Preclinical Studies in Anti-Trypanosomatidae Drug Development.

The trypanosomatid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania are the causative agents of human African trypanosomiasis, Chagas Disease and Leishmaniasis, respectively. These infections primarily affect poor, rural communities in the developing world, and are responsible for trapping sufferers and their families in a disease/poverty cycle. The development of new chemotherapies is a priority given that existing drug treatments are problematic. In our search for novel anti-trypanosomatid agents, we assess the growth-inhibitory properties of >450 compounds from in-house and/or "Pathogen Box" (PBox) libraries against L. infantum, L. amazonensis, L.braziliensis, T. cruzi and T. brucei and evaluate the toxicities of the most promising agents towards murine macrophages. Screens using the in-house series identified 17 structures with activity against and selective toward Leishmania: Compounds displayed 50% inhibitory concentrations between 0.09 and 25 µM and had selectivity index values >10. For the PBox library, ~20% of chemicals exhibited anti-parasitic properties including five structures whose activity against L. infantum had not been reported before. These five compounds displayed no toxicity towards murine macrophages over the range tested with three being active in an in vivo murine model of the cutaneous disease, with 100% survival of infected animals. Additionally, the oral combination of three of them in the in vivo Chagas disease murine model demonstrated full control of the parasitemia. Interestingly, phenotyping revealed that the reference strain responds differently to the five PBox-derived chemicals relative to parasites isolated from a dog. Together, our data identified one drug candidate that displays activity against Leishmania and other Trypanosomatidae in vitro and in vivo, while exhibiting low toxicity to cultured mammalian cells and low in vivo acute toxicity.

Functional characterisation of the methionine sulfoxide reductase repertoire in Trypanosoma brucei.

To combat the deleterious effects that oxidation of the sulfur atom in methionine to sulfoxide may bring, aerobic cells express repair pathways involving methionine sulfoxide reductases (MSRs) to reverse the above reaction. Here, we show that Trypanosoma brucei, the causative agent of African trypanosomiasis, expresses two distinct trypanothione-dependent MSRs that can be distinguished from each other based on sequence, sub-cellular localisation and substrate preference. One enzyme found in the parasite's cytosol, shows homology to the MSRA family of repair proteins and preferentially metabolises the S epimer of methionine sulfoxide. The second, which contains sequence motifs present in MSRBs, is restricted to the mitochondrion and can only catalyse reduction of the R form of peptide-bound methionine sulfoxide. The importance of these proteins to the parasite was demonstrated using functional genomic-based approaches to produce cells with reduced or elevated expression levels of MSRA, which exhibited altered susceptibility to exogenous H2O2. These findings identify new reparative pathways that function to fix oxidatively damaged methionine within this medically important parasite.

Identification of Specific Inhibitors of Trypanosoma cruzi Malic Enzyme Isoforms by Target-Based HTS.

Trypanosoma cruzi is the causative agent of Chagas disease. The lack of an efficient and safe treatment supports the research into novel metabolic targets, with the malic enzyme (ME) representing one such potential candidate. T. cruzi expresses a cytosolic (TcMEc) and a mitochondrial (TcMEm) ME isoform, with these activities functioning to generate NADPH, a key source of reducing equivalents that drives a range of anabolic and protective processes. To identify specific inhibitors that target TcMEs, two independent high-throughput screening strategies using a diversity library containing 30,000 compounds were employed. IC50 values of 262 molecules were determined for both TcMEs, as well as for three human ME isoforms, with the inhibitors clustered into six groups according to their chemical similarity. The most potent hits belonged to a sulfonamide group that specifically target TcMEc. Moreover, several selected inhibitors of both TcMEs showed a trypanocidal effect against the replicative forms of T. cruzi. The chemical diversity observed among those compounds that inhibit TcMEs activity emphasizes the druggability of these enzymes, with a sulfonamide-based subset of compounds readily able to block TcMEc function at a low nanomolar range.

Synthesis and antikinetoplastid activity of a series of N,N'-substituted diamines.

A series of 25 N,N'-substituted diamines were prepared by controlled reductive amination of free aliphatic diamines with different substituted benzaldehydes. The library was screened in vitro for antiparasitic activity on the causative agents of human African trypanosomiasis, Chagas' disease and visceral leishmaniasis. The most potent compounds were derived from a subset of diamines that contained a 4-OBn substitution, having a 50% parasite growth inhibition in the submicromolar (against Trypanosoma cruzi) or nanomolar (against Trypanosoma brucei and Leishmania donovani) range. We conclude that members of this series of N,N'-substituted diamines provide new lead structures that have potential to treat trypanosomal and leishmanial infections.

Insights into the redox biology of Trypanosoma cruzi: Trypanothione metabolism and oxidant detoxification.

