Fig 1.
Sterol metabolic network in N. fowleri.
The pathway is reconstituted based on the metabolites observed in the untreated N. fowleri (shown in bold) and treated with the CYP51 (shown in green), SMT (in blue) and ERG2 (in dark red) inhibitors. Metabolites not observed in the wild-type N. fowleri are depicted in thin lines. The cholesterol arm of the pathway is highlighted by the yellow-green background, the ergosterol arm is highlighted by the blue background. Enclosed in frame are potential SMT substrates. Enzymes interrogated in these studies are labeled in colors corresponding to the accumulating metabolites. Numbering of atoms in sterol molecule is adapted from Nes and Venkatramesh, 1994.[41] Sterol labelling is as in Table 1. ERG enzyme nomenclature is according to the convention for S. cerevisiae.
Table 1.
Sterol flux in N. fowleri.
Fig 2.
Gas chromatography of the total sterol fractions derived from N. fowleri trophozoites treated with (A) 0.5% DMSO, (B) 0.2 μM posaconazole, (C) 5.4 μM epiminolanosterol, and (D) 5.6 μM AY9944. Peaks are labeled with numbers corresponding to metabolites listed in Table 1. Insert in (C) shows deconvolution of the major peak resulting from an overlap of three different sterols: 6, m/z = 349; 7, m/z = 363 and 13, m/z = 400. The color scheme is the same as in Fig 1: CYP51 inhibitor (shown in green), SMT inhibitor (in blue) and ERG2 inhibitor (in dark red).
Table 2.
Conversion of different sterols by N. gruberi SMT.
Table 3.
Isotopic enrichment of N. gruberi sterols incubated with 50 mM U-13C-glucose.
Fig 3.
Dose-response growth inhibition of N. fowleri trophosoites.
Dose-response curves are shown for (A) SMT inhibitors: abafungin, 25-azacycloartenol and 24,25-epiminolanosterol, (B) ERG2 inhibitors: tamoxifen and AY9944, (C) CYP51 inhibitor isavuconazole.
Table 4.
Targeting N. fowleri with inhibitors of known MOA.
Fig 4.
Growth inhibition of N. fowleri as a function of drug combinations.
Heat maps show growth inhibition of N. fowleri by drug combinations: tamoxifen and isavuconazole (A), epiminlanosterol and tamoxifen (B), and epiminolanosterol and isavuconazole (C). Corresponding isobolograms (D, E, F) show the mean values of dose reduction index (DRI) ploted against calculated inhibitor doses required to achieve 95% growth inhibition. Standard deviations for each parameter mean value are shown in S2 Table.
Fig 5.
Synergistic effect of drugs at low concentrations.
The phase contrast microscope images show N. fowleri trophozoites treated for 48 hours with (A) 0.08 μM of isavuconazole and 0.7 μM of epiminolanosterol, (B) 1.9 μM equimolar concentrations of isavuconazole and tamoxifen, (C) 2 μM equimolar concentrations of epiminolanosterol and tamoxifen, and (D) 0.5% DMSO, which served as a negative control. The inhibitor-treated N. fowleri cells visible in the microscope field are rounded, much smaller in size and not viable, whereas DMSO-treated cells are irregularly shaped with visible cytoplasm. Magnification, ×20.
Fig 6.
Sequence alignments between the catalytic domain of sterol Δ8−Δ7 -isomerase (ERG2) and a ligand binding site of non-opioid σ1 receptor (SGMR1) demonstrate high sequence similarity.
Accession numbers in UniProt or AmoebaDB are provided for each sequence. Numbering is according to the Dictyostelium discoideum ERG2 sequence.