A High-Throughput Screening Assay for Small Molecules That Disrupt Yeast Cell Integrity

Abstract

Lead compounds for antifungal drug development are urgently needed because invasive fungal infections are an important cause of morbidity and mortality in immunocompromised patients. Here, a high-throughput screening assay for small molecules that cause yeast cell lysis is described. The assay is based on the detection of the intracellular enzyme adenylate kinase in the culture medium as a reporter of yeast cell lysis. Features of the assay protocol include 1) the ability to detect cell lysis at drug concentrations that cause no apparent growth defect, 2) specificity for fungicidal molecules, 3) a simple 1-plate, add-and-read protocol using a commercially available adenylate kinase assay kit, 4) short, 5-h incubation time, and 5) low cost. The assay is applicable to the model yeast Saccharomyces cerevisiae and to Candida albicans, the most common human fungal pathogen. The adenylate kinase assay is validated in a pilot screen of 4505 compounds. Consistent with its specificity for fungicidal molecules, the largest class of molecules identified in 2 libraries of known bioactive molecules targeted the plasma membrane. Fungistatic compounds are not detected by the assay. Adenylate kinase-based screening appears to be a useful approach to the direct identification of small molecules that kill yeast cells. (Journal of Biomolecular Screening 2008:657-664)

A Repurposing Approach Identifies Off-Patent Drugs with Fungicidal Cryptococcal Activity, a Common Structural Chemotype, and Pharmacological Properties Relevant to the Treatment of Cryptococcosis

“…The starting CFU per well was 1.5 ϫ 10 3 , and the MFC was defined as the lowest concentration of compound that reduced the starting inoculum by 2 log 10 . Propidium iodide (PI) staining was performed as described previously (13). All assays were performed on two or three separate days with independent isolates of H99, and each assay was performed in triplicate.…”

“…The AK assay detects the release of the intracellular enzyme AK into the growth medium by cells that have lost membrane integrity by coupling its conversion of ADP to ATP with a luciferase reporter reaction. We have previously shown that the assay is robust in the HTS setting and is more sensitive than growth assays (8,13). Furthermore, it is specific for molecules that directly kill yeast cells; for example, the fungistatic drug fluconazole is completely inactive in this assay (13).…”

“…We have previously shown that the assay is robust in the HTS setting and is more sensitive than growth assays (8,13). Furthermore, it is specific for molecules that directly kill yeast cells; for example, the fungistatic drug fluconazole is completely inactive in this assay (13).…”

“…We further focused our screen on molecules that directly kill C. neoformans, because drugs with this type of activity have been shown to be more effective in the treatment of C. neoformans disease than molecules that simply inhibit growth. To identify molecules that directly kill C. neoformans cells, we used an AK release assay recently developed in our laboratory (8,13). The AK assay detects the release of the intracellular enzyme AK into the growth medium by cells that have lost membrane integrity by coupling its conversion of ADP to ATP with a luciferase reporter reaction.…”

Role of the Cell Wall Integrity and Filamentous Growth Mitogen-Activated Protein Kinase Pathways in Cell Wall Remodeling during Filamentous Growth

“…6). Although some effective fungicides exist, like capsofungin, which irreversibly inhibits 1,3-ß-glucan synthase (57), lead compounds are needed to attenuate invasive fungal infections of immunocompromised patients (73), and other potential cell wall targets remain largely unexplored (77). Further characterization of filamentation-dependent cell wall remodeling in budding yeast is likely to provide useful insights into the regulatory inputs of cell wall regulation that may pertain to fungal pathogenesis.…”