Voriconazole

Aspergillus fumigatus strain Af293 was used in this study. The strain was cultured in PDB at 37 °C on a rotary shaker at 120 rpm throughout the study. In some experiments, glucose minimal medium (GMM) (1%(w/v) glucose, 0.052% KCl, 0.052% MgSO₄·7H₂O, 0.152% KH₂PO₄, 0.0022% ZnSO₄·7H₂O, 0.0011% H₃BO₃, 0.0005% MnCl₂·4H₂O, 0.0005% FeSO₄·7H₂O, 0.00016% CoCl₂·5H₂O, 0.00016% CuSO₄·5H₂O, 0.00011% (NH₄)6Mo₇O₂₄·4H₂O, 0.005% Na₄EDTA, pH 6.5) containing variable nitrogen sources, such as sodium nitrate, ammonium sulphate, and proline (70 mM) were used, which were designated GMM(NO₃), GMM(NH₄), and GMM(Pro), respectively. The antifungal chemicals used were commercially obtained, as follows: thymol (NACALAI TESQUE, Inc., Kyoto, Japan), farnesol (Sigma-Aldrich Co., St. Louis, MO, USA), citral (NACALAI TESQUE, Inc.), nerol (Tokyo Chemical Industry Co., Ltd, Tokyo, Japan), salicylic acid (NACALAI TESQUE, Inc.), PCA (Wako Pure Chemicals Industries, Osaka, Japan), pyocyanin (Cayman Chemical Company, Ann Arbor, MI, USA), itraconazole (Wako Pure Chemicals Industries), voriconazole (Tokyo Chemical Industry Co., Ltd), and amphotericin B (Sigma-Aldrich Co.). Micafungin was a generous gift from Dr. Keietsu Abe, Tohoku University.

SDS, sorbitol, DTT, NaCl, and LiCl were purchased from Wako Pure Chemical Industries (Osaka, Japan). Fluconazole, voriconazole, hygromycin B, caffeine, and monensin sodium salt were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Nourseothricin was purchased from Jena Bioscience (Dortmund, Germany). Congo red was purchased from Sigma-Aldrich (St. Louis, MO, USA). G418 was purchased from Enzo Life Science, Inc. (Farmingdale, NY, USA). CaCl₂ was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Tunicamycin was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Brefeldin A (BFA) was purchased from Funakoshi Co., Ltd. (Tokyo, Japan). Human serum [(from male AB clotted whole blood), USA origin, sterile-filtered] (Product ID: H6914) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Voriconazole, provided by Tokyo Chemical Industry (Japan), was used to prepare a stock solution of 1.00 g/L in methanol (LC-MS grade from Merck, Germany). This stock solution was further diluted in acetonitrile or deionized pure water to a series of appropriate concentrations (working solution A and B, respectively). Working solution A was used for HPLC-MS/MS analysis, and B was for in vitro sample preparation. Fluconazole (internal standard, IS), provided by Sigma-Aldrich (USA), was used to prepare the IS working solution with acetonitrile (4.00 mg/L). Standard protein solutions were prepared in phosphate-buffered saline (PBS, containing 30 mmol/L sodium dihydrogen phosphate, 70 mmol/L disodium hydrogen phosphate, and 150 mmol/L sodium chloride), with Behring^® (CSL Behring GmbH, Marburg, Germany), α1-Acid Glycoprotein from human plasma (Sigma, Belgium) and Human Immunoglobulin (PH4) for Intravenous Injection (Chengdu Rongsheng pharmaceutical Co. Ltd). All solutions were freshly prepared at the moment of experiments.

Clorgyline, clomiphene, toremifene, tamoxifen, raloxifene, and ospemifene were obtained from Cayman Chemicals (Ann Arbor, MI). Fluconazole was purchased from Fisher Scientific (Pittsburgh, PA). Hexestrol, 2-deoxyGlucose, and cis-stilbene were purchased from Alfa Aesar (Tewksbury, MA). Nile red, itraconazole, voriconazole, and resveratrol were purchased from TCI America (Portland, OR). Glucose and diethylstilbestrol were purchased from Sigma-Aldrich (St. Louis, MO). Gentamicin sulfate was obtained from Chem-Impex International Inc. (Wood Dale, IL).

Sources and descriptions of the fungal strains used in this study are provided in Supplementary Table S1. Yeast-peptone dextrose (YPD) agar and broth were purchased from Becton, and Dickinson Company (Franklin Lakes, NJ). 3-(N-Morpholino) propanesulfonic acid (MOPS) was purchased from Sigma-Aldrich (St. Louis, MO). RPMI 1640 powder, supplemented with glutamine and lacking NaHCO₃, was obtained from Thermo Fisher Scientific (Waltham, MA). Johns Hopkins Clinical Compounds Library (JHCCL) was obtained from the school of medicine at Johns Hopkins University and was used to identify the azole chemosensitizing activity of aprepitant. The library was screened at 16 µM against C. auris AR0390 in the presence or absence of a subinhibitory concentration of Fluconazole (32 µg/ml). Fluconazole was purchased from Fisher Scientific (Pittsburgh, PA). Itraconazole, voriconazole, and aprepitant were purchased from TCI America (Portland, OR). Gentamicin sulfate was obtained from Chem-Impex International Inc. (Wood Dale, IL).