Characterization of two novel neutral sphingomyelinase 2 inhibitors in endosomal sorting and Extracellular Vesicle biogenesis

Sphingomyelinase hydrolyzes the phosphodiester bond of the sphingomyelin to ceramide and phosphorylcholine and have been involved in extracellular vesicle (EV) biogenesis and more recently in membrane repair. Here we describe an initial testing of two recently discovered neutral sphingomyelinase 2 (nSMase2) inhibitors ((R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)-carbamate (PDDC) and 2,6-dimethoxy-4-[4-phenyl-5-(2-thienyl)-1H-imidazol-2-yl]phenol (DPTIP)). PDDC and DPTIP show differential effects on cell viability, and EV marker secretion, indicating that side effects of these inhibitors on lysosomal and autophagic degradation pathways need to be considered. Moreover, similar to commonly used nSMase2 inhibitor GW4869, cell type specificity seems to play a role in the endosomal trafficking routes that can be explored to unravel mechanisms of specific EV biogenesis and secretion pathways.