A series of 9-mer and 13-mer amide-bridged cyclic peptides derived from the linear prototype Ac-PKIIQSVGIS-Nle-K-Nle-NH (Toro et al. ChemBioChem2013) has been designed and synthesized by introduction of the lactam between amino acid side chains that are separated by one helical turn (i, i+4). All of these compounds were tested in vitro as both dimerization and enzyme inhibitors of Leishmania infantum trypanothione reductase (Li-TryR). Three of the 13-mer cyclic peptide derivatives (3, 4 and 6) inhibited the oxidoreductase activity of Li-TryR in the low micromolar range and they also disrupted enzyme dimerization. Cyclic analogues 3 and 4 were more resistant to proteases than was the linear prototype. Furthermore, the most potent TryR inhibitors in the linear and cyclic series displayed potent in vitro activity against Leishmania infantum upon conjugation with cationic cell-penetrating peptides. To date, these conjugated peptides can be considered the first example of TryR dimerization inhibitors that are active in cell culture.

Inhibition of Leishmania infantum trypanothione disulfide reductase ( Li TryR) by disruption of its homodimeric interface has proved to be an alternative and unexploited strategy in the search for novel antileishmanial agents. Proof of concept was first obtained by peptides and peptidomimetics. Building on previously reported dimerization disruptors containing an imidazole-phenyl-thiazole scaffold, we now report a new 1,2,3-triazole-based chemotype that yields noncompetitive, slow-binding inhibitors of Li TryR. Several compounds bearing (poly)aromatic substituents dramatically improve the ability to disrupt Li TryR dimerization relative to reference imidazoles. Molecular modeling studies identified an almost unexplored hydrophobic region at the interfacial domain as the putative binding site for these compounds. A subsequent structure-based design led to a symmetrical triazole analogue that displayed even more potent inhibitory activity over Li TryR and enhanced leishmanicidal activity. Remarkably, several of these novel triazole-bearing compounds were able to kill both extracellular and intracellular parasites in cell cultures.

Disruption of protein-protein interactions of essential oligomeric enzymes by small molecules represents a significant challenge. We recently reported some linear and cyclic peptides derived from an α-helical region present in the homodimeric interface of Leishmania infantum trypanothione reductase (Li-TryR) that showed potent effects on both dimerization and redox activity of this essential enzyme. Here we describe our first steps towards the design of non-peptidic small-molecule Li-TryR dimerization disruptors using a proteomimetic approach. The pyrrolopyrimidine and the 5-6-5 imidazole-phenyl-thiazole α-helix-mimetic scaffolds were suitably decorated with substituents that could mimic three key residues (K, Q and I) of the linear peptide prototype (PKIIQSVGIS-Nle-K-Nle). Extensive optimization of previously described synthetic methodologies was required. A library of 15 compounds bearing different hydrophobic alkyl and aromatic substituents was synthesized. The imidazole-phenyl-thiazole-based analogues outperformed the pyrrolopyrimidine-based derivatives in both inhibiting the enzyme and killing extracellular and intracellular parasites in cell culture. The most active imidazole-phenyl-thiazole compounds 3e and 3f inhibit Li-TryR and prevent growth of the parasites at low micromolar concentrations similar to those required by the peptide prototype. The intrinsic fluorescence of these compounds inside the parasites visually demonstrates their good permeability in comparison with previous peptide-based Li-TryR dimerization disruptors.

Despite the absence of sequences showing significant similarity to any of the members of the Bcl-2 family of proteins in protozoa, experiments carried out in yeast or trypanosomatids have demonstrated that ectopic expression of some of these members alters their response to different death stimuli. Because the BH3 domain is the smallest common signature in all the proteins of this family of apoptosis regulators and also because they are essential for molecular interactions between antagonistic members, we looked for sequences with significant similarity to the BH3 motif in the Leishmania infantum genome. Among the top scoring ones, we found the MYLALQNLGDEV amino-acid stretch at the C terminus of a previously described aquaporin, now renamed as Li-BH3AQP. This motif is highly conserved in homologous proteins from other species of the Leishmania genus. The association of Li-BH3AQP with human Bcl-XL was demonstrated by both co-immunoprecipitation and yeast two-hybrid experiments. Ectopic expression of Li-BH3AQP reduced viability of HeLa cells and this deleterious effect was abrogated by the simultaneous overexpression of Bcl-XL. Although we were not able to demonstrate a reduction in parasite viability when the protein was overexpressed in Leishmania promastigotes, a prodeath effect could be observed when the parasites overexpressing Li-BH3AQP were treated with staurosporine or antimycin A. Surprisingly, these parasites were more resistant, compared with wild-type parasites, to hypotonic stress or nutrient deprivation. The prodeath activity was abolished upon replacement of two highly conserved amino acids in this BH3 domain. Taken together, these results point to Li-BH3AQP as the first non-enzymatic protein ever described in trypanosomatids that is involved in cell death.

The objective of the current study was to enhance the proteolytic stability of peptide-based inhibitors that target critical protein-protein interactions at the dimerization interface of Leishmania infantum trypanothione reductase (Li-TryR) using a backbone modification strategy. To achieve this goal we carried out the synthesis, proteolytic stability studies and biological evaluation of a small library of α/β-peptide foldamers of different length (from 9-mers to 13-mers) and different α→β substitution patterns related to prototype linear α-peptides. We show that several 13-residue α/β-peptide foldamers retain inhibitory potency against the enzyme (in both activity and dimerization assays) while they are far less susceptible to proteolytic degradation than an analogous α-peptide. The strong dependence of the binding affinities for Li-TryR on the length of the α,β-peptides is supported by theoretical calculations on conformational ensembles of the resulting complexes. The conjugation of the most proteolytically stable α/β-peptide with oligoarginines results in a molecule with potent activity against L. infantum promastigotes and amastigotes.

