The repertoire of iron superoxide dismutases from Leishmania infantum as targets in the search for therapeutic agents against leishmaniasis.

Species of Leishmania and Trypanosoma genera are the causative agents of relevant parasitic diseases. Survival inside their hosts requires the existence of a potent antioxidant enzymatic machinery. Four iron superoxide dismutases have been described in trypanosomatids (FeSODA, FeSODB1, FeSODB2, and FeSODC) that hold a potential as therapeutic targets. Nonetheless, very few studies have been developed that make use of the purified enzymes. Moreover, FeSODC remains uncharacterised in Leishmania. In this work, for the first time, we describe the purification and enzymatic activity of recombinant versions of the four Leishmania FeSOD isoforms and establish an improved strategy for developing inhibitors. We propose a novel parameter [(V*cyt. c - Vcyt. c)/Vcyt. c] which, in contrast to that used in the classical cytochrome c reduction assay, correlates linearly with enzyme concentration. As a proof of concept, we determine the IC50 values of two ruthenium carbosilane metallodendrimers against these isoforms.

Next generation of selenocyanate and diselenides with upgraded leishmanicidal activity.

Nowadays, leishmaniasis is still treated with outdated drugs that present several obstacles related to their high toxicity, long duration, parenteral administration, high costs and drug resistance. Therefore, there is an urgent demand for safer and more effective novel drugs. Previous studies indicated that selenium compounds are promising derivatives for innovative therapy in leishmaniasis treatment. With this background, a new library of 20 selenocyanate and diselenide derivatives were designed based on structural features present in the leishmanicidal drug miltefosine. Compounds were initially screened against promastigotes of L. major and L. infantum and their cytotoxicity was evaluated in THP-1 cells. Compounds B8 and B9 were the most potent and less cytotoxic and were further screened for the intracellular back transformation assay. The results obtained revealed that B8 and B9 showed EC50 values of 7.7 µM and 5.7 µM, respectively, in L. major amastigotes, while they presented values of 6.0 µM and 7.4 µM, respectively, against L. infantum amastigotes. Furthermore, they exerted high selectivity (60 < SI > 70) towards bone marrow-derived macrophages. Finally, these compounds exhibited higher TryR inhibitory activity than mepacrine (IC50 7.6 and 9.2 µM, respectively), and induced nitric oxide (NO) and reactive oxygen species (ROS) production in macrophages. These results suggest that the compounds B8 and B9 could not only exert a direct leishmanicidal activity against the parasite but also present an indirect action by activating the microbicidal arsenal of the macrophage. Overall, these new generation of diselenides could constitute promising leishmanicidal drug candidates for further studies.

Predictors of fall risk in older adults using the G-STRIDE inertial sensor: an observational multicenter case-control study.

BACKGROUND: There are a lot of tools to use for fall assessment, but there is not yet one that predicts the risk of falls in the elderly. This study aims to evaluate the use of the G-STRIDE prototype in the analysis of fall risk, defining the cut-off points to predict the risk of falling and developing a predictive model that allows discriminating between subjects with and without fall risks and those at risk of future falls. METHODS: An observational, multicenter case-control study was conducted with older people coming from two different public hospitals and three different nursing homes. We gathered clinical variables ( Short Physical Performance Battery (SPPB), Standardized Frailty Criteria, Speed 4 m walk, Falls Efficacy Scale-International (FES-I), Time-Up Go Test, and Global Deterioration Scale (GDS)) and measured gait kinematics using an inertial measure unit (IMU). We performed a logistic regression model using a training set of observations (70% of the participants) to predict the probability of falls. RESULTS: A total of 163 participants were included, 86 people with gait and balance disorders or falls and 77 without falls; 67,8% were females, with a mean age of 82,63 ± 6,01 years. G-STRIDE made it possible to measure gait parameters under normal living conditions. There are 46 cut-off values of conventional clinical parameters and those estimated with the G-STRIDE solution. A logistic regression mixed model, with four conventional and 2 kinematic variables allows us to identify people at risk of falls showing good predictive value with AUC of 77,6% (sensitivity 0,773 y specificity 0,780). In addition, we could predict the fallers in the test group (30% observations not in the model) with similar performance to conventional methods. CONCLUSIONS: The G-STRIDE IMU device allows to predict the risk of falls using a mixed model with an accuracy of 0,776 with similar performance to conventional model. This approach allows better precision, low cost and less infrastructures for an early intervention and prevention of future falls.

