Role of Heme and Heme-Proteins in Trypanosomatid Essential Metabolic Pathways

Around the world, trypanosomatids are known for being etiological agents of several highly disabling and often fatal diseases like Chagas disease (Trypanosoma cruzi), leishmaniasis (Leishmania spp.), and African trypanosomiasis (Trypanosoma brucei). Throughout their life cycle, they must cope with diverse environmental conditions, and the mechanisms involved in these processes are crucial for their survival. In this review, we describe the role of heme in several essential metabolic pathways of these protozoans. Notwithstanding trypanosomatids lack of the complete heme biosynthetic pathway, we focus our discussion in the metabolic role played for important heme-proteins, like cytochromes. Although several genes for different types of cytochromes, involved in mitochondrial respiration, polyunsaturated fatty acid metabolism, and sterol biosynthesis, are annotated at the Tritryp Genome Project, the encoded proteins have not yet been deeply studied. We pointed our attention into relevant aspects of these protein functions that are amenable to be considered for rational design of trypanocidal agents

New L-proline uptake inhibitors with anti-Trypanosoma cruzi activity

L-Proline is an important amino acid for the pathogenic protists belonging to Trypanosoma and Leishmania genera. In Trypanosoma cruzi, the etiological agent of Chagas disease, this amino acid is involved in fundamental biological processes such as ATP production, differentiation of the insect and intracellular stages, the host cell infection and the resistance to a variety of stresses, including nutritional and osmotic as well as oxidative imbalance. In this study, we explore the L-Proline uptake as a chemotherapeutic target for T. cruzi. For this, we propose a novel rational to design inhibitors containing this amino acid as a recognizable motif. This rational consists of conjugating the amino acid (proline in this case) to a linker and a variable region able to block the transporter. We obtained a series of sixteen 1,2,3-triazolyl-proline derivatives through alkylation and copper(I)-catalyzed azide-alkyne cycloaddition (click chemistry) for in vitro screening against T. cruzi epimastigotes, trypanocidal activity and proline uptake. We successfully obtained inhibitors that are able to interfere with the amino acid uptake, which validated the first example of a rationally designed chemotherapeutic agent targeting a metabolite\u27s transport. Additionally, we designed and prepared fluorescent analogues of the inhibitors that were successfully taken up by T. cruzi, allowing following up their intracellular fate. In conclusion, we successfully designed and produced a series of metabolite uptake inhibitors. This is one of few examples of rationally designed amino acid transporter inhibitor, being the first case where the strategy is applied on the development of chemotherapy against Chagas disease. This unprecedented development is remarkable having in mind that only a small percent of the metabolite transporters has been studied at the structural and/or molecular level

Axial Ligand Modulation of the Electronic Structures of Binuclear Copper Sites: Analysis of Paramagnetic ^1H NMR Spectra of Met160Gln Cu_A

Cu_A is an electron-transfer copper center present in heme-copper oxidases and N_2O reductases. The center is a binuclear unit, with two cysteine ligands bridging the metal ions and two terminal histidine residues. A Met residue and a peptide carbonyl group are located on opposite sides of the Cu2S2 plane; these weaker ligands are fully conserved in all known Cu_A sites. The Met160Gln mutant of the soluble subunit II of Thermus thermophilus ba_3 oxidase has been studied by NMR spectroscopy. In its oxidized form, the binuclear copper is a fully delocalized mixed-valence pair, as are all natural Cu_A centers. The faster nuclear relaxation in this mutant suggests that a low-lying excited state has shifted to higher energies compared to that of the wild-type protein. The introduction of the Gln residue alters the coordination mode of His114 but does not affect His157, thereby confirming the proposal that the axial ligand-to-copper distances influence the copper−His interactions (Robinson, H.; Ang, M. C.; Gao, Y. G.; Hay, M. T.; Lu, Y.; Wang, A. H. Biochemistry 1999, 38, 5677). Changes in the hyperfine coupling constants of the Cys β-CH_2 groups are attributed to minor geometrical changes that affect the Cu−S−C_β−H_β dihedral angles. These changes, in addition, shift the thermally accessible excited states, thus influencing the spectral position of the Cys β-CH_2 resonances. The Cu−Cys bonds are not substantially altered by the Cu−Gln160 interaction, in contrast to the situation found in the evolutionarily related blue copper proteins. It is possible that regulatory subunits in the mitochondrial oxidases fix the relative positions of thermally accessible Cu_A excited states by tuning axial ligand interactions

Heme analogs are selectively imported by <i>T</i>. <i>cruzi</i> epimastigotes but HAs are not imported by trypomastigotes.

<p>Heme analog uptake was evaluated by confocal microscopy images from (A) epimastigotes incubated for 5 minutes and (B) trypomastigotes incubated for 30 minutes, at 28°C with 100 μM of HAs. The results are representative of at least three independent experiments.</p

Heme analogs are transported by amastigotes.

<p>Confocal microscopy images of uninfected Vero cells (A) and infected Vero cells with amastigotes of <i>T</i>. <i>cruzi</i> (B) incubated with 100 μM heme analogs for 30 minutes. The results are representative of at least three independent experiments.</p

TcHTE promotes heme uptake in <i>hem1</i>Δ yeast cells.

<p>Spot growth assay of <i>S</i>. <i>cerevisiae hem1</i>Δ transformed with the p426.MET25 or the vector carrying <i>TcHTE</i>.<i>6HIS</i> or <i>TcHTE</i>.<i>6HIS-GFP</i> constructs. 7 μL of four serial dilutions from the initial cultures of OD = 2 were plated in a solid SC medium with Glucose (A) or Glycerol-Ethanol (B) as a carbon source, and different hemin concentrations, or δ-ALA as control. Western blot of total cell extracts of <i>S</i>. <i>cerevisiae</i> transformed with vector carrying <i>TcHTE</i>.<i>6HIS-GFP</i> with anti-GFP or anti-His antibodies (C). Confocal microscopy images of <i>S</i>. <i>cerevisiae hem1</i>Δ transformed with a vector carrying <i>TcHTE</i>.<i>6HIS-GFP</i> growth in SC medium with Glucose and 0.5 μM hemin (D).</p

TcHTE.6His-GFP negatively affects epimastigote growth under standard heme concentrations.

<p><i>T</i>. <i>cruzi</i> epimastigote (Dm28c.pLEW13) transfected with the p<i>Tc</i>INDEX-GW vector (A) or the <i>TcHTE</i>.<i>6HIS-GFP</i> cloned in p<i>Tc</i>INDEX-GW vector (B) were maintained in LIT 10% FBS, supplemented with 0, 5 or 20 μM hemin with periodic dilutions every 2 days. The results are representative of at least two independent experiments. The experimental data is presented as the mean ± SD.</p

TcHTE.6His-GFP locates in flagellar pocket.

<p>Confocal microscopy images of the epimastigotes expressing <i>TcHTE</i>.<i>6HIS-GFP</i> (A), and isolated flagellar complexes of epimastigotes expressing TcHTE.6His-GFP (green) treated with anti-tubulin antibodies (red) (B).</p