Identification of inhibitors of vacuolar proton-translocating ATPase pumps in yeast by high-throughput screening flow cytometry

• Published in: Analytical biochemistry Vol. 398; no. 2; pp. 203 - 211
• Main Authors: Johnson, Rebecca M., Allen, Chris, Melman, Sandra D., Waller, Anna, Young, Susan M., Sklar, Larry A., Parra, Karlett J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.03.2010
• Subjects: BCECF; Chemical library; Concanamycin A; Disulfiram; Drug Evaluation, Preclinical - methods; Enzyme Inhibitors - pharmacology; Flow Cytometry; Fluoresceins - chemistry; Fluorescence; High throughput; High-Throughput Screening Assays; Hydrogen-Ion Concentration; Macrolides - pharmacology; Pumps; Saccharomyces cerevisiae - enzymology; Spectrometry, Fluorescence; V-ATPase; V-ATPase inhibitors; Vacuolar Proton-Translocating ATPases - antagonists & inhibitors; Vacuoles; Yeast; Yeasts - cytology; Yeasts - drug effects; Yeasts - enzymology; Flow cytometry; Yeast; V-ATPase; Chemical library; BCECF; Fluorescence; High throughput; Pumps; V-ATPase inhibitors; pH; Disulfiram; Vacuoles; Concanamycin A
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2009.12.020

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 μM), 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.