Trypanosoma cruzi is the etiologic agent of Chagas' disease, an infection that affects several million people in Latin America. With no immediate prospect of a vaccine and problems associated with current chemotherapies, the development of new treatments is an urgent priority. Several aspects of the redox metabolism of this parasite differ enough from those in the mammalian host to be considered targets for drug development. Here, we review the information about a trypanosomatid-specific molecule centrally involved in redox metabolism, the dithiol trypanothione, and the main effectors of cellular antioxidant defense. We focus mainly on data from T. cruzi, making comparisons with other trypanosomatids whenever possible. In these parasites trypanothione participates in crucial thiol-disulfide exchange reactions and serves as electron donor in different metabolic pathways, from synthesis of DNA precursors to oxidant detoxification. Interestingly, the levels of several enzymes involved in trypanothione metabolism and oxidant detoxification increase during the transformation of T. cruzi to its mammalian-infective form and the overexpression of some of them has been associated with increased resistance to macrophage-dependent oxidative killing. Together, the evidence suggests a central role of the trypanothione-dependent antioxidant systems in the infection process.

Peroxiredoxins play a major role in protecting Trypanosoma cruzi against macrophage- and endogenously-derived peroxynitrite.

There is increasing evidence that Trypanosoma cruzi antioxidant enzymes play a key immune evasion role by protecting the parasite against macrophage-derived reactive oxygen and nitrogen species. Using T. cruzi transformed to overexpress the peroxiredoxins TcCPX (T. cruzi cytosolic tryparedoxin peroxidase) and TcMPX (T. cruzi mitochondrial tryparedoxin peroxidase), we found that both cell lines readily detoxify cytotoxic and diffusible reactive oxygen and nitrogen species generated in vitro or released by activated macrophages. Parasites transformed to overexpress TcAPX (T. cruzi ascorbate-dependent haemoperoxidase) were also more resistant to H2O2 challenge, but unlike TcMPX and TcCPX overexpressing lines, the TcAPX overexpressing parasites were not resistant to peroxynitrite. Whereas isolated tryparedoxin peroxidases react rapidly (k=7.2 x 10(5) M(-1) x s(-1)) and reduce peroxynitrite to nitrite, our results demonstrate that both TcMPX and TcCPX peroxiredoxins also efficiently decompose exogenous- and endogenously-generated peroxynitrite in intact cells. The degree of protection provided by TcCPX against peroxynitrite challenge results in higher parasite proliferation rates, and is demonstrated by inhibition of intracellular redox-sensitive fluorescence probe oxidation, protein 3-nitrotyrosine and protein-DMPO (5,5-dimethylpyrroline-N-oxide) adduct formation. Additionally, peroxynitrite-mediated over-oxidation of the peroxidatic cysteine residue of peroxiredoxins was greatly decreased in TcCPX overexpressing cells. The protective effects generated by TcCPX and TcMPX after oxidant challenge were lost by mutation of the peroxidatic cysteine residue in both enzymes. We also observed that there is less peroxynitrite-dependent 3-nitrotyrosine formation in infective metacyclic trypomastigotes than in non-infective epimastigotes. Together with recent reports of up-regulation of antioxidant enzymes during metacyclogenesis, our results identify components of the antioxidant enzyme network of T. cruzi as virulence factors of emerging importance.

Mitochondrial superoxide radicals mediate programmed cell death in Trypanosoma cruzi: cytoprotective action of mitochondrial iron superoxide dismutase overexpression.

Trypanosoma cruzi undergo PCD (programmed cell death) under appropriate stimuli, the mechanisms of which remain to be established. In the present study, we show that stimulation of PCD in T. cruzi epimastigotes by FHS (fresh human serum) results in rapid (<1 h) externalization of phosphatidylserine and depletion of the low molecular mass thiols dihydrotrypanothione and glutathione. Concomitantly, enhanced generation of oxidants was established by EPR and immuno-spin trapping of radicals using DMPO (5,5-dimethylpyrroline-N-oxide) and augmentation of the glucose flux through the pentose phosphate pathway. In the early period (<20 min), changes in mitochondrial membrane potential and inhibition of respiration, probably due to the impairment of ADP/ATP exchange with the cytosol, were observed, conditions that favour the generation of O2*-. Accelerated rates of mitochondrial O2*- production were detected by the inactivation of the redox-sensitive mitochondrial aconitase and by oxidation of a mitochondrial-targeted probe (MitoSOX). Importantly, parasites overexpressing mitochondrial FeSOD (iron superoxide dismutase) were more resistant to the PCD stimulus, unambiguously indicating the participation of mitochondrial O2*- in the signalling process. In summary, FHS-induced PCD in T. cruzi involves mitochondrial dysfunction that causes enhanced O(2)(*-) formation, which leads to cellular oxidative stress conditions that trigger the initiation of PCD cascades; moreover, overexpression of mitochondrial FeSOD, which is also observed during metacyclogenesis, resulted in cytoprotective effects.