title: A High-Throughput Genetic Complementation Assay in Yeast Cells Identified Selective Inhibitors of Sphingosine Kinase 1 Not Found Using a Cell-Free Enzyme Assay.
abstract: Sphingosine kinase 1 (SphK1) is a lipid kinase that phosphorylates sphingosine to produce the bioactive sphingolipid, sphingosine-1-phosphate (S1P), and therefore represents a potential drug target for a variety of pathological processes such as fibrosis, inflammation, and cancer. We developed two assays compatible with high-throughput screening to identify small-molecule inhibitors of SphK1: a purified component enzyme assay and a genetic complementation assay in yeast cells. The biochemical enzyme assay measures the phosphorylation of sphingosine-fluorescein to S1P-fluorescein by recombinant human full-length SphK1 using an immobilized metal affinity for phosphochemicals (IMAP) time-resolved fluorescence resonance energy transfer format. The yeast assay employs an engineered strain of Saccharomyces cerevisiae, in which the human gene encoding SphK1 replaced the yeast ortholog and quantitates cell viability by measuring intracellular adenosine 5'-triphosphate (ATP) using a luciferase-based luminescent readout. In this assay, expression of human SphK1 was toxic, and the resulting yeast cell death was prevented by SphK1 inhibitors. We optimized both assays in a 384-well format and screened ∼10(6) compounds selected from the Boehringer Ingelheim library. The biochemical IMAP high-throughput screen identified 5,561 concentration-responsive hits, most of which were ATP competitive and not selective over sphingosine kinase 2 (SphK2). The yeast screen identified 205 concentration-responsive hits, including several distinct compound series that were selective against SphK2 and were not ATP competitive.
authors.affiliation: 1 Small Molecule Discovery Research, Boehringer Ingelheim Pharmaceuticals, Inc. , Ridgefield, Connecticut.
authors.forename: Mohammed A
authors.initials: MA
authors.lastname: Kashem
authors.affiliation: 1 Small Molecule Discovery Research, Boehringer Ingelheim Pharmaceuticals, Inc. , Ridgefield, Connecticut.
authors.forename: Charles A
authors.initials: CA
authors.lastname: Kennedy
authors.affiliation: 1 Small Molecule Discovery Research, Boehringer Ingelheim Pharmaceuticals, Inc. , Ridgefield, Connecticut.
authors.forename: Kylie E
authors.initials: KE
authors.lastname: Fogarty
authors.affiliation: 2 Immunology and Respiratory Research, Boehringer Ingelheim Pharmaceuticals, Inc. , Ridgefield, Connecticut.
authors.forename: Janice R
authors.initials: JR
authors.lastname: Dimock
authors.affiliation: 1 Small Molecule Discovery Research, Boehringer Ingelheim Pharmaceuticals, Inc. , Ridgefield, Connecticut.
authors.forename: Yunlong
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: 1 Small Molecule Discovery Research, Boehringer Ingelheim Pharmaceuticals, Inc. , Ridgefield, Connecticut.
authors.forename: Mary L
authors.initials: ML
authors.lastname: Sanville-Ross
authors.affiliation: 1 Small Molecule Discovery Research, Boehringer Ingelheim Pharmaceuticals, Inc. , Ridgefield, Connecticut.
authors.forename: Donna J
authors.initials: DJ
authors.lastname: Skow
authors.affiliation: 1 Small Molecule Discovery Research, Boehringer Ingelheim Pharmaceuticals, Inc. , Ridgefield, Connecticut.
authors.forename: Steven R
authors.initials: SR
authors.lastname: Brunette
authors.affiliation: 2 Immunology and Respiratory Research, Boehringer Ingelheim Pharmaceuticals, Inc. , Ridgefield, Connecticut.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Swantek
authors.affiliation: 3 CompleGen, Inc. , Seattle, Washington.
authors.forename: Heidi S
authors.initials: HS
authors.lastname: Hummel
authors.affiliation: 3 CompleGen, Inc. , Seattle, Washington.
authors.forename: John
authors.initials: J
authors.lastname: Swindle
authors.affiliation: 1 Small Molecule Discovery Research, Boehringer Ingelheim Pharmaceuticals, Inc. , Ridgefield, Connecticut.
authors.forename: Richard M
authors.initials: RM
authors.lastname: Nelson
mesh_terms: D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D057075:Enzyme Assays / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000201:enzymology*; D000099157:Sphingosine Kinase
chemical_list: D004791:Enzyme Inhibitors; D017853:Phosphotransferases (Alcohol Group Acceptor); D000099157:Sphingosine Kinase