Identification of inhibitors of vacuolar proton-translocating ATPase pumps in yeast by high-throughput screening flow cytometry
Autor: | Susan M. Young, Sandra D. Melman, Larry A. Sklar, Anna Waller, Karlett J. Parra, Rebecca M. Johnson, Chris Allen |
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Rok vydání: | 2010 |
Předmět: |
Vacuolar Proton-Translocating ATPases
ATPase Drug Evaluation Preclinical Biophysics Saccharomyces cerevisiae Vacuole Biochemistry Article Flow cytometry ATP hydrolysis Yeasts medicine V-ATPase Enzyme Inhibitors Molecular Biology biology medicine.diagnostic_test Vacuolar lumen Cell Biology Hydrogen-Ion Concentration Flow Cytometry Fluoresceins Yeast High-Throughput Screening Assays Spectrometry Fluorescence Vacuoles Disulfiram biology.protein Macrolides medicine.drug |
Zdroj: | Analytical Biochemistry. 398:203-211 |
ISSN: | 0003-2697 |
Popis: | Fluorescence intensity of the pH-sensitive carboxyfluorescein derivative 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was monitored by high-throughput flow cytometry in living yeast cells. We measured fluorescence intensity of BCECF trapped in yeast vacuoles, acidic compartments equivalent to lysosomes where vacuolar proton-translocating ATPases (V-ATPases) are abundant. Because V-ATPases maintain a low pH in the vacuolar lumen, V-ATPase inhibition by concanamycin A alkalinized the vacuole and increased BCECF fluorescence. Likewise, V-ATPase-deficient mutant cells had greater fluorescence intensity than wild-type cells. Thus, we detected an increase of fluorescence intensity after short- and long-term inhibition of V-ATPase function. We used yeast cells loaded with BCECF to screen a small chemical library of structurally diverse compounds to identify V-ATPase inhibitors. One compound, disulfiram, enhanced BCECF fluorescence intensity (although to a degree beyond that anticipated for pH changes alone in the mutant cells). Once confirmed by dose-response assays (EC(50)=26 microM), we verified V-ATPase inhibition by disulfiram in secondary assays that measured ATP hydrolysis in vacuolar membranes. The inhibitory action of disulfiram against V-ATPase pumps revealed a novel effect previously unknown for this compound. Because V-ATPases are highly conserved, new inhibitors identified could be used as research and therapeutic tools in cancer, viral infections, and other diseases where V-ATPases are involved. |
Databáze: | OpenAIRE |
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