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Pharmacology

Lead Optimization of Dehydroemetine for Repositioned Use in Malaria

Priyanka Panwar, Kepa K. Burusco, Muna Abubaker, Holly Matthews, Andrey Gutnov, Elena Fernández-Álvaro, Richard A. Bryce, James Wilkinson, Niroshini Nirmalan
Priyanka Panwar
aEnvironment and Life Sciences, University of Salford, Greater Manchester, United Kingdom
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Kepa K. Burusco
bDivision of Pharmacy and Optometry, School of Health Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, United Kingdom
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Muna Abubaker
aEnvironment and Life Sciences, University of Salford, Greater Manchester, United Kingdom
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Holly Matthews
cKeele University, Newcastle-under-Lyme, Staffordshire, United Kingdom
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Andrey Gutnov
dChiroblock GMBH, Wolfen, Germany
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Elena Fernández-Álvaro
eGlaxoSmithKline, Diseases of the Developing World Medicines Development Campus, Tres Cantos, Spain
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Richard A. Bryce
bDivision of Pharmacy and Optometry, School of Health Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, United Kingdom
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James Wilkinson
aEnvironment and Life Sciences, University of Salford, Greater Manchester, United Kingdom
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Niroshini Nirmalan
aEnvironment and Life Sciences, University of Salford, Greater Manchester, United Kingdom
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DOI: 10.1128/AAC.01444-19
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  • FIG 1
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    FIG 1

    Structure of emetine hydrochloride.

  • FIG 2
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    FIG 2

    (a) Overlay of the docked pose of emetine (green) with its enantiomer present in the cryo-EM structure (maroon); observed electron density envelope is also shown (wireframe surface with contour level 0.1542 electrons/Å̂3 [3.50 RMSD]). (b) Interactions of docked emetine (green) with Pf40S residues and comparison with previously modeled interactions of its enantiomer (blue) (16).

  • FIG 3
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    FIG 3

    (a) Overlay of docked poses of (−)-R,S-dehydroemetine (red) and (−)-S,S-dehydroisoemetine (cyan) with emetine (blue). (b) Interactions of (−)-R,S-dehydroemetine (red) and (−)-S,S-dehydroisoemetine (cyan) with the Pf40S binding site. Distances (dotted lines) in Å.

  • FIG 4
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    FIG 4

    (a) Docking through MOE showing the binding site residues for (−)-R,S-dehydroemetine molecule. (b) Docking through MOE showing the binding site residues for (−)-S,S-dehydroisoemetine molecule.

  • FIG 5
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    FIG 5

    (a) The effective dose of (−)-R,S-dehydroemetine (dose range tested in 2-fold serial dilutions from 12.5 nM to 200 nM) on P. falciparum K1 infection after an incubation period of 72 h using SYBR green-based plate reader assay. (b) The effective dose of (−)-S,S-dehydroisoemetine (dose range tested in 2-fold serial dilutions from 0.625 μM to 10 μM) on P. falciparum K1 infection after an incubation period of 72 h using SYBR green-based plate reader assay. The experiments were performed thrice with each concentration of (−)-R,S-dehydroemetine and (−)-S,S-dehydroisoemetine (tested in triplicates). Data were analyzed using GraphPad prism. Error bars show standard deviations.

  • FIG 6
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    FIG 6

    Time course analysis through IC50 speed assay using unsynchronized cultures of Plasmodium falciparum. The graphs show that IC50s could not be reached within 24 h. (−)-R,S-dehydroemetine and (−)-S,S-dehydroisoemetine reached the IC50 by 48 h. Error bars represent the standard errors of the results from experiments performed twice with each concentration of (−)-R,S-dehydroemetine and (−)-S,S-dehydroisoemetine (tested in triplicates). (a) (−)-R,S-dehydroemetine at concentrations of 25, 50, 100, 200, and 400 nM at 24 h, 48 h, and 72 h. (b) (−)-S,S-dehydroisoemetine at concentrations of 0.63, 1.25, 2.5, 5, and 10 μM at 24 h, 48 h, and 72 h.

