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Saccharomyces cerevisiae as a platform for assessing sphingolipid kinase inhibitors

By Yugesh Kharel, Sayeh Agah, Tao Huang, Anna J. Mendelson, Oluwafunmilayo T. Eletu, Peter Barkey-Bircann, James Gesualdi, Jeffrey S. Smith, Webster L. Santos, Kevin R. Lynch

Posted 11 Jun 2017
bioRxiv DOI: 10.1101/148858 (published DOI: 10.1371/journal.pone.0192179)

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 our adaptation of 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.

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