Atovaquone

Aspergillus and Fusarium hyphae were incubated 2 hours with atovaquone, and the zinc chelator TPEN (Sigma-Aldrich) was added at indicated concentrations. To quantify total zinc in the hyphae, 2 μM Zinbo-5 (Santa Cruz Biotechnology, Dallas, TX, USA) in PBS or 25 μM Zinquin (Santa Cruz Biotechnology) in RPMI 1640 were added to wells for 15 minutes. Plates were washed, and fluorescence was read at 358/463 for Zinbo-5 or at 368/490 for Zinquin. For metal shock experiments in which high levels of metals are added to the cultures, conidia were grown to germination in SDB, washed, and RPMI +/− atovaquone +/− ZnSO₄, MnSO₄, or CuSO₄ (Sigma-Aldrich) was added. Hyphae were incubated 18 hours for Aspergillus or 48 hours for Fusarium. Fluorescence was quantified using Calcofluor white, as described above.

B16OVA.HRE and MC38.HRE spheroids were treated with OXPHOS inhibitors 24 h after seeding. The following OXPHOS inhibitors were used for treatment: IACS-010759 (1, 0.33, 0.11 and 0.037 µM, Selleckchem), metformin (9, 3, 1 and 0.33 mM, Sigma-Aldrich), atovaquone (30, 15, 7.5 and 3.75 µM, Sigma-Aldrich), mito₁₀-atovaquone (30, 15, 7.5 and 3.75 µM) [16 (link)], Mito-PEG₅-atovaquone (30, 15, 7.5 and 3.75 µM) [21 ], tamoxifen (5, 2.5, 1.25 and 0.625 µM, Sigma-Aldrich (H7904)) and MitoTam (5, 2.5, 1.25 and 0.625 µM) [27 (link), 28 (link)]. Control spheroids were treated with 0.2% DMSO.

Artesunate, mefloquine hydrochloride, amodiaquine dihydrochloride dihydrate, chloroquine diphosphate, primaquine phosphate, quinine hemisulfate, atovaquone, methylene blue, pyronaridine tetraphosphate, doxycycline hyclate, clindamycin and praziquantel were purchased from Sigma-Aldrich. Ferroquine was obtained from Sanofi-Synthelabo, proguanil and cycloguanil from Jacobus Pharmaceutical Company, tigecycline from Wyeth, and mirincamycin hydrochloride enantiomers from Maldevco [45 (link)]. All compounds were dissolved in sterile DMSO except for quinine for which methanol was used and pure M199 medium (without additives) was used to dissolve proguanil, cycloguanil, clindamycin, and pyronaridine. The stock concentration was 50 mM for Artesunate, amodiaquine, chloroquine, atovaquone, quinine, and primaquine, and 100 mM for praziquantel, proguanil, cycloguanil, methylene blue, pyronaridine, clindamycin, doxycycline, and mirincamycin enantiomers, respectively. Mefloquine was dissolved to 24 mM and Ferroquine to 12.5 mM. All stocks were freshly prepared for the study and stored at -20°C. Maximum concentration of the solvent (DMSO, methanol) in the in vitro assays did not exceed 0.8% and did not interfere with parasite viability.