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Dynasore Blocks Ferroptosis through Combined Modulation of Iron Uptake and Inhibition of Mitochondrial Respiration.

ABSTRACT: Ferroptosis is a form of regulated necrosis characterized by a chain-reaction of detrimental membrane lipid peroxidation following collapse of glutathione peroxidase 4 (Gpx4) activity. This lipid peroxidation is catalyzed by labile ferric iron. Therefore, iron import mediated via transferrin receptors and both, enzymatic and non-enzymatic iron-dependent radical formation are crucial prerequisites for the execution of ferroptosis. Intriguingly, the dynamin inhibitor dynasore, which has been shown to block transferrin receptor endocytosis, can protect from ischemia/reperfusion injury as well as neuronal cell death following spinal cord injury. Yet, it is unknown how dynasore exerts these cell death-protective effects. Using small interfering RNA suppression, lipid reactive oxygen species (ROS), iron tracers and bona fide inducers of ferroptosis, we find that dynasore treatment in lung adenocarcinoma and neuronal cell lines strongly protects these from ferroptosis. Surprisingly, while the dynasore targets dynamin 1 and 2 promote extracellular iron uptake, their silencing was not sufficient to block ferroptosis suggesting that this route of extracellular iron uptake is dispensable for acute induction of ferroptosis and dynasore must have an additional off-target activity mediating full ferroptosis protection. Instead, in intact cells, dynasore inhibited mitochondrial respiration and thereby mitochondrial ROS production which can feed into detrimental lipid peroxidation and ferroptotic cell death in the presence of labile iron. In addition, in cell free systems, dynasore showed radical scavenger properties and acted as a broadly active antioxidant which is superior to N-acetylcysteine (NAC) in blocking ferroptosis. Thus, dynasore can function as a highly active inhibitor of ROS-driven types of cell death via combined modulation of the iron pool and inhibition of general ROS by simultaneously blocking two routes required for ROS and lipid-ROS driven cell death, respectively. These data have important implications for the interpretation of studies observing tissue-protective effects of this dynamin inhibitor as well as raise awareness that off-target ROS scavenging activities of small molecules used to interrogate the ferroptosis pathway should be taken into consideration.

SUBMITTER: Clemente LP

PROVIDER: S-EPMC7650611 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Dynasore Blocks Ferroptosis through Combined Modulation of Iron Uptake and Inhibition of Mitochondrial Respiration.

Clemente Laura Prieto LP Rabenau Malena M Tang Stephan S Stanka Josefina J Cors Eileen E Stroh Jenny J Culmsee Carsten C von Karstedt Silvia S

Cells 20201009 10

Ferroptosis is a form of regulated necrosis characterized by a chain-reaction of detrimental membrane lipid peroxidation following collapse of glutathione peroxidase 4 (Gpx4) activity. This lipid peroxidation is catalyzed by labile ferric iron. Therefore, iron import mediated via transferrin receptors and both, enzymatic and non-enzymatic iron-dependent radical formation are crucial prerequisites for the execution of ferroptosis. Intriguingly, the dynamin inhibitor dynasore, which has been shown ...[more]

PMID: 33050207

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Project description:IntroductionThe increasing morbidity of fungal infections and the prevalence of drug resistance highlighted the discovery of novel antifungal agents and investigation of their modes of action. Iron chelators have been used to treat superficial fungal infections or potentiate the efficacy of certain antifungal drugs. Hinokitiol exhibits potent antifungal activity and iron-chelating ability. However, their relationships have not been established.ObjectivesThis study aims to explore the selectivity of hinokitiol against fungal cells and mammalian cells and determine the role of iron-chelating for the antifungal activity of hinokitiol.MethodsIron probe FeRhonox-1 was used to determine intracellular Fe2+ content. 5-Cyano-2,3-ditolyl tetrazolium chloride probe and Cell Counting Kit-8 were used to detect the mitochondrial respiratory activities. Quantitative real-time PCR and rescue experiments were performed to determine the effect of iron on the antifungal activity of hinokitiol. The effects of hinokitiol on fungal mitochondria were further evaluated using reactive oxygen species probes and several commercial Assay Kits. The ability of hinokitiol to induce resistance in Candida species was carried out using a serial passage method. The in vivo therapeutic effect of hinokitiol was evaluated using Galleria mellonella as an infectious model.ResultsHinokitiol was effective against a panel of Candida strains with multiple azole-resistant mechanisms and persistently inhibited Candida albicans growth. Mechanism investigations revealed that hinokitiol chelated fungal intracellular iron and inhibited the respiration of fungal cells but had minor effects on mammalian cells. Hinokitiol further inhibited the activities of mitochondrial respiratory chain complexes I and II and reduced mitochondrial membrane potential, thereby decreasing intracellular ATP synthesis and increasing detrimental intracellular reductive stress. Moreover, hinokitiol exhibited low potential for inducing resistance in several Candida species and greatly improved the survival of Candida-infected Galleria mellonella.ConclusionsThese findings suggested the potential application of hinokitiol as an iron chelator to treat fungal infections.

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Dynasore Blocks Ferroptosis through Combined Modulation of Iron Uptake and Inhibition of Mitochondrial Respiration.

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Dynasore Blocks Ferroptosis through Combined Modulation of Iron Uptake and Inhibition of Mitochondrial Respiration.

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