Article Figures & Data
Figures
- FIG 1
Efficacy of PC945 as an inhibitor of A. fumigatus sterol 14α-demethylases (CYP51 enzymes). (A) Structure of PC945. (B) Type II azole binding spectra for A. fumigatus CYP51A and CYP51B. Each experiment was performed 4 to 6 times, although data for only one replicate are shown. (C) Azole IC50 determinations for posaconazole (●) and PC945 (○). Mean relative velocity values are shown with standard deviations. (D) Sterol composition of A. fumigatus treated with PC945. The relative levels of lanosterol and eburicol are shown.
- FIG 2
Antifungal activity of PC945 against A. fumigatus in vitro and in vivo. (A) A549 cells treated with PC945 or posaconazole and infected with GFP-expressing A. fumigatus. Green, GFP-expressing A. fumigatus; blue, DAPI-stained nucleus; red, CellTracker-stained cytoplasm. (B) Persistence of action of PC945 and voriconazole on A. fumigatus hyphae. (C) Persistence of action of PC945 and voriconazole on human bronchial cells (BEAS2B cells) infected with A. fumigatus. (D and E) Effects of once-daily intranasal treatment with PC945 (0.56, 2.8, or 14 μg/mouse) and posaconazole (2.8, 14, or 70 μg/mouse) on survival of A. fumigatus-infected immunocompromised mice (n = 8 to 16). (F) Galactomannan levels in BALF. Each horizontal bar shows the mean ± SEM for 8 mice per group. #, dead before day 7; ***, significant difference from no infection (P < 0.001); †††, significant difference from infection control (P < 0.001).
Tables
- TABLE 1
Azole Kd and IC50 determinations versus A. fumigatus CYP51 (AfCYP51) enzymes
Test agent Parameter value (μM)a AfCYP51A AfCYP51B Kd IC50 Kd1 Kd2 IC50 PC945 0.50 0.23 19,298 0.32 0.22 Posaconazole 0.96 0.16 0.012 NA 0.17 ↵a Kd determinations used 4 μM purified AfCYP51A and 4 μM AfCYP51B. IC50 determinations used 0.5 μM AfCYP51A and 0.5 μM AfCYP51B, recovered from the membrane fraction of E. coli expression clones. NA, not applicable.
- TABLE 2
Sterol composition of A. fumigatus treated with either posaconazole or PC945
Sterol Sterol content in cells (%) DMSO-treated cells Posaconazole-treated (μg/ml) cells DMSO-treated cells PC945-treated (μg/ml) cells 0.0001 0.001 0.01 0.1 1 0.0001 0.001 0.01 0.1 1 Ergosterol 100 94.5 87.2 74.7 67.8 67.4 100 95.9 94.7 86.7 80.6 71.3 Ergost-5,7-dienol 0 3.3 3.9 0 0 0 0 4.1 2.5 3.3 0 0 Lanosterol 0 0 3.0 7.0 8.8 8.8 0 0 0 3.6 6.2 9.4 Eburicol 0 2.2 5.9 18.3 23.4 23.8 0 0 2.7 6.5 13.2 19.3 - TABLE 3
Antifungal effects of PC945 and known antifungal agents in azole-susceptible and azole-resistant strains of A. fumigatusa
Strain Resistance mechanism IC50 (IC90) (μg/ml) of indicated agent PC945 Voriconazole Posaconazole Itraconazole Amphotericin B Caspofungin NCPF2010 None 0.0084 (0.010) 0.16 (0.20) 0.0086 (0.014) 0.057 (0.085) 0.23 (0.48) 0.11 (>1) AF294 None 0.0020 (0.0043) 0.082 (0.27) 0.0056 (0.011) 0.041 (0.052) 0.21 (0.79) >1 (>1) AF293 None 0.0012 (0.0041) 0.25 (0.74) 0.010 (0.028) 0.032 (0.23) 0.24 (0.85) >1 (>1) AF72 G54E mutation 0.0061 (0.029) 0.019 (0.062) 0.032 (0.19) 0.43 (>1) 0.18 (0.64) 0.10 (>1) AF91 M220V mutation 0.0081 (0.059) 0.12 (0.38) 0.024 (0.12) 0.26 (>1) 0.42 (>1) 0.072 (>1) TR34/L98H TR34/L98H mutation 0.034 (>1) >1 (>1) 0.086 (0.13) 0.22 (>1) 0.14 (0.29) 0.082 (>1) ↵a IC50 and IC90 values were determined from optical density measurements.
