16 research outputs found
Reliability of working memory assessment in neurocognitive disorders: a study of the Digit Span and Corsi Block-Tapping tasks
Benznidazole biotransformation and multiple targets in <i>Trypanosoma</i> cruzi revealed by metabolomics
<b>Background</b><p></p>
The first line treatment for Chagas disease, a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi, involves administration of benznidazole (Bzn). Bzn is a 2-nitroimidazole pro-drug which requires nitroreduction to become active, although its mode of action is not fully understood. In the present work we used a non-targeted MS-based metabolomics approach to study the metabolic response of T. cruzi to Bzn.<p></p>
<b>Methodology/Principal findings</b><p></p>
Parasites treated with Bzn were minimally altered compared to untreated trypanosomes, although the redox active thiols trypanothione, homotrypanothione and cysteine were significantly diminished in abundance post-treatment. In addition, multiple Bzn-derived metabolites were detected after treatment. These metabolites included reduction products, fragments and covalent adducts of reduced Bzn linked to each of the major low molecular weight thiols: trypanothione, glutathione, Îł-glutamylcysteine, glutathionylspermidine, cysteine and ovothiol A. Bzn products known to be generated in vitro by the unusual trypanosomal nitroreductase, TcNTRI, were found within the parasites, but low molecular weight adducts of glyoxal, a proposed toxic end-product of NTRI Bzn metabolism, were not detected.<p></p>
<b>Conclusions/significance</b><p></p>
Our data is indicative of a major role of the thiol binding capacity of Bzn reduction products in the mechanism of Bzn toxicity against T. cruzi
Using a Non-Image-Based Medium-Throughput Assay for Screening Compounds Targeting N-myristoylation in Intracellular Leishmania Amastigotes
We have refined a medium-throughput assay to screen hit compounds for activity against N-myristoylation in intracellular amastigotes of Leishmania donovani. Using clinically-relevant stages of wild type parasites and an Alamar blue-based detection method, parasite survival following drug treatment of infected macrophages is monitored after macrophage lysis and transformation of freed amastigotes into replicative extracellular promastigotes. The latter transformation step is essential to amplify the signal for determination of parasite burden, a factor dependent on equivalent proliferation rate between samples. Validation of the assay has been achieved using the anti-leishmanial gold standard drugs, amphotericin B and miltefosine, with EC50 values correlating well with published values. This assay has been used, in parallel with enzyme activity data and direct assay on isolated extracellular amastigotes, to test lead-like and hit-like inhibitors of Leishmania Nmyristoyl transferase (NMT). These were derived both from validated in vivo inhibitors of Trypanosoma brucei NMT and a recent high-throughput screen against L. donovani NMT. Despite being a potent inhibitor of L. donovani NMT, the activity of the lead T. brucei NMT inhibitor (DDD85646) against L. donovani amastigotes is relatively poor. Encouragingly, analogues of DDD85646 show improved translation of enzyme to cellular activity. In testing the high-throughput L. donovani hits, we observed macrophage cytotoxicity with compounds from two of the four NMT-selective series identified, while all four series displayed low enzyme to cellular translation, also seen here with the T. brucei NMT inhibitors. Improvements in potency and physicochemical properties will be required to deliver attractive lead-like Leishmania NMT inhibitors
Development of Trypanosoma cruzi in vitro assays to identify compounds suitable for progression in Chagas’ disease drug discovery
Chagas' disease is responsible for significant mortality and morbidity in Latin America. Current treatments display variable efficacy and have adverse side effects, hence more effective, better tolerated drugs are needed. However, recent efforts have proved unsuccessful with failure of the ergosterol biosynthesis inhibitor posaconazole in phase II clinical trials despite promising in vitro and in vivo studies. The lack of translation between laboratory experiments and clinical outcome is a major issue for further drug discovery efforts. Our goal was to identify cell-based assays that could differentiate current nitro-aromatic drugs nifurtimox and benznidazole from posaconazole. Using a panel of T. cruzi strains including the six major lineages (TcI-VI), we found that strain PAH179 (TcV) was markedly less susceptible to posaconazole in vitro. Determination of parasite doubling and cycling times as well as EdU labelling experiments all indicate that this lack of sensitivity is due to the slow doubling and cycling time of strain PAH179. This is in accordance with ergosterol biosynthesis inhibition by posaconazole leading to critically low ergosterol levels only after multiple rounds of division, and is further supported by the lack of effect of posaconazole on the non-replicative trypomastigote form. A washout experiment with prolonged posaconazole treatment showed that, even for more rapidly replicating strains, this compound cannot clear all parasites, indicative of a heterogeneous parasite population in vitro and potentially the presence of quiescent parasites. Benznidazole in contrast was able to kill all parasites. The work presented here shows clear differentiation between the nitro-aromatic drugs and posaconazole in several assays, and suggests that in vitro there may be clinically relevant heterogeneity in the parasite population that can be revealed in long-term washout experiments. Based on these findings we have adjusted our in vitro screening cascade so that only the most promising compounds are progressed to in vivo experiments
Pharmacological interventions for treating heart failure in patients with Chagas cardiomyopathy
Recent Clinical Trials for the Etiological Treatment of Chronic Chagas Disease: Advances, Challenges and Perspectives
Pentamidine exerts in vitro and in vivo anti Trypanosoma cruzi activity and inhibits the polyamine transport in Trypanosoma cruzi
Pentamidine is an antiprotozoal and fungicide drug used in the treatment of leishmaniasis and African trypanosomiasis. Despite its extensive use as antiparasitic drug, little evidence exists about the effect of pentamidine in Trypanosoma cruzi, the etiological agent of Chagas’ disease. Recent studies have shown that pentamidine blocks a polyamine transporter present in Leishmania major; consequently, its might also block these transporters in T. cruzi. Considering that T. cruzi lacks the ability to synthesize putrescine de novo, the inhibition of polyamine transport can bring a new therapeutic target against the parasite. In this work, we show that pentamidine decreases, not only the viability of T. cruzi trypomastigotes, but also the parasite burden of infected cells. In T. cruzi-infected mice pentamidine decreases the inflammation and parasite burden in hearts from infected mice. The treatment also decreases parasitemia, resulting in an increased survival rate. In addition, pentamidine strongly inhibits the putrescine and spermidine transport in T. cruzi epimastigotes and amastigotes. Thus, this study points to reevaluate the utility of pentamidine and introduce evidence of a potential new action mechanism. In the quest of new therapeutic strategies against Chagas disease, the extensive use of pentamidine in human has led to a well-known clinical profile, which could be an advantage over newly synthesized molecules that require more comprehensive trials prior to their clinical use.Fil: DĂaz, MarĂa VerĂłnica. Universidad de Chile; ChileFil: Miranda, Mariana ReneĂ©. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay. Instituto de Investigaciones MĂ©dicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones MĂ©dicas; ArgentinaFil: Campos Estrada, Carolina. Universidad de Chile; ChileFil: Reigada, Chantal. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay. Instituto de Investigaciones MĂ©dicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones MĂ©dicas; ArgentinaFil: Maya, Juan D.. Universidad de Chile; ChileFil: Pereira, Claudio Alejandro. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay. Instituto de Investigaciones MĂ©dicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones MĂ©dicas; ArgentinaFil: LĂłpez Muñoz, Rodrigo. Universidad de Chile; Chil