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

## TL;DR

This study introduces a new method to test antifungal drugs against multiple fungi at once, improving early drug discovery efficiency.

## Contribution

A novel pan-fungal screening platform using optimized media and standardized assays for early-stage antifungal drug discovery.

## Key findings

- Growth in enriched fRPMI medium improved for 7 out of 12 fungal species compared to standard RPMI.
- 23 compounds showed antifungal activity against at least one species in a high-quality pan-fungal screen.
- Five compounds exhibited broad toxicity or interference across fungal assays.

## Abstract

Broad-spectrum activity is a desirable property of novel antifungal drugs, but relevant in vitro 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: Aspergillus fumigatus, Candida albicans, Candida auris, Cryptococcus neoformans, and Nakaseomyces glabratus. Assay quality was assessed using z-factor analysis, and hits were normalized using a standard z-score to identify outliers. All assays achieved a high-quality z-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.