A high-throughput screening assay for small molecules that disrupt yeast cell integrity

Area of Science:

• Biochemistry
• Microbiology
• Drug Discovery

Background:

• Invasive fungal infections pose a significant threat to immunocompromised patients, driving the need for novel antifungal therapies.
• Existing antifungal drug development requires efficient screening methods to identify compounds with fungicidal activity.

Purpose of the Study:

• To develop and validate a high-throughput screening assay for identifying small molecules that induce yeast cell lysis.
• To establish an assay that specifically detects fungicidal activity, distinguishing it from fungistatic effects.

Main Methods:

• A high-throughput screening assay utilizing adenylate kinase release as a reporter for yeast cell lysis.
• The assay employs a commercially available adenylate kinase assay kit in a simple, add-and-read 1-plate format.
• Incubation time was optimized to 5 hours for rapid results.

Main Results:

• The assay successfully detected cell lysis at drug concentrations that did not impair yeast growth.
• The screening demonstrated specificity for fungicidal molecules, with identified compounds targeting the plasma membrane.
• Pilot screening of 4505 compounds validated the assay's utility and specificity for fungicidal agents.

Conclusions:

• Adenylate kinase-based screening is a valuable and direct method for identifying small molecules that kill yeast cells.
• This assay provides a rapid, cost-effective, and specific approach to advance antifungal drug discovery efforts.
• The assay is applicable to both model yeast (Saccharomyces cerevisiae) and the pathogenic fungus (Candida albicans).