Targeting glioblastoma mitochondrial metabolism with S-Gboxin induces cytotoxicity under conditions of the tumor microenvironment

Abstract Glioblastoma (GB) is the most common primary malignant brain tumor in adults. Gboxin, a novel compound that targets oxidative phosphorylation via complex V inhibition, has shown promise in preclinical models of GB. We examined the efficacy of the pharmacokinetically optimized S-Gboxin under conditions replicating the GB microenvironment, including nutrient deprivation and hypoxia. We assessed cytotoxicity and growth-inhibitory effects of S-Gboxin in human GB cell lines, primary GB cultures, as well as immortalized and primary human astrocytes under different nutrient and oxygen deprivation scenarios. Oxygen consumption, cell migration, activation of the integrated stress response (ISR) as well as the relevance of the AMP-activated protein kinase (AMPK) were evaluated as variables under S-Gboxin treatment. S-Gboxin demonstrated cytotoxicity at low micromolar concentrations, with cell death enhanced under nutrient deprivation and hypoxia. S-Gboxin reduced cellular oxygen consumption and uncoupled mitochondria. Cytotoxicity was increased when mitochondrial fuels were the primary energy source. Additionally, S-Gboxin treatment resulted in elevated lactate production and glucose consumption. While the ISR marker ATF4 was induced by S-Gboxin in a dose-dependent manner, ISR inhibition with ISRIB did not affect its cytotoxicity. Conversely, S-Gboxin treatment combined with AMPK inhibition resulted in enhanced tumor cell death. Collectively, these findings demonstrate that S-Gboxin selectively targets cancer-specific metabolic vulnerabilities in GB cells. The synergistic action with AMPK inhibition suggests that this pathway contributes to maintain energy homeostasis in the presence of the drug. Therefore, S-Gboxin is a promising compound for GB therapy, especially in a combinatory approach with AMPK inhibition or other metabolic targeted therapies.