FIG 1.

Antagonism of the azoles to olorofim. (A) Broth dilution assay of olorofim on A. fumigatus MFIG001 to olorofim, following EUCAST methodology and measured by OD₆₀₀ (n = 4). (B) Addition of 10 mM uracil and 10 mM uridine reverses the action of olorofim on A. fumigatus MFIG001 (n = 3). (C) Antagonism on a solid RPMI 1640 plate inoculated with A. fumigatus isolates. Voriconazole (800 mg/L) was inoculated on the disk on the left and olorofim (500 mg/L) on the disk on the right. The disk assay for TR34 L98H contained 8000 mg/L voriconazole to obtain a halo of equal size to MFIG001. (D) Checkerboard assay (n = 3) for CEA10 and the azole resistant TR34 L98H isolate to voriconazole and olorofim. Growth was normalized to RPM-1640 without any antifungal drug. Green denotes full growth, and black denotes no observed growth. (E) Disk assay on solid RPMI 1640 at 800 mg/L voriconazole and 500 mg/L olorofim for the TR34 L98H strain. (F) Dose-response RNA-seq upon itraconazole exposure (0.5× MIC to 4× MIC). The expression of genes of the pyrimidine pathway and upstream pathways were differentially upregulated only in sub-MICs of itraconazole.