Repurposing the Insecticide Flufenerim, the Only Anti- Cryptococcus Compound Identified in the MMV Global Health Priority Box, as a Candidate for the Environmental Control of the Cryptococcosis Agents

Abstract Cryptococcus neoformans and Cryptococcus gattii are environmental fungi that cause cryptococcosis, a severe, life-threatening infection, particularly in immunocompromised individuals. Current treatments are limited by toxicity, restricted availability, and the emergence of resistance, highlighting the need for novel antifungal agents. Here, 240 compounds from the Medicines for Malaria Venture (MMV) Global Health Priority Box were screened for activity against these two species. Flufenerim, originally developed as an insecticide, was the only compound that exhibited inhibitory activity at 1 μM (0.364 μg/mL). Minimum inhibitory concentrations ranged from 0.03 μg/mL to >8 μg/mL across 15 isolates, and the compound exhibited a predominantly fungistatic profile, except for one C. gattii strain. At subinhibitory concentrations, flufenerim significantly reduced capsule thickness in both species, melanization in C. neoformans, and urease production in C. gattii, without affecting phospholipase activity. In silico ADMET predictions indicated predicted toxicological risks requiring further evaluation. Cytotoxicity assays further demonstrated that the compound did not exhibit significant toxicity in murine and human macrophages, nor in human brain endothelial cells (HBECs). Exposure of Galleria mellonella larvae to flufenerim produced sublethal physiological effects without mortality. Due to the unfavorable ADMET profile, we evaluated whether this compound could be used for environmental control of fungal reservoirs. Tree-hollow samples spiked with C. neoformans and treated with flufenerim yielded fewer yeast colonies than untreated controls when plated on Staib’s agar. These findings identify flufenerim as a new anti-Cryptococcus compound and provide preliminary evidence supporting its potential for reducing C. neoformans recovery from environmental substrates. However, the ecological complexity of Cryptococcus reservoirs and the potential effects on nontarget microorganisms require extensive investigation before any environmental application can be considered.