New Phosphoramidates Containing Selenium as Leishmanicidal Agents.

This work reports the synthesis and characterization by Fourier transform infrared spectroscopy (FTIR), 1H, 13C, and 79Se nuclear magnetic resonance (NMR), mass spectrometry, and elemental analysis techniques as well as the in vitro evaluation of the leishmanicidal activity of 13 new selenophosphoramidate derivatives. Among the new compounds, four of them (compounds 1f, 1g, 2f, and 2g), which exhibited the best profiles, were tested against infected macrophages and were selected for further studies related to their leishmanicidal mechanism. In this regard, trypanothione redox system alteration was determined. Compound 1g, under similar conditions, was more effective than the corresponding references. In addition, theoretical calculations showed that this compound also presents most physicochemical and pharmacokinetic properties within the ranges expected for orally available drugs. It is believed that selenophosphoramidate functionalities may represent a scaffold to be explored toward the development of new agents for leishmania treatment.

New Amides Containing Selenium as Potent Leishmanicidal Agents Targeting Trypanothione Reductase.

Two new series of 28 selenocyanate and diselenide derivatives containing amide moieties were designed, synthesized, and evaluated for their leishmanicidal activity against Leishmania infantum axenic amastigotes, and selectivity was assessed in human THP-1 cells. Eleven compounds exhibited excellent leishmanicidal activity with EC50 values lower than the reference drug miltefosine (EC50 = 2.84 µM). In addition, for six of them the selectivity index ranged from 9 to >1,442, greater than both references used. The most potent and selective compounds were compounds 2h, 2k, and 2m that displayed EC50 values of 0.52, 1.19, and 0.50 µM, respectively, and a high selectivity index (SI) when tested against THP-1 monocytic cells (SI = >1,442, >672, and >1,100, respectively). These derivatives showed an efficacy similar to that of the reference drugs but much better SI values. They also showed interesting activity values against infected macrophages. Trypanothione reductase (TryR) activity and intracellular thiol level measurement assays were performed for the three best compounds in an attempt to elucidate their mechanism of action. Despite that the new analogs exhibited comparable or better inhibitory activities than the reference TryR inhibitors, more studies are necessary to confirm this result. In summary, our findings suggest that the three compounds described here could constitute leading leishmanicidal drug candidates.

Pre-clinical evidences of the antileishmanial effects of diselenides and selenocyanates.

A series of thirty-one selenocompounds covering a wide chemical space was assessed for in vitro leishmanicidal activities against Leishmania infantum amastigotes. The cytotoxicity of those compounds was also evaluated on human THP-1 cells. Interestingly most tested derivatives were active in the low micromolar range and seven of them (A.I.3, A.I.7, B.I.1, B.I.2, C.I.7 C.I.8 and C.II.8) stood out for selectivity indexes higher than the ones exhibited by reference compounds mitelfosine and edelfosine. These leader compounds were evaluated against infected macrophages and their trypanothione reductase (TryR) inhibition potency was measured to further approach the mechanism by which they caused their action. Among them diselenide tested structures were pointed out for their ability to reduce infection rates. Three of the leader compounds inhibited TryR effectively, therefore this enzyme may be implicated in the mechanism of action by which these compounds cause their leishmanicidal effect.

Synthesis and Leishmanicidal Activity of Novel Urea, Thiourea, and Selenourea Derivatives of Diselenides.

