title: Chemical validation of trypanothione synthetase: a potential drug target for human trypanosomiasis.
abstract: In the search for new therapeutics for the treatment of human African trypanosomiasis, many potential drug targets in Trypanosoma brucei have been validated by genetic means, but very few have been chemically validated. Trypanothione synthetase (TryS; EC 6.3.1.9; spermidine/glutathionylspermidine:glutathione ligase (ADP-forming)) is one such target. To identify novel inhibitors of T. brucei TryS, we developed an in vitro enzyme assay, which was amenable to high throughput screening. The subsequent screen of a diverse compound library resulted in the identification of three novel series of TryS inhibitors. Further chemical exploration resulted in leads with nanomolar potency, which displayed mixed, uncompetitive, and allosteric-type inhibition with respect to spermidine, ATP, and glutathione, respectively. Representatives of all three series inhibited growth of bloodstream T. brucei in vitro. Exposure to one of our lead compounds (DDD86243; 2 x EC(50) for 72 h) decreased intracellular trypanothione levels to <10% of wild type. In addition, there was a corresponding 5-fold increase in the precursor metabolite, glutathione, providing strong evidence that DDD86243 was acting on target to inhibit TryS. This was confirmed with wild-type, TryS single knock-out, and TryS-overexpressing cell lines showing expected changes in potency to DDD86243. Taken together, these data provide initial chemical validation of TryS as a drug target in T. brucei.
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: Leah S
authors.initials: LS
authors.lastname: Torrie
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: Susan
authors.initials: S
authors.lastname: Wyllie
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: Daniel
authors.initials: D
authors.lastname: Spinks
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: Sandra L
authors.initials: SL
authors.lastname: Oza
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: Stephen
authors.initials: S
authors.lastname: Thompson
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: Justin R
authors.initials: JR
authors.lastname: Harrison
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: Ian H
authors.initials: IH
authors.lastname: Gilbert
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: Paul G
authors.initials: PG
authors.lastname: Wyatt
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: Alan H
authors.initials: AH
authors.lastname: Fairlamb
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom. Electronic address: j.a.frearson@dundee.ac.uk.
authors.forename: Julie A
authors.initials: JA
authors.lastname: Frearson
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects / Q000235:genetics; D019732:Amide Synthases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D000981:Antiprotozoal Agents / Q000737:chemistry / Q000493:pharmacokinetics* / Q000627:therapeutic use; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use; D006801:Humans; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D014353:Trypanosomiasis, African / Q000188:drug therapy* / Q000201:enzymology*
chemical_list: D000981:Antiprotozoal Agents; D004791:Enzyme Inhibitors; D015800:Protozoan Proteins; D019732:Amide Synthases; C064407:trypanothione synthetase