A novel series of thirty-one N-substituted urea, thiourea, and selenourea derivatives containing diphenyldiselenide entities were synthesized, fully characterized by spectroscopic and analytical methods, and screened for their in vitro leishmanicidal activities. The cytotoxic activity of these derivatives was tested against Leishmania infantum axenic amastigotes, and selectivity was assessed in human THP-1 cells. Thirteen of the synthesized compounds showed a significant antileishmanial activity, with 50% effective concentration (EC 50 ) values lower than that for the reference drug miltefosine (EC 50 , 2.84 M). In addition, the derivatives 9, 11, 42, and 47, with EC 50 between 1.1 and 1.95 M, also displayed excellent selectivity (selectivity index ranged from 12.4 to 22.7) and were tested against infected macrophages. Compound 11, a derivative with a cyclohexyl chain, exhibited the highest activity against intracellular amastigotes, with EC 50 values similar to those observed for the standard drug edelfosine. Structure-activity relationship analyses revealed that N-aliphatic substitution in urea and selenourea is recommended for the leishmanicidal activity of these analogs. Preliminary studies of the mechanism of action for the hit compounds was carried out by measuring their ability to inhibit trypanothione reductase. Even though the obtained results suggest that this enzyme is not the target for most of these derivatives, their activity comparable to that of the standards and lack of toxicity in THP-1 cells highlight the potential of these compounds to be optimized for leishmaniasis treatment.

A table of contents entryHelical peptides stabilized via all-hydrocarbon or lactam side-chain bridging were investigated as disruptors of Leishmania infantum trypanothione reductase and the biological results were rationalized by NMR spectroscopy studies and molecular dynamics simulations.Abstract-All-hydrocarbon and lactam-bridged staples linking amino acid side-chains have been used to stabilize the α-helical motif in short 13-mer peptides that target critical protein-protein interactions at the dimerization interface of Leishmania infantum trypanothione reductase (Li-TryR). The design of the best positions for covalent hydrocarbon closure relied on a theoretical prediction of the degree of helicity of the corresponding cyclic peptides in water. Selected (i, i+4) and (i, i+7) hydrocarbon-stapled peptides were prepared by using solid-phase synthesis protocols and optimized ring-closing metathesis reactions under microwave conditions. Structural analysis by NMR spectroscopy confirmed high helical contents in aqueous TFE solutions for both types of helix-constrained cyclic peptides. Remarkably, the ability to prevent Li-TryR dimerization was reduced in both (i, i+4) and (i, i+7) hydrocarbon stapled peptides but was retained in the corresponding (i, i+4) Glu-Lys lactam-bridged analogue, which also showed a higher resistance to proteolytic degradation by proteinase K relative to the linear peptide prototype. In silico studies indicated that the introduction of a hydrocarbon staple vs a lactam bridge likely perturbs critical interactions required for proper binding of the peptide to the Li-TryR monomer.corresponding N-Fmoc-protected amino acid (1.2 equiv), HCTU (1.2 equiv) and DIEA (2.4 equiv) in anhydrous DMF (0.5-1.0 mL) was added over the swollen peptidil-resin (1.0 equiv) in anhydrous DMF in a microwave vial (5-10 mL). The coupling reaction was heated at 40 ºC using microwave radiation for 10 min. Each coupling step was repeated 3 times using freshly prepared Fmoc-amino acid/coupling reagent mixtures. After complete couplings, the resin was drained and washed with DMF/CH 2 Cl 2 /DMF/CH 2 Cl 2 (4 x 0.5 min).Coupling reactions to primary and secondary amines were monitored by the ninhydrin and the chloranil tests, respectively. Ring closing metathesis (RCM) reaction.To the peptidyl-resins 12 and 13 (0.05-0.07 mmol) swollen in anhydrous CH 2 Cl 2 (5 mL) in a microwave vial (5-10 mL) second generation Grubb's catalyst (0.2 equiv) was added and the vial was sealed and gently bubbled with argon. Then, the reaction was heated at 40 ºC using microwave radiation for 30-150 min. The resin was then filtered and washed successively with CH 2 Cl 2 /MeOH.Residual ruthenium impurities were removed by stirring the resin bound peptide with a solution of DMSO (50 equiv relative to the catalyst) in DMF for 12 h. 37

Trypanothione reductase (TryR) is a well-established target in the search for novel antitrypanosomal and antileishmanial agents. We have previously identified linear and lactam-bridged 13-residue peptides derived from an α-helical region making up part of the dimeric interface of Leishmania infantum TryR (Li-TryR) which prevent trypanothione reduction by disrupting enzyme dimerization. We now show that i,i + 4 side-chain cross-linking with an all-hydrocarbon staple stabilizes the helical structure of these peptides and significantly improves their resistance to protease cleavage relative to previous linear and cyclic lactam analogues. Interestingly, replacement of the amide bridge by the hydrocarbon staple at the same cyclization positions generates derivatives (2 and 3) that similarly inhibit oxidoreductase activity of the enzyme but unexpectedly stabilize the TryR homodimer. The most proteolytically stable peptide 2 covalently linked to oligoarginines displayed potent in vitro leishmanicidal activity against L. infantum parasites.