Cellular assay for the characterization of sphingosine-1-phosphate lyase inhibitors
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Billich, Andreas, Beerli, Christian, Bruns, Christian and Loetscher, Erika (2013) Cellular assay for the characterization of sphingosine-1-phosphate lyase inhibitors. Analytical Biochemistry, 434 (2). pp. 247-253. ISSN 0003-2697
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 siRNA or active-site directed inhibitors in cultured mammalian cells does not cause a relevant increase of S1P in the cells as measured by LC-MS/MS. However, 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 upon inhibition or silencing of the enzyme. The assay was optimized with respect to sphingosine concentration, incubation time, and cell density, and was routinely established for use with HEK293 cells. The assay was found suitable for the testing of novel active-site directed S1P lyase inhibitors, providing important information on their relative potency in intact cells.
| Item Type: | Article |
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| Keywords: | Cellular assay, lyase, sphingosine-1-phosphate |
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| Date Deposited: | 13 Oct 2015 13:14 |
| Last Modified: | 13 Oct 2015 13:14 |
| URI: | https://oak.novartis.com/id/eprint/7352 |