- TABLE 5
Antifungal effects of PC945 and known antifungal agents on four itraconazole-susceptible A. fumigatus strains, as determined using CLSI methodology
Test agent MIC (μg/ml)a,c MIC50 (μg/ml)b,c Median Interquartile range Median Interquartile range PC945 0.031* 0.020–0.031 0.011** 0.0083–0.024 Voriconazole 0.5 0.5–0.5 0.14 0.082–0.15 Posaconazole 0.047 0.031–6.0 0.015 0.0095–0.016 - TABLE 6
Antifungal effects of PC945 and posaconazole on other fungal species
Species (strain[s]) No. of strains tested Culture method MIC (μg/ml)a PC945 Voriconazole Posaconazole Aspergillus carbonarius (ATCC 8740) 1 CLSI 4 0.5 0.063 Aspergillus flavus (ATCC 204304) 1 CLSI >8 2 0.13 Aspergillus flavus (AFL8, NRRC3357) 2 EUCAST 6 0.63 0.16 Aspergillus niger (ATCC 1015) 1 EUCAST >8 1 0.20 Aspergillus terreus (AT49, AT7130) 2 EUCAST 0.078 1 0.093 Penicillium chrysogenum (ATCC 9480) 1 CLSI >8 2 0.13 Penicillium citrinum (ATCC 9849) 1 CLSI >8 >8 0.5 Trichophyton rubrum (ATCC 10218) 1 CLSI 0.031 0.063 0.031 Aureobasidium pullulans (ATCC 9348) 1 CLSI >8 >8 1 Cladosporium argillaceum (ATCC 38013) 1 CLSI >8 0.5 0.25 Candida albicansb (20240.047, ATCC 10231) 2 CLSI 0.081 0.14 0.081 AR Candida albicansb,c (20183.073, 20186.025) 2 CLSI 8.25 10 8.13 Candida glabratab (ATCC 36583, R363) 2 CLSI 0.5 8.13 0.5 Candida krusei (ATCC 6258) 1 CLSI 0.125 0.25 0.125 Chaetomium globosum (ATCC 44699) 1 CLSI >8 1 0.25 Gibberella zeae (Fusarium graminearum) (ATCC 16106) 1 CLSI >8 >8 >8 Cryptococcus gattii (clinical isolate) 1 EUCAST 0.25 0.125 0.5 Cryptococcus neoformans (ATCC 24067) 1 CLSI 0.008 0.016 0.016 Lichtheimia corymbifera (ATCC 7909) 1 CLSI >8 >8 >8 Mucor circinelloides (ATCC 8542) 1 CLSI >8 >8 >8 Rhizomucor pusillus (ATCC 16458) 1 CLSI >8 >8 >8 Rhizopus oryzae (ATCC 11145) 1 CLSI 2 >8 >8 - TABLE 7
Potencies and persistence of action of PC945, posaconazole, and voriconazole
Test agent Hyphae BEAS2B cells IC50 (μg/ml)a Fold change IC50 (μg/ml) Fold change No washout Washout No washout Washout PC945 0.00010* 0.000086 0.87 0.0037 0.043 11.5 Voriconazole 0.011 >1 >93 0.054 >1 >18.6 Posaconazole 0.00045 0.0022 4.90 0.0031 0.046 14.7 ↵a *, P < 0.05 for PC945 versus the results for voriconazole (Kruskal-Wallis one-way ANOVA with Dunn's test).
- TABLE 8
In vivo activities of PC945 and posaconazole
Test agent Dose (μg/mouse) No. of survivors on day 7/total no. of mice (%) Median days of survival Log rank (Mantel-Cox test) P valuea Vehicle 3/16 (19) 5 PC945 0.56 4/8 (50) 6 0.14 2.8 5/8 (63) 6.5 0.022* 14 10/16 (63) Undefined 0.0095** Posaconazole 2.8 1/8 (13) 5 0.69 14 7/16 (44) 6 0.050* 70 6/8 (75) Undefined 0.0028** ↵a *, P < 0.05; **, P < 0.01.
Supplemental material
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Supplemental Figures S1 and S2
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