Micafungin

Antifungal susceptibility testing was performed according to the standardized M27-A method approved by the National Committee for Clinical Laboratory Standards (NCCLS) and previously reported methods (Nakai et al., 2003 (link)). Caspofungin (CAS), micafungin (MCFG), amphotericin B (AMB), fluconazole (FLC), itraconazole (ITC), and vorconazole (VOC) were purchased from Med Chem Express (New Jersey, NJ, United States). Stock solutions were made with sterile distilled water (MCFG and AMB) or 100% dimethyl sulfoxide (CAS, FLC, ITC and VOC). The stock solutions were diluted in RPMI 1640 medium prepared according to the Clinical and Laboratory Standards Institute (CLSI) standards and then further serially diluted twofold. The final concentration ranges of the antifungals were 0.0625 to 32 μg/ml (CAS and MCFG), 0.0156 to 8 μg/ml (AMB), 0.125 to 64 μg/ml (FLC), and 0.0013 to 1 μg/ml (ITC and VOC). Wild-type and ΔcnaA mutant strains were incubated in the presence or absence of drug for 48 h at 25°C and 37°C, and all experiments were performed in triplicate. Candida parapsilosis ATCC22019 served as a control.

Venetoclax, rifampin, ritonavir, cobicistat, ketoconazole, fluconazole, itraconazole voriconazole, posaconazole, isavuconazole, micafungin, caspofungin, and anidulafungin were purchased from MedChemExpress (Monmouth Junction, NJ, USA). The major itraconazole metabolites, hydroxy-itraconazole and keto-itraconazole were kindly provided by Dr. Nina Isoherranen (University of Washington, Seattle, WA, USA). [³H]Estradiol-17β-D-glucuronide (EβG; specific activity, 50.1 Ci/mmol), a positive control substrate for OATP1B, was purchased from American Radiolabeled Chemicals (Saint Louis, MO, USA). [³H]Venetoclax (specific activity, 29.3 Ci/mmol) was obtained from ViTrax (Placentia, CA, USA). PEG400 was purchased from Sigma-Aldrich (Burlington, MA, USA). Dimethyl sulfoxide (DMSO), LC-MS-grade formic acid, methanol, and acetonitrile were purchased from Fisher Scientific (Fair Lawn, NJ, USA), and 8-acetoxy-trisulfopyrene was obtained from Carbosynth Limited (Compton, Berkshire, UK). Human recombinant CYP3A4 Supersomes (456207) was purchased from Corning (Glendale, AZ, USA). NADPH RapidStart Regenerating System was purchased from SEKISUI XenoTech (Kansas City, KS, USA).