Identification of Novel Chemical Scaffolds Inhibiting Trypanothione Synthetase from Pathogenic Trypanosomatids

Abstract

BackgroundThe 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 FindingA 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/SignificanceNovel 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.

4-Aroyl-3-hydroxy-5-phenyl-1H-pyrrol-2(5H)-ones as N-formyl peptide receptor 1 (FPR1) antagonists

“…For example, 5-aryl-4-benzoyl-3-hydroxy-1-(2-arylethyl)-2 H -pyrrol-2-ones have been identified as competitive and specific vasopressin-2 receptor (V2R) antagonists [54]. Certain 4-aroyl-3-hydroxy-5-phenyl-1 H -pyrrol-2(5 H )-one derivatives showed specificity for targeting trypanothione synthetase in Leishmania infantum [55]. Previously, a series of 4-aroyl-3-hydroxy-5-phenyl-1 H -pyrrol-2(5 H )-ones were reported as inhibitors of the annexin A2–S100A10 protein interaction [34].…”

5-Substituted 3-chlorokenpaullone derivatives are potent inhibitors of Trypanosoma brucei bloodstream forms

“…33 Moreover, previous studies involving paullones as anti-TryS scaffolds revealed that substitution of 4-azapaullones in position 9 or 11 with alkyl or aryl groups is detrimental for target inhibition 34 , whereas paullones harboring a methyl ethylendiamine side chain at the N(5)-position (e.g., 2) inhibited both recombinant L. infantum TryS (LiTryS) and the in vitro growth of L. infantum promastigotes (insect stage). 19,20 The most potent derivative (IC 50 0.15 lM and EC 50 12 lM), containing halogen substituents in position 3 (chloro) and 9 (bromo), produced a significant depletion of trypanothione in L. infantum, which confirmed its on-target effect. 19,20 We here report an approach to develop new paullones with potent growth inhibitory activity against the infective form of T. brucei.…”

“…19,20 The most potent derivative (IC 50 0.15 lM and EC 50 12 lM), containing halogen substituents in position 3 (chloro) and 9 (bromo), produced a significant depletion of trypanothione in L. infantum, which confirmed its on-target effect. 19,20 We here report an approach to develop new paullones with potent growth inhibitory activity against the infective form of T. brucei. The new compounds were also screened for their anti-TryS activity against the enzyme from three major pathogenic species of trypanosomatids: T. brucei, T. cruzi and L. infantum.…”

“…In a more general context, these data add value to a recent biochemical study with TryS from trypanosomatids performed by our groups, which proposed the existence of species-specific differences between TryS to explain the selective interaction of the enzymes with different ligands (substrates, inhibitors). 20 While the tests on isolated TryS enzymes produced disappointing results, several compounds of series 3 exhibited potent antiparasitic activity on cultivated T. brucei bloodstream forms with single digit or submicromolar EC 50 values. This biological activity was clearly related to the structure of the congeners, since the three compounds 3j-l with a pyridylmethyl moiety as part of the side chain in 5-position were less potent compared to the derivatives with a benzyl structure (3a-3i), except for compound 3c as discussed below.…”

“…Recently, N(5)-substituted paullones were identified as inhibitors of TryS. 19,20 Paullones (Chart 1) are a group of 7,12-dihydroindolo [3,2-d][1]benzazepin-6(5H)-ones which besides other biological properties [21][22][23] display inhibitory activities against mammalian protein kinases of the CMGC superfamily, e.g., cyclin-dependent kinases (CDKs) [24][25][26] and glycogen synthase kinase-3 (GSK-3). 27,28 Kenpaullone (1a), 29 the prototype of the series, is used as a standard GSK-3 inhibitor which displays selectivity versus a wide array of protein kinases (e.g., ERK1, DYRK1A, VEGF-R2, PLK1, INS-R).…”