Identification of Antifungal H⁺-ATPase Inhibitors with Effect on Plasma Membrane Potential

The plasma membrane H⁺-ATPase (Pma1) is an essential fungal protein and a proposed target for new antifungal medications. The compounds in a small-molecule library containing ∼191,000 commercially available compounds were screened for their ability to inhibit Saccharomyces cerevisiae plasma membranes containing Pma1. The overall hit rate was 0.2%, corresponding to 407 compounds. These hit compounds were further evaluated for ATPase selectivity and broad-spectrum antifungal activity. Following this work, one Pma1 inhibitor series based on compound 14 and analogs was selected for further evaluation. This compound series was able to depolarize the membrane and inhibit extracellular acidification in intact fungal cells concomitantly with a significant increase in intracellular ATP levels. Collectively, we suggest that these effects may be a common feature of Pma1 inhibitors. Additionally, the work uncovered a dual mechanism for the previously identified cationic peptide BM2, revealing fungal membrane disruption, in addition to Pma1 inhibition. The methods presented here provide a solid platform for the evaluation of Pma1-specific inhibitors in a drug development setting. The present inhibitors could serve as a starting point for the development of new antifungal agents with a novel mode of action.