High-Throughput Screening of Neutral Sphingomyelinase 2 Inhibitors

The library of pharmacologically active compounds (LOPAC, Sigma) and a subset of the Spectrum Collection (Food and Drug Administration, MicroSource Discovery Systems) were screened against human nSMase2. This screening was done in 384-well format at a single concentration (10 μM) in duplicate using the fluorescence-based activity assay. Positive control inhibitors known to block nSMase2 activity were used in all assays: 150 μM GW4869 (Sigma), 100 μM manumycin A (Enzo Lifescience) and 100 μM altenusin (Enzo Lifescience). A negative control, 100 μM zoledronic acid (Enzo Lifescience), known to inhibit acid but not neutral sphingomyelinase, was also included in all plates. Compounds that showed at least 50% inhibitory activity selective for nSMase2 were considered hits. The compound physicochemical properties and drug-like features were used to evaluate the hits and select those to be tested in the confirmatory direct activity assay using ¹⁴C-labeled substrate.