FIG 1.

The trisubstituted isoxazole MMV688766 has fungicidal activity against yeasts and molds. (A) Summary of Pathogen Box screen (left). Each of the 400 Pathogen Box compounds was screened at 25 μM in RPMI at 30°C for 48 h against a C. auris clinical isolate (CDC0387) in 96-well plates. Growth was monitored by optical density at 600 nm (OD₆₀₀). Percent growth of treated samples was calculated by normalizing the OD₆₀₀ of treated wells to the average OD₆₀₀ of DMSO solvent controls (16 per plate). Single-agent antifungal hits (red) were identified by a >80% reduction in growth compared to DMSO solvent controls. MMV688766 is indicated in solid red. Amphotericin B control is indicated in black. Structures of each hit compound are shown at right. (B) The five hits were validated for bioactivity using dose-response assays in RPMI at 30°C. Growth was measured using absorbance at 600 nm after 48 h. Technical duplicates were averaged, and measurements were normalized to no-drug controls. Relative growth is quantitatively represented by color (see scale bar at bottom). Fungicidal activity was assessed by removing 3 μL from each well from the dose-response assay after the 48-h incubation, and spotting the inoculum onto drug-free YPD agar. Plates were incubated for 24 h at 30°C. (C) MMV688766 activity was tested against a panel of C. auris isolates from each geographically distinct clade (1 to 4) as described in panel B. (D) MMV688766 activity was tested against C. albicans, C. glabrata, and C. neoformans as described in B. For A. fumigatus, MMV688766 activity was assessed using a 2-fold dose-response assay in RPMI at 37°C for 48 h. Relative viable cell number was quantified by incubating cells with alamarBlue (0.03% wt/vol) after 48 h followed by an additional 24-h incubation at 37°C. Growth of A. fumigatus was also observed microscopically using an IncuCyte live cell imager after 48-h incubation with compound at ×20 magnification (scale bar = 200 μm). All dose-response data are plotted using Java TreeView3; see color bars.