Erastin-like anti-Warburg compounds X1 and X4 are GLS2-selective covalent glutaminase inhibitors

The Warburg effect is a metabolic alteration in cancer cells characterized by aerobic glycolysis and lactate production. A recent phenotypic screen for compounds that reverse Warburg metabolism identified two compounds, X1 and X4, that restore the mitochondrial membrane potential, decrease lactate production, and increase the level of reactive oxygen species in cancer cells. Here we show that X1 and X4 are GLS2-selective covalent glutaminase inhibitors. Glutaminase enzymes hydrolyze glutamine to glutamate, which supports cancer cell metabolism through TCA cycle anaplerosis and glutathione biosynthesis. The GLS1 glutaminase isozyme has well-established roles in cancer cell metabolism. Conversely, GLS2 is an enigmatic enzyme with reported roles in both tumor promotion and tumor suppression and remains an underdeveloped drug target. This finding suggests roles for GLS2 in supporting Warburg metabolism and managing oxidative stress in cancer cells. X1 and X4 may accelerate the development of high-quality inhibitors of GLS2 to clarify its unique roles in cancer cell metabolism.