Biology subjects

Miranda, M. R.

Publications and source records attributed to Miranda, M. R..

4 recordsLinked to original sources

bioRxiv2020

Capsaicin inhibits arginine kinase and exerts anti-Trypanosoma cruzi activity

Trypanosoma cruzi is the causative agent of Chagas disease, considered within the list of twenty neglected diseases according to the World Health Organization. There are only two therapeutic drugs for Chagas disease, both of them unsuitable for the chronic phase, therefore the development of new drugs is a priority. T. cruzi arginine kinase (TcAK) is a promising drug target since it is absent in humans and it is involved in cellular stress responses. In a previous study from our laboratory, possible TcAK inhibitors were identified through computer simulations, resulting in the best-scoring compounds cyanidin derivatives and capsaicin. Considering these results, in this work we evaluate the effect of capsaicin on TcAK activity and its trypanocidal effect. Although capsaicin produced a weak inhibition on the recombinant TcAK activity (IC50 {approx} 800 {micro}M), it had a strong trypanocidal effect on epimastigotes and trypomastigotes (IC50 = 6.26 {micro}M and 0.26 {micro}M, respectively) being 20-fold more active on trypomastigotes than mammalian cells. Epimastigotes that overexpress TcAK were 37% more resistant to capsaicin than wild type parasites, suggesting that trypanocidal activity could be due, in part, to the enzyme inhibition. However, the difference between the concentrations at which the enzyme is inhibited and the parasite death is caused implies the presence of other targets. In this sense, the prohibitin-2 and calmodulin were identified as other possible capsaicin targets. Capsaicin is a strong and selective trypanocidal agent active in nanomolar concentrations, with an IC50 57-fold lower than benznidazole, the drug currently used for treating Chagas disease.

microbiology↗

bioRxiv2020

Trypanocidal activity of the anthocyanidin delphinidin, a non-competitive inhibitor of arginine kinase

The enzyme arginine kinase from Trypanosoma cruzi (TcAK) catalyzes the interconversion of arginine and phosphoarginine to maintain the ATP/ADP cell balance, and is involved in the parasites energetic homeostasis and stress responses. Using virtual screening approaches, some plant-derived polyphenolic pigments such as anthocyanidins, were predicted to inhibit TcAK activity. In this work, it was demonstrated that the anthocyanidin delphinidin showed a non-competitive inhibition mechanism of TcAK in vitro (Ki arginine = 1.32 M and Ki ATP = 500 M). Molecular docking simulations predicted that delphinidin occupies a hydrophobic pocket close to the ATP/ADP binding site. Delphinidin also exerted trypanocidal activity over T. cruzi trypomastigotes with a calculated IC50 of 19.51 M. Anthocyanidins are low-toxicity natural products which can be exploited for the development of trypanocidal drugs with less secondary effects than those currently used for the treatment of Chagas disease.

microbiology↗

bioRxiv2019

Role of Trypanosoma cruzi nucleoside diphosphate kinase 1 in DNA-damage responses

NME23/NDPK proteins are well conserved proteins found in all living organism. Besides their catalytic activity of nucleoside diphosphate kinase (NDPK) they are considered multifunctional, which were first characterized as non-metastatic proteins in mammalian cells. Later, increasing evidences placed NME/NDPK as proteins involved in DNA stability such as gene regulation and DNA-repair. TcNDPK1 is the canonical NDPK isoform present in the parasite Trypanosoma cruzi, orthologous to NME23-H1/H2 which has been shown to have in vitro nuclease activity and DNA-binding properties. In the present study we investigate the role of TcNDPK1 in DNA-damage responses using heterologous gene expression systems and over-expression in epimastigote cells. We found that different strains of bacteria, WT and ndk-mutants, expressing the enzyme decreased about 5 fold and 18 fold the spontaneous mutation rate, respectively. In addition, yeasts lacking the endogenous gene YNK1 (YNK1-) and expressing TcNDPK1, were significantly more resistant to different concentrations of hydrogen peroxide and were less sensible to UV radiation than controls. Parasites over-expressing TcNDPK1 were able to withstand different genotoxic stresses caused by hydrogen peroxide, phleomycin and hidroxyurea. In addition, under oxidative damage, TcNDPK1 over-expressing parasites presented lesser genomic damage and augmented levels of poly(ADP)ribose and poly(ADP)ribose polymerase, an enzyme involved in DNA repair. These results strongly suggest that TcNDPK1 is involved in the maintenance of parasite genomic-DNA integrity, thus, giving rise to a novel function.

microbiology↗

bioRxiv2019

In silico identification of putative Trypanosoma cruzi enolase inhibitors

Enolase is a glycolytic enzyme that catalyzes the interconversion between 2-phosphoglycerate and phosphoenolpyruvate. In trypanosomatids enolase was proposed as a key enzyme after in silico and in vivo analysis and it was validated as a protein essential for the survival of the parasite. Therefore, enolase constitutes an interesting enzyme target for the identification of drugs against Chagas disease. In this work, a combined virtual screening strategy was implemented, employing similarity virtual screening, molecular docking and molecular dynamics. First, two known enolase inhibitors and the enzyme substrates were used as queries for the similarity screening on the Sweetlead database using five different algorithms. Compounds retrieved in the top 10 of at least three search algorithms were selected for further analysis, resulting in six compounds of medical use (etidronate, pamidronate, fosfomycin, acetohydroximate, triclofos, and aminohydroxybutyrate). Molecular docking simulations predicted acetohydroxamate and triclofos would not bind to the active site of the enzyme, and a re-scoring of the obtained poses signaled fosfomycin and aminohydroxybutyrate as bad enzyme binders. Docking poses obtained for etidronate, pamidronate, and PEP, were used for molecular dynamics calculations to describe their mode of binding. From the obtained results, we propose etidronate as a possible TcENO inhibitor, and describe desirable and undesirable molecular motifs to be taken into account in the repurposing or design of drugs aiming this enzyme active site.

microbiology↗