  • FIG 7
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    FIG 7

    Stage-specific profiling using synchronized cultures of Plasmodium falciparum. The decrease in growth of trophozoites/schizonts was observed to be more marked than for rings. The drug effects are expressed as percentages of growth of rings relative to growth of trophozoites/schizonts 24 h after the postexposure wash. Error bars represent the standard errors of the results from experiments performed twice with each concentration of (−)-R,S-dehydroemetine and (−)-S,S-dehydroisoemetine (tested in triplicates). (a) Cultures were exposed to a serial dilution of (−)-R,S-dehydroemetine (from 132.81 nM to 8,500 nM) for 24 h. (b) Cultures were exposed to a serial dilution of (−)-S,S-dehydroisoemetine (from 3.13 μM to 200 μM) for 24 h.

  • FIG 8
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    FIG 8

    Forty-eight-hour MTT assays. Cells were seeded at 5,000 cells per well. Cell viability was determined using a standard MTT assay. Data were analyzed using GraphPad prism. Error bars represent the standard deviations of the results from experiments performed thrice with each concentration of emetine, cisplatin, (−)-R,S-dehydroemetine, and (−)-S,S-dehydroisoemetine (tested in triplicate). Forty-eight-hour MTT assays for emetine, tested in 2-fold serial dilutions from 31.25 nM to 2,000 nM (a), cisplatin, tested in 2-fold serial dilutions from 0.78 μM to 25 μM (b), (−)-R,S-dehydroemetine, tested in 2-fold serial dilutions from 11.72 nM to 750 nM (c), and (−)-S,S-dehydroisoemetine, tested in 2-fold serial dilutions from 0.312 μM to 10 μM (d).

  • FIG 9
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    FIG 9

    Staining of parasites with rhodamine 123 and Draq5. Merge with bright-field image shows localization of the dye within the parasite. P. falciparum K1 strain trophozoites stained with 10 μM Draq5 (a) and 200 nM rhodamine 123 (b), Draq5 and rhodamine merge (c), and Draq5, rhodamine 123, and brightfield merge (d). Visualized under fluorescence microscope at ×100 magnification.

  • FIG 10
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    FIG 10

    Disruption of mitochondrial membrane potential. Changes in mitochondrial membrane potential observed on treatment with IC50s of atovaquone, emetine, and (−)-R,S-dehydroemetine. (a) Graphical representation of changes in fluorescence intensity on application of drugs at IC50s. (b) Table showing changes in fluorescence intensity (FITC-A mean) on application of drugs at IC50s and 10× IC50s. FITC-A mean value of infected blood without drugs was considered to be the base value (0), and FITC-A mean values of the compounds are represented as percentages of deviation from the base value.

  • FIG 11
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    FIG 11

    CalcuSyn-based drug interactivity analysis for atovaquone and proguanil. CalcuSyn-based median effect plot (a), isobologram (b), and dose-effect curve analyzed through GraphPad Prism (c) for drug interactivity between atovaquone and proguanil. The combination of atovaquone and proguanil (1:7,000) was found to be strongly synergistic at the IC50 (combination index [CI] = 0.21) and IC75 (CI = 0.34) and synergistic at the IC90 (CI = 0.57). fa, fraction affected; fu, fraction unaffected.

  • FIG 12
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    FIG 12

    CalcuSyn-based drug interactivity analysis for (−)-R,S-dehydroemetine–atovaquone combination. Dose-effect curve analyzed through GraphPad Prism for drug interactivity between (−)-R,S-dehydroemetine and atovaquone. The combination of atovaquone and (−)-R,S-dehydroemetine (1:25) was found to display slight synergy at the IC50 (CI = 0.88), IC75 (CI = 0.88), and IC90 (CI = 0.89).

  • FIG 13
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    FIG 13

    CalcuSyn drug interactivity analysis for (−)-R,S-dehydroemetine–proguanil combination. Dose-effect curve analyzed through GraphPad Prism for drug interactivity between proguanil and (−)-R,S-dehydroemetine. The combination of proguanil and (−)-R,S-dehydroemetine (140:1) was found to be synergistic at the IC50 (CI = 0.67), nearly additive at the IC75 (CI = 1.04), and antagonistic at the IC90 (CI = 1.62).

