Abstract Background Viruses are dependent on cellular energy metabolism for their replication, the drug Nitazoxanide (Alinia) was shown to interfere with both. An effect of Alinia on cellular energy metabolism is the uncoupling of mitochondrial oxidative phosphorylation (OXPHOS). Our hypothesis was that uncoupling grounds the antiviral properties of Alinia. Methods Alinia or an unrelated uncoupler were applied to a viral releasing cell line to obtain the same increasing levels of uncoupling hence identical interference with OXPHOS. Findings Decrease in infectious viral particles release reflected the intensity of interference irrespective of the nature of the drug and was significant with modest deviation (≤25%) from normal. Interpretations A mild interference on cellular energy metabolism impacts significantly on viral replication cycle. This would explain Alinia’s antiviral properties in vitro moreover antiviral action of Alinia is supported by clinical trials. Perspectives Altogether this indicates that moderate interference with mitochondrial bioenergetics should be considered as a ground for a therapeutic effect. In addition, Alinia would constitute example for a safe therapeutical use of an uncoupler, which deserves consideration for a wider range of applications.
Introduction of the extracellular flux analyzer “Seahorse” boosted evaluation of cellular oxidative metabolism. The largest part of experiments implies a standard “Cell Mito Stress” protocol, which includes the determination of a maximal intensity of cellular oxygen consumption rate (OCRmax) by stimulation of cellular respiration with uncoupler. However, this actually takes place after full inhibition of ATP production by mitochondrial oxidative phosphorylation (Oxphos). Firstly, if cells are heavily dependent on Oxphos their viability would be jeopardized when stimulation by uncoupler takes place. Secondly, the dependence of OCR on uncoupler concentration has a bell shape meaning that optimal concentrations of uncoupler should be determined, they may differ between the cell types compared in the experiment. Titration with increasing doses is then required but the Mitostress protocol allows only two additions of uncoupler. These limitations find reflection in available experiment reports. Inclusion of a second type of experiments with increasing dosage of uncoupler and excluding other interventions improves determination of OCRmax. Complications are limited and absent if determination of optimal uncoupler dosage for Mitostress is performed in separate experiments. Oxphos is not nullified when stimulation by uncoupler starts but declines as uncoupler concentration increases, so that cell viability would be less impacted than with the standard Cell Mito Stress protocol.
Abstract Viruses are dependent on cellular energy metabolism for their replication, and the drug nitazoxanide (Alinia) was shown to interfere with both processes. Nitazoxanide is an uncoupler of mitochondrial oxidative phosphorylation (OXPHOS). Our hypothesis was that mitochondrial uncoupling underlies the antiviral effects of nitazoxanide. Tizoxanide (the active metabolite of nitazoxanide), its derivative RM4848 and the uncoupler CCCP were applied to a virus-releasing cell line to obtain the same increasing levels of mitochondrial uncoupling, hence identical interference with OXPHOS. A decrease in infectious viral particle release was observed and reflected the intensity of interference with OXPHOS, irrespective of the nature of the drug. The antiviral effect was significant although the impact on OXPHOS was modest (≤ 25%), and disappeared when a high concentration (25 mM) of glucose was used to enhance glycolytic generation of ATP. Accordingly, the most likely explanation is that moderate interference with mitochondrial OXPHOS induced rearrangement of ATP use and acquisition of infective properties of the viral particles be highly sensitive to this rearrangement. The antiviral effect of nitazoxanide has been supported by clinical trials, and nitazoxanide is considered a safe drug. However, serious adverse effects of the uncoupler dinitrophenol occurred when used to increase significantly metabolic rate with the purpose of weight loss. In addition, dinitrophenol is known to interfere with mitochondrial ATP transport while we demonstrate that nitazoxanide does not. Taken together, while impairment of mitochondrial bioenergetics is an unwanted drug effect, moderate interference should be considered as a basis for therapeutic efficacy.
