitors for in vitro liver microsomal stability(Bioorganic and Medicinal Chemistry Letters, 2013-09) Choi, Sungwoon; Keys, Heather; Staples, Richard J.; Yuan, Junying; Degterev, Alexei; Cuny, Gregory D.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., ?-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.Item Pyrido-pyrimidinone Derivatives as RIPK2 Kinase and NOD Signaling Inhibitors(2018-08) Nikhar, Sameershivnath; Cuny, Gregory D.; Das, Joydip; Ruan, Ke-He; Degterev, Alexei; May, Jeremy A.Receptor interacting protein kinase-2 (RIPK2) is an enzyme that transduces pro-inflammatory signaling from the nucleotide binding oligomerization containing proteins (NOD1/2) during bacterial invasion in immune cells. However, NOD 1/2 mutations cause uncontrolled activation of RIPK2, resulted in excessive signaling and cytokine production. Such dysregulated signaling has been associated with inflammatory bowel (Crohn’s disease) and neuro-inflammatory diseases (Multiple Sclerosis), making RIPK2 an important target. Although, several RIPK2 kinase inhibitors have been reported in the literature, they possess limitations including off-target effects, reduced cellular potency, compromised in vivo efficacy and hERG/CYP inhibition. Thus, novel types of RIPK2 inhibitors are needed to address such problems. To this end we have identified a pyrido-pyrimidine based inhibitor (UH15) that has shown potent RIPK2 inhibition in enzymes and cells, but also showed non-selective inhibition of structurally similar activin like kinase-2 (ALK2). Nevertheless, molecular docking studies of UH15 in RIPK2 and ALK2 identified key differences that have been used to optimize the compound towards potent and selective RIPK2 inhibitors. The docking model-based optimization of UH15 identified two sets of analogs that differentiate RIPK2 inhibition in cells based upon size and position of substituents at a specific site on the inhibitor. Compounds that occupy the shallow hydrophobic pocket between the β3/β5 strands and the α-C helix in the kinase domain, showed effective RIPK2 cellular inhibition. The behavior of this subset of UH15 inhibitors correlates to the recently identified CSLP compounds that have shown RIPK2 cellular potency by interfering with RIPK2-XIAP interaction after occupying the hydrophobic pocket. The most potent and selective RIPK2 inhibitor (UH15-15) has shown optimal absorption, distribution, metabolism and excretion (ADME) and pharmacokinetic (PK) properties, making it a suitable probe for delineating the role of RIPK2 in disease models. Furthermore, modifications of the central pyrido-pyrimidine scaffold of UH15 series resulted in several other distinct structural classes of RIPK2 inhibitors. Overall, the identified RIPK2 inhibitors show mechanism based cellular potency, selectivity verses the structurally similar ALK2 kinase and promising pharmacokinetic properties. These compounds will allow investigation of RIPK2 function in disease models and will provide the basis for further optimization and advancement of RIPK2 inhibitors.Item Structure activity relationship study of [1,2,3]thiadiazole necroptosis inhibitors(Bioorganic and Medicinal Chemistry Letters, 2008-12) Teng, Xin; Keys, Heather; Jeevanandam, Arumugasamy; Porco, John A. Jr.; Degterev, Alexei; Yuan, Junying; Cuny, Gregory D.Necroptosis is a regulated caspase-independent cell death mechanism that results in morphological features resembling non-regulated necrosis. This form of cell death can be induced in an array of cell types in apoptotic deficient conditions with death receptor family ligands. A series of [1,2,3]thiadiazole benzylamides was found to be potent necroptosis inhibitors (called necrostatins). A structure activity relationship study revealed that small cyclic alkyl groups (i.e. cyclopropyl) and 2,6-dihalobenzylamides at the 4- and 5-positions of the [1,2,3]thiadiazole, respectively, were optimal. In addition, when a small alkyl group (i.e. methyl) was present on the benzylic position all the necroptosis inhibitory activity resided with the (S)-enantiomer. Finally, replacement of the [1,2,3]thiadiazole with a variety of thiophene derivatives was tolerated, although some erosion of potency was observed.Item Structure Guided Design of Potent and Selective Ponatinib-Based Hybrid Inhibitors for RIPK1(Cell Reports, 2015-03) Najjar, Malek; Suebsuwong, Chalada; Ray, Soumya S.; Thapa, Roshan J.; Maki, Jenny L.; Nogusa, Shoko; Shah, Saumil; Saleh, Danish; Gough, Peter J.; Bertin, John; Yuan, Junying; Balachandran, Siddharth; Cuny, Gregory D.; Degterev, AlexeiRIPK1 and RIPK3, two closely related RIPK family members, have emerged as important regulators of pathologic cell death and inflammation. In the current work, we report that the Bcr-Abl inhibitor and anti-leukemia agent ponatinib is also a first-in-class dual inhibitor of RIPK1 and RIPK3. Ponatinib potently inhibited multiple paradigms of RIPK1- and RIPK3-dependent cell death and inflammatory tumor necrosis factor alpha (TNF-?) gene transcription. We further describe design strategies that utilize the ponatinib scaffold to develop two classes of inhibitors (CS and PN series), each with greatly improved selectivity for RIPK1. In particular, we detail the development of PN10, a highly potent and selective “hybrid” RIPK1 inhibitor, capturing the best properties of two different allosteric RIPK1 inhibitors, ponatinib and necrostatin-1. Finally, we show that RIPK1 inhibitors from both classes are powerful blockers of TNF-induced injury in vivo. Altogether, these findings outline promising candidate molecules and design approaches for targeting RIPK1- and RIPK3-driven inflammatory pathologies.Item Structure–activity relationship and liver microsome stability studies of pyrrole necroptosis inhibitors(Bioorganic and Medicinal Chemistry Letters, 2009-06) Teng, Xin; Keys, Heather; Yuan, Junying; Degterev, Alexei; Cuny, Gregory D.Necroptosis is a regulated caspase-independent cell death pathway resulting in morphology reminiscent of passive non-regulated necrosis. Several diverse structure classes of necroptosis inhibitors have been reported to date, including a series of [1,2,3]thiadiazole benzylamide derivatives. However, initial evaluation of mouse liver microsome stability indicated that this series of compounds was rapidly degraded. A structure–activity relationship (SAR) study of the [1,2,3]thiadiazole benzylamide series revealed that increased mouse liver microsome stability and increased necroptosis inhibitory activity could be accomplished by replacement of the 4-cyclopropyl-[1,2,3]thiadiazole with a 5-cyano-1-methylpyrrole. In addition, the SAR and the cellular activity profiles, utilizing different cell types and necroptosis-inducing stimuli, of representative [1,2,3]thiadiazole and pyrrole derivatives were very similar suggesting that the two compound series inhibit necroptosis in the same manner.