A novel pan-fungal screening platform for antifungal drug discovery: proof of principle study

Broad-spectrum activity is a desirable property of novel antifungal drugs, but relevant <i>in vitro</i> testing is complicated by differential nutritional requirements and growth dynamics of fungal pathogens. Many screens for novel drugs are initiated against individual species or genera, with hit compounds later tested for "pan-fungal" activity. Hypothesizing that an optimized pan-fungal methodology would enhance the efficiency of early-stage drug discovery, a standardized assay was developed for a selection of World Health Organization-defined critical and high-priority fungal pathogens. Instead of using the standard susceptibility testing broth RPMI, an enriched media "fungal RPMI" (fRPMI), including multiple additional fungal growth-enhancing nutrients, was utilized. To assess utility for pan-fungal growth assessments, growth in fRPMI was compared to RPMI medium for 12 fungal pathogens. Growth was significantly improved in 7/12 species in fRPMI after 24 and/or 48 hours. For our proof-of-principle study, 500 chemical fragments from the Maybridge Ro3 Fragment library were screened at concentrations of 0.1 or 1 mM against five fungal pathogens: <i>Aspergillus fumigatus</i>, <i>Candida albicans</i>, <i>Candida auris</i>, <i>Cryptococcus neoformans</i>, and <i>Nakaseomyces glabratus</i>. Assay quality was assessed using <i>z</i>-factor analysis, and hits were normalized using a standard <i>z</i>-score to identify outliers. All assays achieved a high-quality <i>z</i>-factor (≥0.5) with readings at ≤24 hours, allowing the identification of 23 compounds with antifungal activity against at least one fungal species. From these, five compounds were identified as having pan-assay interference or broadly toxic properties. In conclusion, hits identified from pan-fungal phenotypic growth-based assays demonstrate reproducibility in all fungal species tested with carefully optimized conditions and precise timing.