3 years ago

Saccharomyces cerevisiae as a platform for assessing sphingolipid kinase inhibitors

Mendelson, W. L., O. T., S., Agah, Santos, Gesualdi, Kharel, Lynch, Smith, J., A. J., Y., K. R., J. S., Eletu
Successful medicinal chemistry campaigns to discover and improve sphingosine kinase inhibitors require a robust assay for screening chemical libraries and for determining rank order potencies. Existing assays for these enzymes are laborious, expensive and/or low throughput. The toxicity of excessive levels of phosphorylated sphingoid bases for the budding yeast, Saccharomyces cerevisiae, affords an assay wherein inhibitors added to the culture media rescue growth in a dose-dependent fashion. Herein, we describe a simple, inexpensive, and high throughput assay for assessing inhibitors of sphingosine kinase types 1 and 2 as well as ceramide kinase and for testing enzymatic activity of sphingosine kinase type 2 mutants. The assay was validated using recombinant enzymes and generally agrees with rank order of potencies of existing inhibitors.

Publisher URL: http://biorxiv.org/cgi/content/short/148858v1

DOI: 10.1101/148858

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