Analogs of the Natural Product Chamuvarinin Target the Trypanosomatid FoF1-ATP Synthase Mitochondrial Complex V

Stefanie K. Menzies (Lead / Corresponding author), Lindsay B. Tulloch, Andrew L. Fraser, Eoin R. Gould, Elizabeth F. King, Marija K. Zacharova, Gordon J. Florence, Terry K. Smith

Research output: Contribution to journalMeeting abstract

4 Citations (Scopus)

Abstract

Current treatments for trypanosomatid diseases are outdated, increasingly ineffective and associated with severe adverse effects. We recently reported trypanocidal activity of a series of novel synthetic bistetrahydropyran 1,4-triazole (B-THP-T) analogs based on the framework of the natural product chamuvarinin, an acetogenin first synthesized by
our groups. Acetogenins are potent inhibitors of the human mitochondrial Complex I, however our compounds show potent inhibitory activity against bloodstream form Trypanosoma brucei, in which Complex I is absent, thus chamuvarinin must target another protein in the trypanosomatids. The mode of action of our B-THP-T compounds is unknown so this work aimed to identify their target in Leishmania major and T. cruzi. We synthesized a series of B-THP-T analogs for use in photo-affinity labeling (PAL), to covalently tag our target for protein target identification in vivo. We subsequently identified the FoF1-ATP synthase (mitochondrial complex V) as a potential target of our compounds using pull-down experiments. Next we undertook a series of biological analyses to validate this pulldown. By labeling B-THP-T-tagged proteins with the Cy5.5 fluorophore we confirmed that the target is mitochondrial by fluorescence co-localization. Using a luciferase-based ATP quantitation assay we show that B-THP-T compounds inhibit cellular ATP production and that they ablate oxidative phosphorylation. Taken together, these data indicate that our B-THP-T compounds are trypanocidal through their mitochondrial targeting of the FoF1-ATP synthase. We are using genetic manipulation of T. cruzi and L. major FoF1 ATP synthase subunits to validate these findings. With the
target of our bis-tetrahydropyran 1,4-triazoles identified as mitochondrial complex V a structure-based approach can be used to optimize inhibitor potency and specificity.
Original languageEnglish
Article number520
Pages (from-to)164
Number of pages1
JournalAmerican Journal of Tropical Medicine and Hygiene
Volume95
Issue number5 Supplement
Early online date28 Mar 2017
DOIs
Publication statusPublished - 2017
EventASTMH 65th Annual Meeting - Atlanta, United States
Duration: 13 Nov 201617 Nov 2016

Fingerprint

Mitochondrial Proton-Translocating ATPases
Triazoles
Biological Products
Acetogenins
Adenosine Triphosphate
Leishmania major
Trypanosoma brucei brucei
Proteins
Oxidative Phosphorylation
chamuvarinin
oligomycin sensitivity-conferring protein
Luciferases
Fluorescence

Cite this

Menzies, S. K., Tulloch, L. B., Fraser, A. L., Gould, E. R., King, E. F., Zacharova, M. K., ... Smith, T. K. (2017). Analogs of the Natural Product Chamuvarinin Target the Trypanosomatid FoF1-ATP Synthase Mitochondrial Complex V. American Journal of Tropical Medicine and Hygiene, 95(5 Supplement), 164. [520]. https://doi.org/10.4269/ajtmh.abstract2016
Menzies, Stefanie K. ; Tulloch, Lindsay B. ; Fraser, Andrew L. ; Gould, Eoin R. ; King, Elizabeth F. ; Zacharova, Marija K. ; Florence, Gordon J. ; Smith, Terry K. / Analogs of the Natural Product Chamuvarinin Target the Trypanosomatid FoF1-ATP Synthase Mitochondrial Complex V. In: American Journal of Tropical Medicine and Hygiene. 2017 ; Vol. 95, No. 5 Supplement. pp. 164.
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abstract = "Current treatments for trypanosomatid diseases are outdated, increasingly ineffective and associated with severe adverse effects. We recently reported trypanocidal activity of a series of novel synthetic bistetrahydropyran 1,4-triazole (B-THP-T) analogs based on the framework of the natural product chamuvarinin, an acetogenin first synthesized byour groups. Acetogenins are potent inhibitors of the human mitochondrial Complex I, however our compounds show potent inhibitory activity against bloodstream form Trypanosoma brucei, in which Complex I is absent, thus chamuvarinin must target another protein in the trypanosomatids. The mode of action of our B-THP-T compounds is unknown so this work aimed to identify their target in Leishmania major and T. cruzi. We synthesized a series of B-THP-T analogs for use in photo-affinity labeling (PAL), to covalently tag our target for protein target identification in vivo. We subsequently identified the FoF1-ATP synthase (mitochondrial complex V) as a potential target of our compounds using pull-down experiments. Next we undertook a series of biological analyses to validate this pulldown. By labeling B-THP-T-tagged proteins with the Cy5.5 fluorophore we confirmed that the target is mitochondrial by fluorescence co-localization. Using a luciferase-based ATP quantitation assay we show that B-THP-T compounds inhibit cellular ATP production and that they ablate oxidative phosphorylation. Taken together, these data indicate that our B-THP-T compounds are trypanocidal through their mitochondrial targeting of the FoF1-ATP synthase. We are using genetic manipulation of T. cruzi and L. major FoF1 ATP synthase subunits to validate these findings. With thetarget of our bis-tetrahydropyran 1,4-triazoles identified as mitochondrial complex V a structure-based approach can be used to optimize inhibitor potency and specificity.",
author = "Menzies, {Stefanie K.} and Tulloch, {Lindsay B.} and Fraser, {Andrew L.} and Gould, {Eoin R.} and King, {Elizabeth F.} and Zacharova, {Marija K.} and Florence, {Gordon J.} and Smith, {Terry K.}",
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Menzies, SK, Tulloch, LB, Fraser, AL, Gould, ER, King, EF, Zacharova, MK, Florence, GJ & Smith, TK 2017, 'Analogs of the Natural Product Chamuvarinin Target the Trypanosomatid FoF1-ATP Synthase Mitochondrial Complex V', American Journal of Tropical Medicine and Hygiene, vol. 95, no. 5 Supplement, 520, pp. 164. https://doi.org/10.4269/ajtmh.abstract2016