Cellular bioenergetics requires an intense ATP turnover that is increased further by hypermetabolic states caused by cancer growth or inflammation. Both are associated with metabolic alterations and, notably, enhancement of the Warburg effect (also known as aerobic glycolysis) of poor efficiency with regard to glucose consumption when compared to mitochondrial respiration. Therefore, beside this efficiency issue, other properties of these two pathways should be considered to explain this paradox: (1) biosynthesis, for this only indirect effect should be considered, since lactate release competes with biosynthetic pathways in the use of glucose; (2) ATP production, although inefficient, glycolysis shows other advantages when compared to mitochondrial respiration and lactate release may therefore reflect that the glycolytic flux is higher than required to feed mitochondria with pyruvate and glycolytic NADH; (3) Oxygen supply becomes critical under hypermetabolic conditions, and the ATP/O2 ratio quantifies the efficiency of oxygen use to regenerate ATP, although aerobic metabolism remains intense the participation of anaerobic metabolisms (lactic fermentation or succinate generation) could greatly increase ATP/O2 ratio; (4) time and space constraints would explain that anaerobic metabolism is required while the general metabolism appears oxidative; and (5) active repression of respiration by glycolytic intermediates, which could ensure optimization of glucose and oxygen use.
Viruses are dependent on cellular energy metabolism for their replication, and the drug nitazoxanide (Alinia) was shown to interfere with both processes. Nitazoxanide is an uncoupler of mitochondrial oxidative phosphorylation (OXPHOS). Our hypothesis was that mitochondrial uncoupling underlies the antiviral effects of nitazoxanide. Tizoxanide (the active metabolite of nitazoxanide), its derivative RM4848 and the uncoupler CCCP were applied to a virus-releasing cell line to obtain the same increasing levels of mitochondrial uncoupling, hence identical impact on OXPHOS. A decrease in infectious viral particle release was observed and reflected the intensity of impact on OXPHOS, irrespective of the nature of the drug. The antiviral effect was significant although the impact on OXPHOS was modest (≤ 25%), and disappeared when a high concentration (25 mM) of glucose was used to enhance glycolytic generation of ATP. Accordingly, the most likely explanation is that moderate interference with mitochondrial OXPHOS induced rearrangement of ATP use and acquisition of infective properties of the viral particles be highly sensitive to this rearrangement. The antiviral effect of nitazoxanide has been supported by clinical trials, and nitazoxanide is considered a safe drug. However, serious adverse effects of the uncoupler dinitrophenol occurred when used to increase significantly metabolic rate with the purpose of weight loss. Taken together, while impairment of mitochondrial bioenergetics is an unwanted drug effect, moderate interference should be considered as a basis for therapeutic efficacy.
Nous avons etudie les relations entre les differentes voies du metabolisme energetique lors de la mise en place des effets Crabtree et Warburg. L’effet du glucose sur le metabolisme energetique de S. cerevisiae se traduit dans un premier temps par une inhibition cinetique du metabolisme oxydatif (effet Crabtree). Apres l’ajout de glucose aux cellules, nous avons mis en evidence l’accumulation d’un intermediaire de la glycolyse, le F1,6bP. Ceci induit une diminution drastique du rapport G6P/F1,6bP. Or, il a ete montre que le G6P stimule et le F1,6bP inhibe l’activite de la chaine respiratoire mitochondriale « in-situ ». L’utilisation de mutants et la modulation de ce rapport nous a permis de montrer que l’induction de l’effet Crabtree chez la levure Saccharomyces cerevisiae est du a une diminution du rapport G6P/F1,6bP. Parallelement, le glucose induit un rearrangement genetique qui a terme conduit a un effet Warburg. Nous avons mis en evidence une diminution, au cours du temps du contenu mitochondrial par effet de dilution, suite a un arret de la biogenese mitochondriale (repression de HAP4). Nous avons pu montrer que cette diminution quantitative des OXPHOS est sans effet sur la synthese d’ATP cellulaire. Ceci est du a une augmentation du flux de synthese d’ATP glycolytique. L’utilisation de mutants HAP4, nous a permis de montrer qu’il n’y a pas de lien simple entre proliferation et repression des OXPHOS. Bien que le flux glycolytique diminue dans les conditions de maintien des OXPHOS, ceci est sans effet notoire sur la vitesse de proliferation. Ceci est un rare exemple d’une situation biologique ou l’on observe un decouplage entre metabolisme energetique et proliferation.
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