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.

Chemical Inhibitors of Dynamin Exert Differential Effects in VEGF Signaling

“…As they offer the advantage of a rapid effect, these drugs avoid the manifestation of compensatory trafficking pathways [ 56 ]. However, despite their wide use and their undoubtful inhibitory effect on dynamin, the fact that they are small molecules that could bind to multiple targets raises concerns about possible off-target effects [ 18 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ]. The most compelling evidence in favor of this concern comes from experiments in dynamin triple knockout cells, where, although lack of all three dynamins did not affect fluid-phase endocytosis and membrane ruffling, dynasore and dyngo robustly inhibited these two processes both in wild-type and in the triple knockout cells [ 22 ].…”

“…In addition to dynasore, other dynamin inhibitors, i.e., dyngo 4a and dynole 34-2 [ 19 ], have been widely used to block dynamin-dependent endocytosis, especially when cells are resistant to transfection methods. However, since small molecule inhibitors may bind to multiple targets, it cannot be excluded that these inhibitors exert off-target effects [ 20 , 21 ], similarly to dynasore [ 18 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ]. Here, by comparing the effect of the above drugs in VEGF-induced internalization of VEGFR2 and downstream signaling to ERK1/2 and Akt, we suggest that these inhibitors, besides exerting their specific effect in dynamin-dependent endocytosis, cause inhibitory effects on signaling that are independent of endocytosis; i.e., they are due to off-target effects.…”

Prescription drugs and mitochondrial metabolism

“…Indirect interactions through biogenesis or degradation have been shown to require a period of several hours in vitro , and thus, should reflect timescales that differ from direct interactions with mitochondrial energy transduction which are typically much faster [ 93 ]. Supporting evidence for the notion that any of the aforementioned compounds function purely through indirect effects is limited, and follow-up studies have demonstrated some direct effects including inhibitory activity against ETS enzymes and antioxidant activities [ 94 , 95 ]. This highlights that caution should be taken when classifying xenobiotics as indirect effectors of mitochondrial metabolism unless the possibility of direct effects has been ruled out.…”

Radiotherapy-Induced Digestive Injury: Diagnosis, Treatment and Mechanisms

“…Similarly, ferroptosis inhibitors decrease ROS and inflammatory cytokine levels in radiation-induced lung injury (211). Other ferroptosis inhibitors, such as p53, PEBP1, ENPP2, and phospholipase iPLA (2) b, may also serve as radiation protectors (212)(213)(214)(215)(216)(217)(218)(219)(220)(221)(222)(223)(224)(225)(226)(227)(228)(229). Ferroptosis inducers have the potential to be effective radiosensitizers for radiotherapy (230-248) (Figure 4).…”