Analogs of the Natural Product Chamuvarinin Target the Trypanosomatid FoF1-ATP Synthase Mitochondrial Complex V. / Menzies, Stefanie K. (Lead / Corresponding author); Tulloch, Lindsay B.; Fraser, Andrew L.; Gould, Eoin R.; King, Elizabeth F.; Zacharova, Marija K.; Florence, Gordon J.; Smith, Terry K.

In: American Journal of Tropical Medicine and Hygiene, Vol. 95, No. 5 Supplement, 520, 2017, p. 164.

Research output: Contribution to journalMeeting abstract

TY - JOUR

T1 - Analogs of the Natural Product Chamuvarinin Target the Trypanosomatid FoF1-ATP Synthase Mitochondrial Complex V

AU - Menzies, Stefanie K.

AU - Tulloch, Lindsay B.

AU - Fraser, Andrew L.

AU - Gould, Eoin R.

AU - King, Elizabeth F.

AU - Zacharova, Marija K.

AU - Florence, Gordon J.

AU - Smith, Terry K.

PY - 2017

Y1 - 2017

N2 - Current treatments for trypanosomatid diseases are outdated, increasingly ineffective and associated with severe adverse effects. We recently reported trypanocidal activity of a series of novel synthetic bistetrahydropyran 1,4-triazole (B-THP-T) analogs based on the framework of the natural product chamuvarinin, an acetogenin first synthesized byour groups. Acetogenins are potent inhibitors of the human mitochondrial Complex I, however our compounds show potent inhibitory activity against bloodstream form Trypanosoma brucei, in which Complex I is absent, thus chamuvarinin must target another protein in the trypanosomatids. The mode of action of our B-THP-T compounds is unknown so this work aimed to identify their target in Leishmania major and T. cruzi. We synthesized a series of B-THP-T analogs for use in photo-affinity labeling (PAL), to covalently tag our target for protein target identification in vivo. We subsequently identified the FoF1-ATP synthase (mitochondrial complex V) as a potential target of our compounds using pull-down experiments. Next we undertook a series of biological analyses to validate this pulldown. By labeling B-THP-T-tagged proteins with the Cy5.5 fluorophore we confirmed that the target is mitochondrial by fluorescence co-localization. Using a luciferase-based ATP quantitation assay we show that B-THP-T compounds inhibit cellular ATP production and that they ablate oxidative phosphorylation. Taken together, these data indicate that our B-THP-T compounds are trypanocidal through their mitochondrial targeting of the FoF1-ATP synthase. We are using genetic manipulation of T. cruzi and L. major FoF1 ATP synthase subunits to validate these findings. With thetarget of our bis-tetrahydropyran 1,4-triazoles identified as mitochondrial complex V a structure-based approach can be used to optimize inhibitor potency and specificity.

AB - Current treatments for trypanosomatid diseases are outdated, increasingly ineffective and associated with severe adverse effects. We recently reported trypanocidal activity of a series of novel synthetic bistetrahydropyran 1,4-triazole (B-THP-T) analogs based on the framework of the natural product chamuvarinin, an acetogenin first synthesized byour groups. Acetogenins are potent inhibitors of the human mitochondrial Complex I, however our compounds show potent inhibitory activity against bloodstream form Trypanosoma brucei, in which Complex I is absent, thus chamuvarinin must target another protein in the trypanosomatids. The mode of action of our B-THP-T compounds is unknown so this work aimed to identify their target in Leishmania major and T. cruzi. We synthesized a series of B-THP-T analogs for use in photo-affinity labeling (PAL), to covalently tag our target for protein target identification in vivo. We subsequently identified the FoF1-ATP synthase (mitochondrial complex V) as a potential target of our compounds using pull-down experiments. Next we undertook a series of biological analyses to validate this pulldown. By labeling B-THP-T-tagged proteins with the Cy5.5 fluorophore we confirmed that the target is mitochondrial by fluorescence co-localization. Using a luciferase-based ATP quantitation assay we show that B-THP-T compounds inhibit cellular ATP production and that they ablate oxidative phosphorylation. Taken together, these data indicate that our B-THP-T compounds are trypanocidal through their mitochondrial targeting of the FoF1-ATP synthase. We are using genetic manipulation of T. cruzi and L. major FoF1 ATP synthase subunits to validate these findings. With thetarget of our bis-tetrahydropyran 1,4-triazoles identified as mitochondrial complex V a structure-based approach can be used to optimize inhibitor potency and specificity.

U2 - 10.4269/ajtmh.abstract2016

DO - 10.4269/ajtmh.abstract2016

M3 - Meeting abstract

VL - 95

SP - 164

JO - American Journal of Tropical Medicine and Hygiene

JF - American Journal of Tropical Medicine and Hygiene

SN - 0002-9637

IS - 5 Supplement

M1 - 520

ER -