Bafilomycin-A1 and ML9 Exert Different Lysosomal Actions to Induce Cell Death

Objective: Bafilomycin-A1 and ML9 are lysosomotropic agents, irrespective of cell types. However, the mechanisms of lysosome targeting either bafilomycin-A1 or ML9 are unclear. Methods: The present research has been carried out by different molecular and biochemical analyses like western blot, confocal imaging and FACS studies, as well as molecular docking. Results: Our data shows that pre-incubation of neonatal cardiomyocytes with ML9 for 4h induced cell death, whereas a longer period of time (24h) with bafilomycin-A1 was required to induce an equivalent effect. Neither changes in ROS nor ATP production is associated with such death mechanisms. Flow cytometry, LC3-II expression levels, and LC3-GFP puncta formation revealed a similar lysosomotropic effect for both compounds. We used a molecular docking approach, that predicts a stronger inhibitory activity against V-ATPase-C1 and C2 domains for bafilomycin-A1 in comparison to ML9. Conclusion: Bafilomycin-A1 and ML9 are lysosomotropic agents, involved in cell death events. But such death events are not associated with ATP and ROS production. Furthermore, both the drugs target lysosomes through different mechanisms. For the latter, cell death is likely due to lysosomal membrane permeabilization and release of lysosomal proteases into the cytosol.