  • FIG 14
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    FIG 14

    Synthesis of 2, 3-dehydroemetine. Conditions were as follows. (a) (i) Iodomethane, ethanol, room temperature (RT), 24 h, (ii) compound 2, KOAc, reflux, 3 h, 72%; (b) malononitrile, ammonium acetate/acetic acid, toluene, reflux, 2.5 h, 69%; (c) (i) 20% HCl, reflux, 5 h, (ii) methanolic HCl, RT, 18 h, 45%; (d) (+)-dibenzoyl tartrate, methanol, two recrystallizations, 25%; (e) (i) 3 M HCl, reflux, 90 min, (ii) 3,4-dimethoxyphenethylamine, xylenes, reflux, 18 h, (iii) POCl3, benzene, reflux, 1 h, 28%; (f) NaBH4, methanol, RT, 1 h, 81% of a 1:1 diastereomeric mixture. Compound 7, (S)-2-((R)-6,7-dimethoxy-1,2,3,4-tetrahydro-isoquinolin-1-ylmethyl)-3-ethyl-9,10-dimethoxy-1,6,7,11b-tetrahydro-4H-pyrido[2,1-a]isoquinoline * dihydrobromide. Compound 8, (S)-2-((S)-6,7-dimethoxy-1,2,3,4-tetrahydro-isoquinolin-1-ylmethyl)-3-ethyl-9,10-dimethoxy-1,6,7,11b-tetrahydro-4H-pyrido[2,1-a]isoquinoline * dihydrobromide.

Tables

  • Figures
  • TABLE 1

    Analysis of results of 3H-hypoxanthine incorporation assay to determine cross-resistance

    TABLE 1
    • ↵a In vitro IC50s of emetine dihydrochloride, (−)-R,S-dehydroemetine, and (−)-S,S-dehydroisoemetine in sensitive P. falciparum strain 3D7A and resistant P. falciparum strains Dd2 and W2, as well as ratios of in vitro cross-resistance of emetine dihydrochloride and (−)-R,S-dehydroemetine in both resistant strains (Dd2 and W2), using strain 3D7A as reference. ND, not determined.

  • TABLE 2

    In vitro IC50s of (−)-R,S-dehydroemetine and (−)-S,S-dehydroisoemetine against male and female gametocytes in P. falciparum strain NF54

    TABLE 2
    • ↵a In vitro IC50s of (−)-R,S-dehydroemetine and (−)-S,S-dehydroisoemetine in P. falciparum dual gamete formation assay.

  • TABLE 3

    hERG channel inhibition assay results for (−)-R,S-dehydroemetine and (−)-S,S-dehydroisoemetinea

    TABLE 3
    • ↵a This experiment was outsourced to Cyprotex, UK. Each value is the mean of triplicate values.

  • TABLE 4

    Dose series used for the combination of existing antimalarials with (−)-R,S-dehydroemetine

    TABLE 4
    • ↵a The ratios for combination with (−)-R,S-dehydroemetine were 1:25 for atovaquone and 140:1 for proguanil.

  • TABLE 5

    CalcuSyn-based drug interaction analysisa

    TABLE 5
    • ↵a The combination ratios and CalcuSyn determined the combination index (CI) values for the assays performed. The r value represents the linear correlation coefficient for the median effect plot and indicates conformity to the mass action law.

  • TABLE 6

    Classification of synergism or antagonism using CI values generated by the Chou-Talalay method (CalcuSyn manual, Biosoft [38])

    TABLE 6
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Lead Optimization of Dehydroemetine for Repositioned Use in Malaria
Priyanka Panwar, Kepa K. Burusco, Muna Abubaker, Holly Matthews, Andrey Gutnov, Elena Fernández-Álvaro, Richard A. Bryce, James Wilkinson, Niroshini Nirmalan
Antimicrobial Agents and Chemotherapy Mar 2020, 64 (4) e01444-19; DOI: 10.1128/AAC.01444-19

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Lead Optimization of Dehydroemetine for Repositioned Use in Malaria
Priyanka Panwar, Kepa K. Burusco, Muna Abubaker, Holly Matthews, Andrey Gutnov, Elena Fernández-Álvaro, Richard A. Bryce, James Wilkinson, Niroshini Nirmalan
Antimicrobial Agents and Chemotherapy Mar 2020, 64 (4) e01444-19; DOI: 10.1128/AAC.01444-19
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KEYWORDS

malaria
antimalarial drug interactions
SYBR green flow cytometry
emetine
dehydroemetine
drug discovery
repositioning

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