Development of a FRET-Based Assay for Human Neutral Sphingomyelinase 2

Neutral sphingomyelinase 2 (nSMase2) is a membrane-bound enzyme that hydrolyzes sphingomyelin (SM) into ceramide and phosphocholine. By generating the bioactive lipid ceramide, nSMase2 plays a critical role in cell stress responses and in regulating exosomes that package and transfer pathogenic factors, including tau protein and amyloid β. Thus, nSMase2 has been implicated in Alzheimer's disease and other neurological disorders. However, current tools to measure nSMase2 activity are limited, in particular those that could be used in therapeutic development. Here we developed a high-throughput assay to measure human nSMase2 activity. The assay uses a sphingomyelin substrate analogue with a FRET donor and acceptor pair attached to the headgroup and acyl-chain, respectively. Using recombinant human nSMase2, we sensitively detected both wild-type and mutant activity and demonstrated inhibition by the known nSMase2 inhibitor GW4869. The assay captures the major hallmarks of nSMase2 regulation by anionic lipids and displays sensitivity capable of detecting nSMase2 activity from cell lysates. Together, this assay enables rapid screening of nSMase2 inhibitors to support future therapeutic development.