Related Experiment Video
Updated: Apr 1, 2026
08:12
High-throughput Screening for Protein-based Inheritance in S. cerevisiae
Published on: August 8, 2017
6.8K
A High-Throughput Genetic Complementation Assay in Yeast Cells Identified Selective Inhibitors of Sphingosine Kinase
Mohammed A Kashem1, Charles A Kennedy1, Kylie E Fogarty1
11 Small Molecule Discovery Research, Boehringer Ingelheim Pharmaceuticals, Inc. , Ridgefield, Connecticut.
Assay and Drug Development Technologies
|October 2, 2015
Summary
We developed two high-throughput screening assays to find inhibitors of sphingosine kinase 1 (SphK1), a target for diseases like cancer. The yeast assay successfully identified selective SphK1 inhibitors not competitive with ATP.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Sphingosine kinase 1 (SphK1) phosphorylates sphingosine to sphingosine-1-phosphate (S1P).
- SphK1 is implicated in pathological processes including fibrosis, inflammation, and cancer.
- SphK1 is a potential drug target for various diseases.
Purpose of the Study:
- To develop and validate high-throughput screening (HTS) assays for identifying small-molecule inhibitors of SphK1.
- To screen a large compound library for SphK1 inhibitors using the developed assays.
Main Methods:
- Developed a biochemical assay using immobilized metal affinity for phosphochemicals (IMAP) and time-resolved fluorescence resonance energy transfer (TR-FRET).
- Developed a genetic complementation assay in Saccharomyces cerevisiae using a luciferase-based readout for cell viability.
- Screened approximately 10^6 compounds using both optimized 384-well format assays.
Main Results:
- The IMAP biochemical assay identified 5,561 concentration-responsive hits, primarily ATP-competitive and lacking SphK2 selectivity.
- The yeast genetic assay identified 205 concentration-responsive hits.
- The yeast assay yielded distinct compound series selective for SphK1 over SphK2 and not ATP-competitive.
Conclusions:
- The developed yeast assay is effective for identifying SphK1 inhibitors with desirable selectivity profiles.
- This study provides a foundation for discovering novel therapeutics targeting SphK1-mediated diseases.
