Fluorogenic Assays for Measuring the Activity of Ceramidase and Sphingomyelinase

DESCRIPTION (provided by applicant): Sphingolipids are a class of lipids that have important roles as structural components of cell membranes yet are also key signaling molecules involved in numerous cellular processes such as apoptosis, cell survival, dif ferentiation, motility, and growth. The levels of the various sphingolipids are kept in balance by metabolizing enzymes and disruptions by abnormal levels of these enzymes are known to occur in diseases such as cancer, diabetes, arthersclerosis, and autoim mune disorders. In this application, fluorogenic assays for two important sphingolipid metabolizing enzymes, ceramidase and sphingomyelinase, will be developed. The fluorescence dequenching assays utilize internally quenched fluorescent substrates which, w hen hydrolyzed by the enzymes, fluoresce due to the quencher no longer being in close proximity to the fluor. Labeled analogs of ceramide and sphingomyelin will be synthesized, evaluated as substrates with their respective enzymes, and the assay conditions will be optimized. These assays can be employed to further basic research and be adapted to high throughput screening for therapeutic compounds. PUBLIC HEALTH RELEVANCE: The overall goal of this project is to develop assays for two important lipid metabolizing enzymes: ceramidase and sphingomyelinase. These assays will help to further basic research in this growing field and potentially be used for developing new drugs for diseases such as cancer, diabetes, and Alzheimer's disease.