title: Cellular assay for the characterization of sphingosine-1-phosphate lyase inhibitors.
abstract: Sphingosine-1-phosphate (S1P) lyase represents a target for therapeutic intervention in immune regulation. Inhibitors of the lyase can be identified by established biochemical assays, but a cellular test system for such inhibitors has not been described so far. We found that silencing or inhibition of S1P lyase with short interfering RNA (siRNA) or active site-directed inhibitors in cultured mammalian cells does not cause a relevant increase of S1P in the cells as measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, the addition of sphingosine to cultures of cell lines or primary cells provides a source of intracellular S1P that is susceptible to degradation by the lyase and, hence, increases on inhibition or silencing of the enzyme. The assay was optimized with respect to sphingosine concentration, incubation time, and cell density and was established for routine use with HEK293 cells. The assay was found to be suitable for the testing of novel active site-directed S1P lyase inhibitors, providing important information on their relative potency in intact cells.
authors.affiliation: Novartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland. andreas.billich@novartis.com
authors.forename: Andreas
authors.initials: A
authors.lastname: Billich
authors.forename: Christian
authors.initials: C
authors.lastname: Beerli
authors.forename: Reinhard
authors.initials: R
authors.lastname: Bergmann
authors.forename: Christian
authors.initials: C
authors.lastname: Bruns
authors.forename: Erika
authors.initials: E
authors.lastname: Loetscher
mesh_terms: D000818:Animals; D001681:Biological Assay; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D008246:Lysophospholipids / Q000037:antagonists & inhibitors* / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D015394:Molecular Structure; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000235:genetics
chemical_list: D004791:Enzyme Inhibitors; D008246:Lysophospholipids; C060506:sphingosine 1-phosphate; D013110:Sphingosine