A novel series of thirty-one N-substituted urea, thiourea, and selenourea derivatives containing diphenyldiselenide entities were synthesized, fully characterized by spectroscopic and analytical methods, and screened for their in vitro leishmanicidal activities. The cytotoxic activity of these derivatives was tested against Leishmania infantum axenic amastigotes, and selectivity was assessed in human THP-1 cells. Thirteen of the synthesized compounds showed a significant antileishmanial activity, with 50% effective concentration (EC50) values lower than that for the reference drug miltefosine (EC50, 2.84 µM). In addition, the derivatives 9, 11, 42, and 47, with EC50 between 1.1 and 1.95 µM, also displayed excellent selectivity (selectivity index ranged from 12.4 to 22.7) and were tested against infected macrophages. Compound 11, a derivative with a cyclohexyl chain, exhibited the highest activity against intracellular amastigotes, with EC50 values similar to those observed for the standard drug edelfosine. Structure-activity relationship analyses revealed that N-aliphatic substitution in urea and selenourea is recommended for the leishmanicidal activity of these analogs. Preliminary studies of the mechanism of action for the hit compounds was carried out by measuring their ability to inhibit trypanothione reductase. Even though the obtained results suggest that this enzyme is not the target for most of these derivatives, their activity comparable to that of the standards and lack of toxicity in THP-1 cells highlight the potential of these compounds to be optimized for leishmaniasis treatment.

Discovery of new organoselenium compounds as antileishmanial agents.

We report new organoselenium compounds bearing the sulfonamide moiety as effective inhibitors of the ß-isoform of Carbonic Anhydrase from the unicellular parasitic protozoan L. donovani chagasi. All derivatives were evaluated in vitro for their leishmanicidal activities against Leishmania infantum amastigotes along with their cytotoxicities in human THP-1 cells. Compounds 3e-g showed their activity in the low micromolar range with IC50 values spanning from 0.72 to 0.81 µM and selectivity indexes (SI) > 8 (for 3g SI > 30), thus much higher than those observed for the reference drugs miltefosine and edelfosine. This is the first study which reports new selenoderivatives with promising leishmanicidal properties and acting as Carbonic Anhydrase inhibitors too thus paving the way to the development of innovative agents for the treatment of neglected diseases such as leishmaniasis.

Structure-based domain assignment in Leishmania infantum EndoG: characterization of a pH-dependent regulatory switch and a C-terminal extension that largely dictates DNA substrate preferences.

Mitochondrial endonuclease G from Leishmania infantum (LiEndoG) participates in the degradation of double-stranded DNA (dsDNA) during parasite cell death and is catalytically inactive at a pH of 8.0 or above. The presence, in the primary sequence, of an acidic amino acid-rich insertion exclusive to trypanosomatids and its spatial position in a homology-built model of LiEndoG led us to postulate that this peptide stretch might act as a pH sensor for self-inhibition. We found that a LiEndoG variant lacking residues 145-180 is indeed far more active than its wild-type counterpart at pH values >7.0. In addition, we discovered that (i) LiEndoG exists as a homodimer, (ii) replacement of Ser211 in the active-site SRGH motif with the canonical aspartate from the DRGH motif of other nucleases leads to a catalytically deficient enzyme, (iii) the activity of the S211D variant can be restored upon the concomitant replacement of Ala247 with Arg and (iv) a C-terminal extension is responsible for the observed preferential cleavage of single-stranded DNA (ssDNA) and ssDNA-dsDNA junctions. Taken together, our results support the view that LiEndoG is a multidomain molecular machine whose nuclease activity can be subtly modulated or even abrogated through architectural changes brought about by environmental conditions and interaction with other binding partners.

Comparing the Performance of Indoor Localization Systems through the EvAAL Framework.

