Targeting the energy metabolism of melanoma cells: FX-11 acts as a mitochondrial uncoupler

In a compound screen on melanoma cells, we identified FX-11 as one of the compounds inhibiting the growth of sensitive and Encorafenib/Binimetinib-resistant 624Mel and Wm3248 melanoma cells. FX-11, reported to be a lactate dehydrogenase inhibitor (LDHi), did not decrease the NAD + /NADH ratio, glucose uptake, or lactate secretion of melanoma cells, unlike other LDH inhibitors we used as controls. Instead, FX-11 increased both the oxygen consumption and the extracellular acidification rate of cells in Seahorse assays, behaving similarly to the mitochondrial uncouplers FCCP and BAM15. FX-11 could also be used as an uncoupler in Mito Stress tests. In addition, FX-11 and BAM15 drastically decreased the mitochondrial membrane potential, in contrast to the LDH inhibitors LDH-IN-I and GNE-140. Finally, FX-11 treatment induced an increase in AMPK and acetyl-CoA carboxylase (ACC) phosphorylation and a decrease in phosphorylation of the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), indicative of AMPK activation by decreased ATP/AMP ratio. The observed AMPK activation seems to be involved in FX-11-mediated cell death since knock-down of AMPKα1 reduced cell death. Specific activation of AMPK by MK8722, on the other hand, increased cell death. Taken together, we provide evidence that FX-11 inhibits the growth of melanoma cells, including drug-resistant ones, through an AMPK-dependent mechanism by acting as a mitochondrial uncoupler. Our data do not support that FX-11 acts as an LDH inhibitor.