title: Identification of Novel Chemical Scaffolds Inhibiting Trypanothione Synthetase from Pathogenic Trypanosomatids.
abstract: BACKGROUND
  The search for novel chemical entities targeting essential and parasite-specific pathways is considered a priority for neglected diseases such as trypanosomiasis and leishmaniasis. The thiol-dependent redox metabolism of trypanosomatids relies on bis-glutathionylspermidine [trypanothione, T(SH)2], a low molecular mass cosubstrate absent in the host. In pathogenic trypanosomatids, a single enzyme, trypanothione synthetase (TryS), catalyzes trypanothione biosynthesis, which is indispensable for parasite survival. Thus, TryS qualifies as an attractive drug target candidate.
  
  
  RESULTS
  A library composed of 144 compounds from 7 different families and several singletons was screened against TryS from three major pathogen species (Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum). The screening conditions were adjusted to the TryS´ kinetic parameters and intracellular concentration of substrates corresponding to each trypanosomatid species, and/or to avoid assay interference. The screening assay yielded suitable Z' and signal to noise values (≥0.85 and ~3.5, respectively), and high intra-assay reproducibility. Several novel chemical scaffolds were identified as low μM and selective tri-tryp TryS inhibitors. Compounds displaying multi-TryS inhibition (N,N'-bis(3,4-substituted-benzyl) diamine derivatives) and an N5-substituted paullone (MOL2008) halted the proliferation of infective Trypanosoma brucei (EC50 in the nM range) and Leishmania infantum promastigotes (EC50 = 12 μM), respectively. A bis-benzyl diamine derivative and MOL2008 depleted intracellular trypanothione in treated parasites, which confirmed the on-target activity of these compounds.
  
  
  CONCLUSIONS
  Novel molecular scaffolds with on-target mode of action were identified as hit candidates for TryS inhibition. Due to the remarkable species-specificity exhibited by tri-tryp TryS towards the compounds, future optimization and screening campaigns should aim at designing and detecting, respectively, more potent and broad-range TryS inhibitors.
authors.affiliation: Laboratory Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Montevideo, Uruguay.
authors.forename: Diego
authors.initials: D
authors.lastname: Benítez
authors.affiliation: Laboratory Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Montevideo, Uruguay.|Departamento de Bioquímica, Universidad de la República, Montevideo, Uruguay.
authors.forename: Andrea
authors.initials: A
authors.lastname: Medeiros
authors.affiliation: Laboratory Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Montevideo, Uruguay.
authors.forename: Lucía
authors.initials: L
authors.lastname: Fiestas
authors.affiliation: Instituto de Química Rosario-CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.
authors.forename: Esteban A
authors.initials: EA
authors.lastname: Panozzo-Zenere
authors.affiliation: Institut für Medizinische und Pharmazeutische Chemie, Technische Universität Braunschweig, Braunschweig, Germany.
authors.forename: Franziska
authors.initials: F
authors.lastname: Maiwald
authors.affiliation: Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, Athens, Greece.
authors.forename: Kyriakos C
authors.initials: KC
authors.lastname: Prousis
authors.affiliation: Laboratory of Organic Chemistry, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
authors.forename: Marina
authors.initials: M
authors.lastname: Roussaki
authors.affiliation: Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, Athens, Greece.
authors.forename: Theodora
authors.initials: T
authors.lastname: Calogeropoulou
authors.affiliation: Laboratory of Organic Chemistry, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Detsi
authors.affiliation: German Centre for Infection Research, Braunschweig, Germany.
authors.forename: Timo
authors.initials: T
authors.lastname: Jaeger
authors.affiliation: Department of the Biochemistry of Xenobiotics Institute of Biochemistry, Vilnius University, Vilnius, Lithuania.
authors.forename: Jonas
authors.initials: J
authors.lastname: Šarlauskas
authors.affiliation: Department for Medicinal Chemistry, Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Lucíja
authors.initials: L
authors.lastname: Peterlin Mašič
authors.affiliation: Institut für Medizinische und Pharmazeutische Chemie, Technische Universität Braunschweig, Braunschweig, Germany.
authors.forename: Conrad
authors.initials: C
authors.lastname: Kunick
authors.affiliation: Instituto de Química Rosario-CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.
authors.forename: Guillermo R
authors.initials: GR
authors.lastname: Labadie
authors.affiliation: Departamento de Bioquímica, Universidad de la República, Montevideo, Uruguay.|Department of Molecular Medicine, Università degli Studi di Padova, Padova, Italy.
authors.forename: Leopold
authors.initials: L
authors.lastname: Flohé
authors.affiliation: Laboratory Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Montevideo, Uruguay.
authors.forename: Marcelo A
authors.initials: MA
authors.lastname: Comini
mesh_terms: D019732:Amide Synthases / Q000037:antagonists & inhibitors*; D000981:Antiprotozoal Agents / Q000302:isolation & purification* / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D018314:Leishmania infantum / Q000187:drug effects* / Q000201:enzymology; D014346:Trypanosoma brucei brucei / Q000187:drug effects* / Q000201:enzymology; D014349:Trypanosoma cruzi / Q000187:drug effects* / Q000201:enzymology
chemical_list: D000981:Antiprotozoal Agents; D004791:Enzyme Inhibitors; D019732:Amide Synthases; C064407:trypanothione synthetase