In recent years, indoor localization systems have been the object of significant research activity and of growing interest for their great expected social impact and their impressive business potential. Application areas include tracking and navigation, activity monitoring, personalized advertising, Active and Assisted Living (AAL), traceability, Internet of Things (IoT) networks, and Home-land Security. In spite of the numerous research advances and the great industrial interest, no canned solutions have yet been defined. The diversity and heterogeneity of applications, scenarios, sensor and user requirements, make it difficult to create uniform solutions. From that diverse reality, a main problem is derived that consists in the lack of a consensus both in terms of the metrics and the procedures used to measure the performance of the different indoor localization and navigation proposals. This paper introduces the general lines of the EvAAL benchmarking framework, which is aimed at a fair comparison of indoor positioning systems through a challenging competition under complex, realistic conditions. To evaluate the framework capabilities, we show how it was used in the 2016 Indoor Positioning and Indoor Navigation (IPIN) Competition. The 2016 IPIN competition considered three different scenario dimensions, with a variety of use cases: (1) pedestrian versus robotic navigation, (2) smartphones versus custom hardware usage and (3) real-time positioning versus off-line post-processing. A total of four competition tracks were evaluated under the same EvAAL benchmark framework in order to validate its potential to become a standard for evaluating indoor localization solutions. The experience gained during the competition and feedback from track organizers and competitors showed that the EvAAL framework is flexible enough to successfully fit the very different tracks and appears adequate to compare indoor positioning systems.

Novel Heteroaryl Selenocyanates and Diselenides as Potent Antileishmanial Agents.

A series of new selenocyanates and diselenides bearing interesting bioactive scaffolds (quinoline, quinoxaline, acridine, chromene, furane, isosazole, etc.) was synthesized, and their in vitro leishmanicidal activities against Leishmania infantum amastigotes along with their cytotoxicities in human THP-1 cells were determined. Interestingly, most tested compounds were active in the low micromolar range and led us to identify four lead compounds (1h, 2d, 2e, and 2f) with 50% effective dose (ED50) values ranging from 0.45 to 1.27 µM and selectivity indexes of >25 for all of them, much higher than those observed for the reference drugs. These active derivatives were evaluated against infected macrophages, and in order to gain preliminary knowledge about their possible mechanism of action, the inhibition of trypanothione reductase (TryR) was measured. Among these novel structures, compounds 1h (3,5-dimethyl-4-isoxazolyl selenocyanate) and 2d [3,3'-(diselenodiyldimethanediyl)bis(2-bromothiophene)] exhibited good association between TryR inhibitory activity and antileishmanial potency, pointing to 1h, for its excellent theoretical ADME (absorption, distribution, metabolism, and excretion) properties, as the most promising lead molecule for leishmancidal drug design.

Probing the dimerization interface of Leishmania infantum trypanothione reductase with site-directed mutagenesis and short peptides.

Binding at the interface: We tested the inhibitory activity of a set of peptide sequences derived from an α-helix of the dimeric trypanothione reductase from Leishmania infantum. Replacement of a glutamic acid residue with a lysine promoted monomer dissociation and enzyme inhibition.

Evaluation of new Toxocara canis chimeric antigens as an alternative to conventional TES-Ag for anti-Toxocara antibodies detection.

Human toxocariasis is one of the neglected helminthiases and it is caused by the zoonotic roundworm species Toxocara canis and Toxocara cati. Diagnosis of human toxocariasis is based on the combination of clinical, parasitological, and epidemiological criteria, as well as serology tests that detect anti-Toxocara antibodies. Notwithstanding, due to the absence of pathognomonic symptoms and signs of the disease, serology is the key evidence to support a conclusive diagnosis. TES-ELISA is the most widely used serological test for diagnosis. However, cross-reaction of TES antigens with antibodies produced to other helminth antigens is a major drawback for its application in countries with high parasitic prevalence. T. canis recombinant antigens have been described as an alternative to native TES for diagnosis. Nevertheless, the selection of antigenic proteins is a complex process that requires validation. In this paper, we developed an eGFP carrier-based system to express and purify blocks of recombinant polypeptides of T. canis antigenic proteins. Intense cross-reaction polypeptides were detected by Immunoblot and avoided to finally produce a chimeric prototype protein. Additionally, a control chimeric protein that harbors the complete tested proteins was produced. Purified chimeric antigens were tested in ELISA and Immunoblot assays with 310 sera samples of negative and positive control individuals. Our results showed that chimeric rCHITC0 and rCHITC1 antigens (with sensitivities of 62% 58%, 38% and 16% in IB-rCHITC0, ELISA-rCHITC0, ELISA-rCHITC1 and IB-rCHITC1 respectively for OLMS) can perform better in terms of specificity (being 91%, 89%, 87% and 76% for ELISA-rCHITC1, IB-rCHITC1, ELISA-rCHITC0 and IB-rCHITC0 respectively for OLMS) than T. canis TES-ELISA (with 61% specificity), giving a higher signal with serum samples of infected individuals as well the possibility to discriminate false positive cases with other parasitic infections. Our data suggest that T. canis chimeric proteins, represent candidate antigens for phase II studies.

