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Mechanisms of Action: Physiological Effects

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

Richard J. Wall, Sonia Moniz, Michael G. Thomas, Suzanne Norval, Eun-Jung Ko, Maria Marco, Timothy J. Miles, Ian H. Gilbert, David Horn, Alan H. Fairlamb, Susan Wyllie
Richard J. Wall
aWellcome Trust Centre for Anti-Infectives Research, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, United Kingdom
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Sonia Moniz
aWellcome Trust Centre for Anti-Infectives Research, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, United Kingdom
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Michael G. Thomas
aWellcome Trust Centre for Anti-Infectives Research, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, United Kingdom
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Suzanne Norval
aWellcome Trust Centre for Anti-Infectives Research, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, United Kingdom
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Eun-Jung Ko
aWellcome Trust Centre for Anti-Infectives Research, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, United Kingdom
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Maria Marco
bDiseases of the Developing World, GlaxoSmithKline, Madrid, Spain
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Timothy J. Miles
bDiseases of the Developing World, GlaxoSmithKline, Madrid, Spain
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Ian H. Gilbert
aWellcome Trust Centre for Anti-Infectives Research, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, United Kingdom
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David Horn
aWellcome Trust Centre for Anti-Infectives Research, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, United Kingdom
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Alan H. Fairlamb
aWellcome Trust Centre for Anti-Infectives Research, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, United Kingdom
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Susan Wyllie
aWellcome Trust Centre for Anti-Infectives Research, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, United Kingdom
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DOI: 10.1128/AAC.00235-18
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  • FIG 1
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    FIG 1

    Chemical structures and activities of three 8-HNT compounds. Chemical structures of 8-HNT compound 1 (DDD01007886) (TCMDC-143180; GSK1363355A), compound 2 (DDD01011700) (GSK3454797A), and compound 3 (DDD01012232) (GSK3454397A) are shown. Potencies of compounds against L. donovani promastigotes (pro), L. donovani intramacrophage amastigotes, T. brucei bloodstream forms (bsf), THP-1 cells, and HepG2 cells are shown; data are from ≥3 independent replicates.

  • FIG 2
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    FIG 2

    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.

  • FIG 3
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    FIG 3

    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.

  • FIG 4
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    FIG 4

    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.

  • FIG 5
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    FIG 5

    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+).

  • FIG 6
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    FIG 6

    Proposed structure of the 2:1 complex formed between 8-HNT compounds and Zn2+.

Tables

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  • TABLE 1

    EC50s for 8-HNT compounds against T. brucei in the presence and absence of divalent transition metalsa

    Divalent cationCompound 1Compound 2Compound 3
    Mean EC50 (μM) ± SDShift (fold)Mean EC50 (μM) ± SDShift (fold)Mean EC50 (μM) ± SDShift (fold)
    WTPlus cationWTPlus cationWTPlus cation
    CaCl20.3 ± 0.80.4 ± 0.11.11.2 ± 0.041.3 ± 0.061.00.8 ± 0.10.7 ± 0.040.9
    CuCl20.3 ± 0.080.4 ± 0.0051.11.4 ± 0.071.3 ± 0.060.90.8 ± 0.050.8 ± 0.061.1
    FeCl20.2 ± 0.011.8 ± 0.28.00.7 ± 0.061.6 ± 0.052.40.9 ± 0.033.5 ± 0.33.7
    MgCl20.4 ± 0.010.4 ± 0.031.10.8 ± 0.050.9 ± 0.021.10.8 ± 0.050.7 ± 0.050.9
    MnCl20.4 ± 0.010.4 ± 0.021.01.4 ± 0.091.2 ± 0.050.80.8 ± 0.051.01 ± 0.041.3
    ZnCl20.4 ± 0.012.8 ± 0.18.00.8 ± 0.0515.6 ± 0.719.70.5 ± 0.036.3 ± 0.212.0
    • ↵a EC50s represent the means ± standard deviations for at least 3 experiments.

Additional Files

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      Supplemental Figures S1 to S4, Table S4, Table S5, and legends to Tables S1 to S3

      PDF, 301K

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      Tables S1 to S3

      XLSX, 52K

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Antitrypanosomal 8-Hydroxy-Naphthyridines Are Chelators of Divalent Transition Metals
Richard J. Wall, Sonia Moniz, Michael G. Thomas, Suzanne Norval, Eun-Jung Ko, Maria Marco, Timothy J. Miles, Ian H. Gilbert, David Horn, Alan H. Fairlamb, Susan Wyllie
Antimicrobial Agents and Chemotherapy Jul 2018, 62 (8) e00235-18; DOI: 10.1128/AAC.00235-18

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Antitrypanosomal 8-Hydroxy-Naphthyridines Are Chelators of Divalent Transition Metals
Richard J. Wall, Sonia Moniz, Michael G. Thomas, Suzanne Norval, Eun-Jung Ko, Maria Marco, Timothy J. Miles, Ian H. Gilbert, David Horn, Alan H. Fairlamb, Susan Wyllie
Antimicrobial Agents and Chemotherapy Jul 2018, 62 (8) e00235-18; DOI: 10.1128/AAC.00235-18
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KEYWORDS

chelator
drug discovery
kinetoplastids
mechanisms of action
transition metals

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