Identification of Novel Chemical Scaffolds Inhibiting Trypanothione Synthetase from Pathogenic Trypanosomatids

PLoS Negl Trop Dis. 2016 Apr 12;10(4):e0004617. doi: 10.1371/journal.pntd.0004617. eCollection 2016 Apr.

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.

Methodology/principal finding: 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/significance: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amide Synthases / antagonists & inhibitors*
  • Antiprotozoal Agents / isolation & purification*
  • Antiprotozoal Agents / metabolism
  • Drug Evaluation, Preclinical
  • Enzyme Inhibitors / isolation & purification*
  • Enzyme Inhibitors / metabolism
  • Leishmania infantum / drug effects*
  • Leishmania infantum / enzymology
  • Trypanosoma brucei brucei / drug effects*
  • Trypanosoma brucei brucei / enzymology
  • Trypanosoma cruzi / drug effects*
  • Trypanosoma cruzi / enzymology

Substances

  • Antiprotozoal Agents
  • Enzyme Inhibitors
  • Amide Synthases
  • trypanothione synthetase

Grant support

The financial support of Agencia Nacional de Investigación e Innovación (ANII, Uruguay) is gratefully acknowledged by DB (POS_NAC_2013_1_114477), LFi (POS_NAC_2011_1_3284) and MAC (project DCI-ALA/2007/19.040). MAC acknowledges the support of MERCOSUR Structural Convergence Fund (FOCEM) and Institut Pasteur ACIP call 2015 (project ACIP 17-2015). AM acknowledges the support of Comisión Sectorial de Investigación Científica, Universidad de la Republica Uruguay (project nr. 3404). TC, AD, JŠ, LPM, CK, LFl and MAC acknowledge the support of the European Cooperation in the field of Technical and Scientific Research (COST), Action CM0801. FM and CK acknowledge the support from the German Bundesministerium für Bildung und Forschung (KMU-innovativ 5, Förderkennzeichen 0315814). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.