Identification of Novel Chemical Scaffolds Inhibiting Trypanothione Synthetase from Pathogenic Trypanosomatids.

International audienceBACKGROUND: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

Inhibitors of tritryp trypanothione synthetase (TryS) identified in this work and their biological activity.

<p>Inhibitors of tritryp trypanothione synthetase (TryS) identified in this work and their biological activity.</p

Kinetic parameters for recombinant tritryp trypanothione synthetase (TryS).

<p>Kinetic parameters for recombinant tritryp trypanothione synthetase (TryS).</p

Biological activity of compounds against infective <i>Trypanosoma brucei brucei</i> with downregulated expression of trypanothione synthetase (TryS).

<p><b>A)</b> Western blot analysis of cell extracts from 2x10⁷ <i>T</i>. <i>b</i>. <i>brucei</i> from the wildtype (WT), 48 h tetracycline-induced (+) and non-induced (-) TryS-RNAi cell line. Two hundred ng of recombinant <i>Tb</i>TryS was loaded as control. Bands from the molecular weight marker are indicated on left. The picture at the bottom shows the abundance of TryS for each condition as estimated by densitometry and expressed relative to the level of the WT cell line. <b>B)</b> Ponceau staining of the Western blot membrane that served as normalization control of protein load for each condition. <b>C)</b> Cytotoxicity (%) ± S.D. (n = 2) for tetracycline-induced (+) and non-induced (-) TryS-RNAi <i>T</i>. <i>b</i>. <i>brucei</i> treated with 5 μM nifurtimox or 100 nM EAP1-47.</p

Screening work flow.

<p>The different steps, the most relevant assay conditions and the go/no-go criteria of the screening campaign are indicated in boxes. The figures on the right refer to the number of compounds screened and that subsequently advanced during the campaign. From 144 compounds, 22 compounds lowered assay signal ≥ 45% for at least one TryS. From these 22, 7 BDA were false positive and the remaining 15 compounds were confirmed as enzyme inhibitors. Two of them are <b>AI</b> with potency in the submicromolar range against <i>Li</i>TryS. AI (P), 4,5-dihydroazepino[4,5-<i>b</i>]indol-2(1<i>H</i>,3<i>H</i>,6<i>H</i>)-one derivatives (P, paullone); APPDA, 6-arylpyrido[2,3-<i>d</i>]pyrimidine-2,7-diamine derivatives; BZ, benzofuroxan derivatives; BDA, <i>N</i>,<i>N'</i>-bis(3,4-substituted-benzyl) diamine derivatives.</p

Structure of compounds affecting tritryp trypanothione synthetase activity.

<p><b>AI (P)</b>, 4,5-dihydroazepino[4,5-<i>b</i>]indol-2(1<i>H</i>,3<i>H</i>,6<i>H</i>)-one derivatives, paullones derivatives, (FS-554 and MOL2008), five APPDA, 6-arylpyrido[2,3-<i>d</i>]pyrimidine-2,7-diamine derivatives (ZEA10, ZEA35, ZEA40, ZEA41 and ZVR159), eight BDA, <i>N</i>,<i>N'</i>-bis(3,4-substituted-benzyl) diamine derivatives (EAP1-47, EAP1-63, APC1-67, APC1-87, APC1-89, APC1-99, APC1-101 and APC1-111), seven BBHPP, 1-(benzo[<i>d</i>]thiazol-2-yl)-4-benzoyl-3-hydroxy-5-phenyl-1<i>H</i>-pyrrol-2(5<i>H</i>)-one derivatives (AD81, AD84, ADMRC158, ADKPN160, ADKPN161, ADKPN164 and ADKPN165), three BZ, benzofuroxan derivatives (J18, J20 and J31) and one PD, 1<i>H</i>-purine-2,6(3<i>H</i>,7<i>H</i>)-dione derivatives [(<i>Z</i>)-8-(2-(2,4-dihydroxybenzylidene)hydrazinyl)-7-(2-hydroxy-3-phenoxy propyl)-1,3-dimethyl-1<i>H</i>-purine-2,6(3<i>H</i>,7<i>H</i>)-dione, TC227]. iPr, tBu, OBn, Mo and Ph, correspond to an isopropyl, tert-butyl, O-benzyl, 4 -morpholinyl and phenyl substitution, respectively.</p

Trypanothione dependent redox metabolism.

<p>The chemical structure of trypanothione (<i>N</i>¹,<i>N</i>⁸-bis(glutathionyl)spermidine; T(SH)₂) is depicted at the center. Synthesis: trypanothione synthetase catalyzes the ligation of two molecules of gluthatione to one of spermidine using the energy provided by two ATP molecules. Regeneration: trypanothione reductase maintains trypanothione in the reduced state at expenses of NADPH, which can be supplied by the oxidative phase of the pentose phosphate pathway <i>via</i> glucose 6-phosphate dehydrogenase. Utilization: reduced trypanothione is involved in multiple functions such as the detoxification of xenobiotics, cell proliferation, defense against oxidants and protein thiol-redox homeostasis. The multipurpose oxidoreductase tryparedoxin plays an important role catalyzing electron transfer from T(SH)₂ to different molecular targets (e.g. peroxidases, ribonucleotide reductase and protein disulfides). G6P: glucose-6-phosphate, 6PGL: 6-phosphogluconolactone, T(SH)₂: reduced trypanothione, TS_2[ox]: oxidized trypanothione, NDPs: nucleosides diphosphate, dNDP: deoxinucleosides diphosphate, E^-: electrophilic species, TS-E: trypanothione-electrophile adduct, ROOH: hydroperoxide, ONOOH: peroxynitrite, NO₂^-: nitrite.</p

Inhibition plot of <i>Leishmania infantum</i> trypanothione synthetase (TryS) by paullones.

<p>The data are presented as mean TryS activity (%) ± 2 S.D. (n = 4) as function of Log₁₀ concentration (nM) of compound adjusted to a four-parameter Boltzmann sigmoidal equation. Representative dose-response plot obtained for MOL2008 (IC₅₀ = 0.14 ± 0.03 μM; slope plot 0.38 ± 0.03) and for FS-554 (IC₅₀ = 0.32 ± 0.73 μM; slope plot 0.37 ± 0.03).</p