A sensitive cell-based method to screen for selective inhibitors of SMS1 or SMS2 using HPLC and a fluorescent substrate.
Recent studies have revealed that sphingomyelin (SM) is involved in metabolic syndrome and is a new target of an anti-metabolic syndrome drug. Deficiencies in the enzyme SM synthase 1 (SMS1) result in severe abnormalities, whereas deficiencies in SMS2 do not. SMS1 and SMS2 synthesize SM under similar conditions, so their respective activities cannot be measured separately. We report here on a sensitive, high-throughput and reliable cell-based method to separately measure each SMS activity and to screen for SMS-specific inhibitors, using HPLC and fluorescent ceramide (Cer) analogs. We isolated SMS-null cells and stably transfected them with SMS1 or SMS2. Using these cells, individual SMS activities could be measured separately. Fluorescent Cer, SM, and glucosylceramide analogs could be separated within 4 min by HPLC using an NH(2) column. SMS activities of SMS1- or SMS2-expressing cells seeded in a single well of a 96-well plate could be measured using HPLC and fluorescent Cer analogs. This method clearly demonstrated that treatment of the cells with their respective siRNA or D609, an inhibitor of SMS, resulted in a significant decrease in each SMS activity. These results indicate that our newly developed method can be utilized for screening therapeutics against metabolic syndrome that target SMS2.
Department of Biomembrane and Biofunctional Chemistry and Developmental Biotechnology, Faculty of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan.
Kota
K
Zama
Susumu
S
Mitsutake
Ken
K
Watanabe
Toshiro
T
Okazaki
Yasuyuki
Y
Igarashi
D000818:Animals; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D002518:Ceramides / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D055808:Drug Discovery / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology*; D005456:Fluorescent Dyes / Q000737:chemistry*; D057166:High-Throughput Screening Assays; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D017855:Transferases (Other Substituted Phosphate Groups) / Q000037:antagonists & inhibitors* / Q000235:genetics
D002518:Ceramides; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; C556686:Sgms1 protein, mouse; D017855:Transferases (Other Substituted Phosphate Groups); C543123:Sgms2 protein, mouse