Identification of inhibitors of vacuolar proton-translocating ATPase pumps in yeast by high-throughput screening flow cytometry.
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.
Johnson
Rebecca M
RM
Department of Biochemistry and Molecular Biology, University of New Mexico School of Medicine, Albuquerque, NM, USA.
Allen
Chris
C
Melman
Sandra D
SD
Waller
Anna
A
Young
Susan M
SM
Sklar
Larry A
LA
Parra
Karlett J
KJ
D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005434:Flow Cytometry; D005452:Fluoresceins / Q000737:chemistry; D057166:High-Throughput Screening Assays; D006863:Hydrogen-Ion Concentration; D018942:Macrolides / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors*; D014617:Vacuoles; D015003:Yeasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*
D004791:Enzyme Inhibitors; D005452:Fluoresceins; D018942:Macrolides; C057433:2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester; C036978:concanamycin A; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D025262:Vacuolar Proton-Translocating ATPases