Identification of L. infantum trypanothione synthetase inhibitors with leishmanicidal activity from a (non-biased) in-house chemical library.

Redox homeostasis in trypanosomatids is based on the low-molecular-weight trypanothione, an essential dithiol molecule that is synthetized by trypanothione synthetase (TryS) and maintained in its reduced state by trypanothione disulfide reductase (TryR). The fact that both enzymes are indispensable for parasite survival and absent in the mammalian hosts makes them ideal drug targets against leishmaniasis. Although many efforts have been directed to developing TryR inhibitors, much less attention has been focused on TryS. The screening of an in-house library of 144 diverse molecules using two parallel biochemical assays allowed us to detect 13 inhibitors of L. infantum TryS. Compounds 1 and 3 were characterized as competitive inhibitors with Ki values in the low micromolar range and plausible binding modes for them were identified by automated ligand docking against refined protein structures obtained through computational simulation of an entire catalytic cycle. The proposed binding site for both inhibitors overlaps the polyamine site in the enzyme and, additionally, 1 also occupies part of the ATP site. Compound 4 behaves as a mixed hyperbolic inhibitor with a Ki of 0.8 µM. The activity of 5 is clearly dependent on the concentration of the polyamine substrate, but its kinetic behavior is clearly not compatible with a competitive mode of inhibition. Analysis of the activity of the six best inhibitors against intracellular amastigotes identified 5 as the most potent leishmanicidal candidate, with an EC50 value of 0.6 µM and a selectivity index of 35.

Pyridazino-pyrrolo-quinoxalinium salts as highly potent and selective leishmanicidal agents targeting trypanothione reductase.

Fifteen pyridazino-pyrrolo-quinoxalinium salts were synthesized and tested for their antiprotozoal activity against Leishmania infantum amastigotes. Eleven of them turned out to be leishmanicidal, with EC50 values in the nanomolar range, and displayed low toxicity against the human THP-1 cell line. Selectivity indices for these compounds range from 10 to more than 1000. Compounds 3b and 3f behave as potent inhibitors of the oxidoreductase activity of the essential enzyme trypanothione disulfide reductase (TryR). Interestingly, binding of 3f is not affected by high trypanothione concentrations, as revealed by the noncompetitive pattern of inhibition observed when tested in the presence of increasing concentrations of this substrate. Furthermore, when analyzed at varying NADPH concentrations, the characteristic pattern of hyperbolic uncompetitive inhibition supports the view that binding of NADPH to TryR is a prerequisite for inhibitor-protein association. Similar to other TryR uncompetitive inhibitors for NADPH, 3f is responsible for TryR-dependent reduction of cytochrome c in a reaction that is typically inhibited by superoxide dismutase.

Identification of 1,2,3-triazolium salt-based inhibitors of Leishmania infantum trypanothione disulfide reductase with enhanced antileishmanial potency in cellulo and increased selectivity.

N-methylation of the triazole moiety present in our recently described triazole-phenyl-thiazole dimerization disruptors of Leishmania infantum trypanothione disulfide reductase (LiTryR) led to a new class of potent inhibitors that target different binding sites on this enzyme. Subtle structural changes among representative library members modified their mechanism of action, switching from models of classical competitive inhibition to time-dependent mixed noncompetitive inhibition. X-ray crystallography and molecular modeling results provided a rationale for this distinct behavior. The remarkable potency and selectivity improvements, particularly against intracellular amastigotes, of the LiTryR dimerization disruptors 4c and 4d reveal that they could be exploited as leishmanicidal agents. Of note, L. infantum promastigotes treated with 4c significantly reduced their low-molecular-weight thiol content, thus providing additional evidence that LiTryR is the main target of this novel compound.

