The mutation G133D on Leishmania guyanensis AQP1 is highly destabilizing as revealed by molecular modeling and hypo-osmotic shock assay.

The Leishmania aquaglyceroporin 1 (AQP1) plays an important role in osmoregulation and antimony (Sb) uptake, being determinant for resistance to antimony. We have previously demonstrated that G133D mutation on L. guyanensis AQP1 (LgAQP1) leads to reduced Sb uptake. Here, we investigated the effects of G133D mutation on LgAQP1 structure, associated with Sb uptake and alterations in osmoregulation capacity. High confidence molecular models of wild-type LgAQP1 as well as the LgAQP1::G133D mutant were constructed and optimized via comparative homology modeling. Computational methods from the mCSM platform were used to evaluate the effects on protein stability and on its ability to bind to glycerol. Functional validation of the disruptive effect of the mutation on LgAQP1 was done by challenging the parasites with hypo-osmotic chock. Glycine 133 is on transmembrane helix 3, buried in the membrane in both open and closed conformation. G133D mutation was predicted to be highly destabilizing, as it alters the helical bundling arrangement in order to accommodate the aspartic acid side chain. The shift in helices also resulted in fewer favorable contacts with glycerol in the channel, which would explain the reduced affinity for similar small molecules as SbO3. Under hypo-osmotic condition, L. guyanensis AQP1G133D presented a 3-fold increase in cellular volume and pronounced delay to recover osmosis homeostasis when compared to the wild-type, a profile that was enhanced in LgAQP1-/- mutants. In conclusion, G133D is a highly disruptive mutation that will destabilize the monomer, compromise tetramer formation and alter pore conformation, leading to reduced Sb uptake and deficient osmoregulation.

Unveiling six potent and highly selective antileishmanial agents via the open source compound collection 'Pathogen Box' against antimony-sensitive and -resistant Leishmania braziliensis.

Despite all efforts to provide new chemical entities to tackle leishmaniases, we are still dependent on a the limited drug arsenal, together with drawbacks like toxicity and drug-resistant parasites. Collaborative drug discovery emerged as an option to speed up the way to find alternative antileishmanial agents. This is the case of Medicines for Malaria Ventures - MMV, that promotes an open source drug discovery initiative to fight diseases worldwide. Here, we screened 400 compounds from 'Pathogen Box' (PBox) collection against Leishmania braziliensis, the main etiological agent of cutaneous leishmaniasis in Brazil. Twenty-three compounds were able to inhibit ≥ 80 % L. braziliensis growth at 5 µM. Six out of the PBox selected 23 compounds were found to be highly selective against L. braziliensis intracellular amastigotes with selectivity index varying from > 104 to > 746 and IC50s ranging from 47 to 480 nM. The compounds were also active against antimony-resistant L. braziliensis isolated from the field or laboratory selected mutants, revealing the potential on treating patients infected with drug resistant parasites. Most of the selected compounds were known to be active against kinetoplastids, however, two compounds (MMV688703 and MMV676477) were part of toxoplasmosis and tuberculosis 'PBox' disease set, reinforcing the potential of phenotyping screening to unveil drug repurposing. Here we applied a computational prediction of pharmacokinetic properties using the ADMET predictor pkCSM (http://biosig.unimelb.edu.au/pkcsm/). The tool offered clues on potential drug development needs and can support further in vivo studies. Molecular docking analysis identified CRK3 (LbrM.35.0660), CYP450 (LbrM.30.3580) and PKA (LbrM.18.1180) as L. braziliensis targets for MMV676604, MMV688372 and MMV688703, respectively. Compounds from 'Pathogen Box' thus represents a new hope for novel (or repurposed) small molecules source to tackle leishmaniases.

Preclinical Gold Complexes as Oral Drug Candidates to Treat Leishmaniasis Are Potent Trypanothione Reductase Inhibitors.

The drugs currently used to treat leishmaniases have limitations concerning cost, efficacy, and safety, making the search for new therapeutic approaches urgent. We found that the gold(I)-derived complexes were active against L. infantum and L. braziliensis intracellular amastigotes with IC50 values ranging from 0.5 to 5.5 µM. All gold(I) complexes were potent inhibitors of trypanothione reductase (TR), with enzyme IC50 values ranging from 1 to 7.8 µM. Triethylphosphine-derived complexes enhanced reactive oxygen species (ROS) production and decreased mitochondrial respiration after 2 h of exposure, indicating that gold(I) complexes cause oxidative stress by direct ROS production, by causing mitochondrial damage or by impairing TR activity and thus accumulating ROS. There was no cross-resistance to antimony; in fact, SbR (antimony-resistant mutants) strains were hypersensitive to some of the complexes. BALB/c mice infected with luciferase-expressing L. braziliensis or L. amazonensis and treated orally with 12.5 mg/kg/day of AdT Et (3) or AdO Et (4) presented reduced lesion size and parasite burden, as revealed by bioimaging. The combination of (3) and miltefosine allowed for a 50% reduction in miltefosine treatment time. Complexes 3 and 4 presented favorable pharmacokinetic and toxicity profiles that encourage further drug development studies. Gold(I) complexes are promising antileishmanial agents, with a potential for therapeutic use, including in leishmaniasis caused by antimony-resistant parasites.

Planarians as models to investigate the bioactivity of gold(I) complexes in vivo.

Gold(I)-containing complexes are used in drug discovery research for rheumatoid arthritis, cancer, and parasitic infections. In this study, we tested the bioactivity of gold(I) complexes in vivo using planarians. The planarian Schmidtea mediterranea possesses orthologues of tumor suppressor genes, such as p53, that, when silenced, cause deregulation of cell proliferation and apoptosis. In this context, we tested two triethylphosphine-gold(I) complexes (AdO and AdT) to determine if they can attenuate phenotypes that result from p53 inhibition. First, we identified the drug concentration that did not affect survival or regeneration and evaluated the drug's effect on cell division and apoptosis. We found that AdT treatment decreased the number of mitotic cells and that all drug treatments increased the number of apoptotic cells. We then performed p53(RNAi) and drug treatments concomitantly and observed the phenotype progression. Drug treatment increased survival three-fold and decreased apoptosis, which resulted in an attenuated phenotype. Our results indicate that planarians can be treated with gold(I) complexes, and that this treatment can diminish the p53(RNAi) phenotype and extend survival. In this work we show that planarians can be used as a model to study the in vivo effect of gold(I) complexes and to further investigate their mechanisms of action.