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Photo-affinity labelling and biochemical analyses identify the target of trypanocidal simplified natural product analogues
Current drugs to treat African sleeping sickness are inadequate and new therapies are urgently required. As part of a medicinal chemistry programme based upon the simplification of acetogenin-type ether scaffolds, we previously reported the promising trypanocidal activity of compound 1, a bis-tetrah...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608556/ https://www.ncbi.nlm.nih.gov/pubmed/28873407 http://dx.doi.org/10.1371/journal.pntd.0005886 |
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author | Tulloch, Lindsay B. Menzies, Stefanie K. Fraser, Andrew L. Gould, Eoin R. King, Elizabeth F. Zacharova, Marija K. Florence, Gordon J. Smith, Terry K. |
author_facet | Tulloch, Lindsay B. Menzies, Stefanie K. Fraser, Andrew L. Gould, Eoin R. King, Elizabeth F. Zacharova, Marija K. Florence, Gordon J. Smith, Terry K. |
author_sort | Tulloch, Lindsay B. |
collection | PubMed |
description | Current drugs to treat African sleeping sickness are inadequate and new therapies are urgently required. As part of a medicinal chemistry programme based upon the simplification of acetogenin-type ether scaffolds, we previously reported the promising trypanocidal activity of compound 1, a bis-tetrahydropyran 1,4-triazole (B-THP-T) inhibitor. This study aims to identify the protein target(s) of this class of compound in Trypanosoma brucei to understand its mode of action and aid further structural optimisation. We used compound 3, a diazirine- and alkyne-containing bi-functional photo-affinity probe analogue of our lead B-THP-T, compound 1, to identify potential targets of our lead compound in the procyclic form T. brucei. Bi-functional compound 3 was UV cross-linked to its target(s) in vivo and biotin affinity or Cy5.5 reporter tags were subsequently appended by Cu(II)-catalysed azide-alkyne cycloaddition. The biotinylated protein adducts were isolated with streptavidin affinity beads and subsequent LC-MSMS identified the F(o)F(1)-ATP synthase (mitochondrial complex V) as a potential target. This target identification was confirmed using various different approaches. We show that (i) compound 1 decreases cellular ATP levels (ii) by inhibiting oxidative phosphorylation (iii) at the F(o)F(1)-ATP synthase. Furthermore, the use of GFP-PTP-tagged subunits of the F(o)F(1)-ATP synthase, shows that our compounds bind specifically to both the α- and β-subunits of the ATP synthase. The F(o)F(1)-ATP synthase is a target of our simplified acetogenin-type analogues. This mitochondrial complex is essential in both procyclic and bloodstream forms of T. brucei and its identification as our target will enable further inhibitor optimisation towards future drug discovery. Furthermore, the photo-affinity labeling technique described here can be readily applied to other drugs of unknown targets to identify their modes of action and facilitate more broadly therapeutic drug design in any pathogen or disease model. |
format | Online Article Text |
id | pubmed-5608556 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-56085562017-10-09 Photo-affinity labelling and biochemical analyses identify the target of trypanocidal simplified natural product analogues Tulloch, Lindsay B. Menzies, Stefanie K. Fraser, Andrew L. Gould, Eoin R. King, Elizabeth F. Zacharova, Marija K. Florence, Gordon J. Smith, Terry K. PLoS Negl Trop Dis Research Article Current drugs to treat African sleeping sickness are inadequate and new therapies are urgently required. As part of a medicinal chemistry programme based upon the simplification of acetogenin-type ether scaffolds, we previously reported the promising trypanocidal activity of compound 1, a bis-tetrahydropyran 1,4-triazole (B-THP-T) inhibitor. This study aims to identify the protein target(s) of this class of compound in Trypanosoma brucei to understand its mode of action and aid further structural optimisation. We used compound 3, a diazirine- and alkyne-containing bi-functional photo-affinity probe analogue of our lead B-THP-T, compound 1, to identify potential targets of our lead compound in the procyclic form T. brucei. Bi-functional compound 3 was UV cross-linked to its target(s) in vivo and biotin affinity or Cy5.5 reporter tags were subsequently appended by Cu(II)-catalysed azide-alkyne cycloaddition. The biotinylated protein adducts were isolated with streptavidin affinity beads and subsequent LC-MSMS identified the F(o)F(1)-ATP synthase (mitochondrial complex V) as a potential target. This target identification was confirmed using various different approaches. We show that (i) compound 1 decreases cellular ATP levels (ii) by inhibiting oxidative phosphorylation (iii) at the F(o)F(1)-ATP synthase. Furthermore, the use of GFP-PTP-tagged subunits of the F(o)F(1)-ATP synthase, shows that our compounds bind specifically to both the α- and β-subunits of the ATP synthase. The F(o)F(1)-ATP synthase is a target of our simplified acetogenin-type analogues. This mitochondrial complex is essential in both procyclic and bloodstream forms of T. brucei and its identification as our target will enable further inhibitor optimisation towards future drug discovery. Furthermore, the photo-affinity labeling technique described here can be readily applied to other drugs of unknown targets to identify their modes of action and facilitate more broadly therapeutic drug design in any pathogen or disease model. Public Library of Science 2017-09-05 /pmc/articles/PMC5608556/ /pubmed/28873407 http://dx.doi.org/10.1371/journal.pntd.0005886 Text en © 2017 Tulloch et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Tulloch, Lindsay B. Menzies, Stefanie K. Fraser, Andrew L. Gould, Eoin R. King, Elizabeth F. Zacharova, Marija K. Florence, Gordon J. Smith, Terry K. Photo-affinity labelling and biochemical analyses identify the target of trypanocidal simplified natural product analogues |
title | Photo-affinity labelling and biochemical analyses identify the target of trypanocidal simplified natural product analogues |
title_full | Photo-affinity labelling and biochemical analyses identify the target of trypanocidal simplified natural product analogues |
title_fullStr | Photo-affinity labelling and biochemical analyses identify the target of trypanocidal simplified natural product analogues |
title_full_unstemmed | Photo-affinity labelling and biochemical analyses identify the target of trypanocidal simplified natural product analogues |
title_short | Photo-affinity labelling and biochemical analyses identify the target of trypanocidal simplified natural product analogues |
title_sort | photo-affinity labelling and biochemical analyses identify the target of trypanocidal simplified natural product analogues |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608556/ https://www.ncbi.nlm.nih.gov/pubmed/28873407 http://dx.doi.org/10.1371/journal.pntd.0005886 |
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