Fig. 6: The sterol C4-methyl oxidase inhibitor PF1163A preferentially targets Erg251 and reduces C. albicans virulence.

a Representative disk diffusion assay results of PF1163A (structure shown) against WT, erg251, and erg25 strains. Quantified size of inhibition zones was shown by the AR in millimeter (lines: medians of biological quadruplicates). ns not significant (p-values: 0.6; 0.89; 0.2) b Gene expression was measured from log-phase cultures treated with DMSO or 20 μM PF1163A for 75 min. c Disk diffusion assay with the same layout as in (a). d, e Sensitivity of Candida ERG251 and ERG25 orthologs to PF1163A and 1181-0519. C. albicans strains expressing p251-driven fungal ERG251 or ERG25 as the sole source of sterol C4-methyl oxidase were tested for susceptibility to 10.5 nmol PF1163A (d) or 50 nmol 1181-0519 (e) in disk diffusion assays. f SC5314 was tested in a dual-disk diffusion assay with PF1163A and 1181-0519 at the indicated dose. The experiment was repeated three times with comparable results. g Strains were grown in 200 µL of RPMI + 10% FBS at 37 °C for 24 h and treated with DMSO or PF1163A. Metabolic activity of biofilm was measured using by XTT reduction. h Co-culture of C. albicans and luciferized human HEK293T (HEK293T-fLuc) cells were treated with DMSO or PF1163A for 24 h. Fungal burden was determined by OD and HEK293T-fLuc survival by luminescence. i J774A.1 cells infected with C. albicans were treated with DMSO or PF1163A. Macrophage death stained by propidium iodide (PI) was quantified normalized to DMSO controls. Macrophage Survival = 1- normalized PI area. j C. albicans infected C. elegans (AU37, glp-4 sek-1) was treated with either PF1163A or DMSO and survival monitored for 96 h. ns not significant (p-value: 0.68). b, d, and e show mean ± SD of biological triplicates. g–i show mean ± SD of biological triplicates and curves fit by nonlinear regression. Statistical tests used: Mann–Whitney tests (a, d, and e); two-tailed p-values reported), unpaired student t-tests (b; two-tailed p-values reported), and Mantel–Cox tests (j). In (d, e, g–i), SDs were not shown if smaller than the limit for proper display.