Antitrypanosomal 8-Hydroxy-Naphthyridines Are Chelators of Divalent Transition Metals

Genome-wide RNAi screening of 8-HNT compounds to identify putative cation transporters in T. brucei. (A) Genome-wide map indicating RIT-seq hits from screening of compound 1. Multiple RIT-seq fragments represent primary hits, indicated in green. Other loci with mapped reads are indicated in gray. RPKM, reads per kilobase of transcript per million mapped reads. (B) Hits on individual genes (indicated in green). Other protein-coding sequences are indicated as black bars. Red peaks, forward reads with RNAi construct barcodes; blue peaks, reverse reads with RNAi construct barcodes; gray peaks, all other reads. (C) Dose-response curves of compound 1 against uninduced (●) and induced (○) Tb927.11.15050 and Tb927.11.1910 knockdown bloodstream trypanosomes. Results are the means ± standard deviations of data from three independent experiments.

Putative cation transporters share homology with mammalian ZnT transporters and are localized to the Golgi apparatus. (A) Phylogenetic analysis of the proteins encoded by Tb927.11.15050 and Tb927.11.1910 and their relationship to members of the human ZnT zinc transporter family. (B) Myc tags (12×) were introduced to the C termini of each putative cation transporter (Tb927.11.15050 and Tb927.11.1910). Tagged versions of these putative transporters (green) were found to colocalize with GRASP (red), a known marker of the Golgi apparatus of bloodstream trypanosomes. Cells were also stained with 4′,6-diamidino-2-phenylindole (DAPI). Bars, 5 μm.

ZnCl₂ or FeCl₂ protects parasites, and intracellular Zn²⁺ levels are decreased in the presence of 8-HNT compounds. (A and B) Dose-response curves of compound 1 in the presence (○) and absence (●) of ZnCl₂ (A) or FeCl₂ (B) in T. brucei bloodstream-form parasites. In the presence of ZnCl₂ (200 μM), EC₅₀s shifted from 0.35 ± 0.01 μM to 2.8 ± 0.12 μM. In the presence of FeCl₂ (10 μM), EC₅₀s shifted from 0.22 ± 0.01 μM to 1.8 ± 0.18 μM. (C and D) Dose-response curves of compound 1 in the presence (○) and absence (●) of ZnCl₂ (C) or FeCl₂ (D) in L. donovani promastigotes. In the presence of ZnCl₂ (100 μM), EC₅₀s shifted from 0.76 ± 0.008 μM to 8.3 ± 0.32 μM. In the presence of FeCl₂ (100 μM), EC₅₀s shifted from 0.78 ± 0.03 μM to 1.6 ± 0.05 μM. All EC₅₀s represent the means ± standard deviations from three independent experiments. (E and F) Intracellular measurement of zinc as a percentage in T. brucei bloodstream parasites normalized to the dimethyl sulfoxide (DMSO) control (100%) (E) and intracellular measurement of zinc as a percentage in L. donovani promastigotes normalized to the DMSO control (100%) (F). Levels of intracellular zinc were measured by using the fluorescent zinc reporter FluoZin-3 (final concentration, 5 μM), as described in Materials and Methods, in the presence of the zinc chelator TPEN (8 μM), exogenous ZnCl₂ (100 μM), or compounds 1 to 3 (5 μM). All values represent the means ± standard errors of the means for at least 3 experiments.

Characterization of Zn²⁺ binding to 8-HNT compounds in a cell-free system. (A) Levels of zinc were measured by using the fluorescent zinc reporter FluoZin-3 salt (final concentration, 5 μM), as described in Materials and Methods, in the presence of the zinc chelator TPEN (8 μM), exogenous ZnCl₂ (100 μM), DFMO (1, 10, and 100 μM), or compounds 1 to 3 (1, 10, and 100 μM). Data were normalized to Zn²⁺ levels in the presence of a DMSO control (100%). All values represent the means ± standard errors of the means for 3 independent experiments. (B) UV-Vis spectra of 50 μM compound 1 (red), compound 2 (green), and compound 3 (blue). (C to E) Job's plots of Δ absorbance at 475 nm for compound 1, 390 nm for compound 2, and 375 nm for compound 3 relative to fixed molar ratios of compound to Zn²⁺. The intercepts at molar fractions of 0.34 for compound 1, 0.31 for compound 2, and 0.35 for compound 3, illustrated by dashed red lines, are indicative of a 2:1 stoichiometry between 8-HNT compounds and Zn²⁺, respectively. (F to H) The Δ absorbance values at fixed molar ratios of compound to cation of 0.33 were plotted for each 8-HNT compound and a range of divalent cations (Cu²⁺, Fe²⁺, Mg²⁺, Mn²⁺, and Zn²⁺).