Voriconazole

Antifungal susceptibility testing was performed using a broth microdilution method as described by Leong et al. (10 (link)). Briefly, 200× drug stock dilutions were prepared at a 2× concentration in fresh OptiMAL medium. Amphotericin B, terbinafine, clotrimazole, miconazole, itraconazole, fluconazole, voriconazole, and ketoconazole were purchased from Sigma-Aldrich, Singapore. Stock and drug plate dilutions were prepared in accordance with CLSI and EUCAST guidelines. Yeast inocula were obtained from 4- to 7-day-old strains of Malassezia spp. A 50-μl yeast inoculum was added to 50 μl of 2× concentrated antifungals to achieve a final cell density of 5 × 10³ to 5 × 10⁴ CFU/ml. A 10-μl portion of 2× yeast inoculum that had been diluted 10 times was also plated onto a modified Leeming-Notman agar plate and incubated for 4 to 7 days at 35°C for postverification of the CFU inoculum (10 to 100 colonies per 10 μl). Each assay was performed in triplicate plates for a single culture at every individual time point or reading.

The clinical isolate was subjected to antifungal susceptibility testing according to CLSI guidelines (M38-A document), as previously described [3 (link), 18 (link)], and the MICs of antifungal combinations were determined according to previously described methods [3 (link), 18 (link)]. Itraconazole (Xian-Janssen Pharmaceutical Ltd. Xi’ an, China), terbinafine (Beijing Novartis Pharmaceutical Ltd. (Beijing, China)) and voriconazole (Sigma, USA) were dissolved in 100% DMSO as a stock solution (3200 μg/mL). Drugs were diluted to obtain final concentrations, with Itraconazole and voriconazole from 0.008 to 8 μg/mL and terbinafine from 0.008 to 0.5 μg/mL. Isolates were sub-cultured, and spores from colonies were collected and adjusted with saline to achieve an inoculum concentration of 10⁶ conidia/mL. Each suspension was diluted 1:50–100 with RPMI 1640 to obtain the final test inoculum (0.4–5×10⁴ conidia/mL). Conidial suspension of each of the tested strains were cultivated on RPMI 1640 medium for 7 days at 35°C. Candida parapsilosis ATCC22019 (CBS604), obtained from Centraalbureau voor Schimmelcultures (CBS, the Netherlands), was used as a quality control. The final test inoculum concentration was 0.5–2.5×10³ conidia/mL.

The OJT007 (purity > 90%) was purchased from MolPort (Riga, Latvia). The voriconazole, formic acid, ascorbic acid and dimethyl sulfoxide (DMSO) were procured from Sigma-Aldrich (St. Louis, MO, USA). LC-MS grade water and acetonitrile were purchased from J.T. Baker Chemical Co. (Phillipsburg, NJ, USA). LC-MS/UPLC grade Methanol was acquired from Mallinckrodt Baker (Phillipsburg, NJ, USA). The blank rat plasma was purchased from Innovative Research (Novi, MI, USA). Male Sprague–Dawley (SD) rats were purchased from Envigo RMS (Indianapolis, IN, USA). All of the other chemicals and reagents were used as received.

All C. africana isolates obtained herein were tested for in vitro susceptibility to amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, and posaconazole (Sigma, St. Louis, MO, USA), caspofungin (Merck & Co., Inc.), and micafungin (Astellas Pharma) by using broth microdilution method as described in CLSI document M27-A3 and M27-S4. The MIC values were read following 24 h of incubation and determined visually as the lowest concentration in which prominent decrease in turbidity was observed excepting for amphotericin B which is defined as the lowest concentration in which the absence of turbidity is observed. The recently revised CLSI clinical breakpoints (CBPs) values for C. albicans were used as reference [28 (link)]. Quality control was performed as recommended in CLSI documents using C. parapsilosis ATCC22019 and C. krusei ATCC 6258.

Amphotericin B, ketoconazole and voriconazole were purchased from Sigma. All other CYP51 inhibitors were synthesized in-house (see supplementary methods and [34 (link),35 (link),36 (link)]). Stationary phase promastigotes (8x10⁵/mL) were treated for 72 h with two-fold dilution of inhibitors in 384 well plate format. Resazurin (0.025 mg/mL, Santa Cruz) was added for 5 h, cells were fixed, and fluorescence measured at 490 nm excitation and 595 nm emission wavelengths. Data was normalized to the Amphotericin B positive control and DMSO vehicle negative control for each plate, and EC50 values calculated using Collaborative Drug Discovery Vault software. T. cruzi cell-based activity was determined by high content screening in triplicate, as previously described [36 (link)].