Mechanistic insight into the catalytic activity of ßßα-metallonucleases from computer simulations: Vibrio vulnificus periplasmic nuclease as a test case.

Using information from wild-type and mutant Vibrio vulnificus nuclease (Vvn) and I-PpoI homing endonuclease co-crystallized with different oligodeoxynucleotides, we have built the complex of Vvn with a DNA octamer and carried out a series of simulations to dissect the catalytic mechanism of this metallonuclease in a stepwise fashion. The distinct roles played in the reaction by individual active site residues, the metal cation and water molecules have been clarified by using a combination of classical molecular dynamics simulations and quantum mechanical calculations. Our results strongly support the most parsimonious catalytic mechanism, namely one in which a single water molecule from bulk solvent is used to cleave the phosphodiester bond and protonate the 3'-hydroxylate leaving group.

Antileishmanial activity of imidothiocarbamates and imidoselenocarbamates.

In the present study, a family of 15 imidothio- and imidoselenocarbamates (1-15) analogs have been synthesized and screened for their in vitro antileishmanial potential against Leishmania infantum promastigotes. The six most active ones (2, 4, 7, 13, 14, and 15) were also tested in an axenic amastigote model. In order to establish their selectivity indexes (SI) the cytotoxic effect of each compound was also assayed against Jurkat and THP-1 cell lines. Compounds 2 and 4, both with a pyridine moiety, showed a moderate antileishmanial activity with an IC(50) value of 4.68 ± 0.46 and 3.03 ± 0.24 µM, respectively, in the amastigote model. The activity was compared with that of standard drugs, edelfosine (IC50 = 0.82 ± 0.13 µM) and miltefosine (IC50 = 2.84 ± 0.10 µM). Related to selectivity, the SI of both compounds are similar to those of the standard drugs when compared against the THP-1 cell line. Moreover, compound 4 was able to reduce the number of amastigote-infected THP-1 cells to 40% of that observed in untreated controls after a 96-h period of treatment. These derivatives thus represent two new leads for further studies aimed at establishing their mechanism of action.

Efficient Dimerization Disruption of Leishmania infantum Trypanothione Reductase by Triazole-phenyl-thiazoles.

Inhibition of Leishmania infantum trypanothione disulfide reductase (LiTryR) by disruption of its homodimeric interface has proved to be an alternative and unexploited strategy in the search for novel antileishmanial agents. Proof of concept was first obtained by peptides and peptidomimetics. Building on previously reported dimerization disruptors containing an imidazole-phenyl-thiazole scaffold, we now report a new 1,2,3-triazole-based chemotype that yields noncompetitive, slow-binding inhibitors of LiTryR. Several compounds bearing (poly)aromatic substituents dramatically improve the ability to disrupt LiTryR dimerization relative to reference imidazoles. Molecular modeling studies identified an almost unexplored hydrophobic region at the interfacial domain as the putative binding site for these compounds. A subsequent structure-based design led to a symmetrical triazole analogue that displayed even more potent inhibitory activity over LiTryR and enhanced leishmanicidal activity. Remarkably, several of these novel triazole-bearing compounds were able to kill both extracellular and intracellular parasites in cell cultures.

Small Molecule-Peptide Conjugates as Dimerization Inhibitors of Leishmania infantum Trypanothione Disulfide Reductase.

Trypanothione disulfide reductase (TryR) is an essential homodimeric enzyme of trypanosomatid parasites that has been validated as a drug target to fight human infections. Using peptides and peptidomimetics, we previously obtained proof of concept that disrupting protein-protein interactions at the dimer interface of Leishmania infantum TryR (LiTryR) offered an innovative and so far unexploited opportunity for the development of novel antileishmanial agents. Now, we show that linking our previous peptide prototype TRL38 to selected hydrophobic moieties provides a novel series of small-molecule-peptide conjugates that behave as good inhibitors of both LiTryR activity and dimerization.