Publications by authors named "Marcelo A Comini"

46 Publications

CUTie2: The Attack of the Cyclic Nucleotide Sensor Clones.

Front Mol Biosci 2021 11;8:629773. Epub 2021 Mar 11.

BioMolecular Simulation Group, Institut Pasteur de Montevideo, Montevideo, Uruguay.

The detection of small molecules in living cells using genetically encoded FRET sensors has revolutionized our understanding of signaling pathways at the sub-cellular level. However, engineering fluorescent proteins and specific binding domains to create new sensors remains challenging because of the difficulties associated with the large size of the polypeptides involved, and their intrinsically huge conformational variability. Indeed, FRET sensors' design still relies on vague structural notions, and trial and error combinations of linkers and protein modules. We recently designed a FRET sensor for the second messenger cAMP named CUTie (yclic nucleotide Universal ag for maging xperiments), which granted sub-micrometer resolution in living cells. Here we apply a combination of sequence/structure analysis to produce a new-generation FRET sensor for the second messenger cGMP based on Protein kinase G I (PKGI), which we named CUTie2. Coarse-grained molecular dynamics simulations achieved an exhaustive sampling of the relevant spatio-temporal coordinates providing a quasi-quantitative prediction of the FRET efficiency, as confirmed by experiments. Moreover, biochemical characterization showed that the cGMP binding module maintains virtually the same affinity and selectivity for its ligand thant the full-length protein. The computational approach proposed here is easily generalizable to other allosteric protein modules, providing a cost effective-strategy for the custom design of FRET sensors.
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http://dx.doi.org/10.3389/fmolb.2021.629773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7991088PMC
March 2021

Glucose 6-Phosphate Dehydrogenase from Trypanosomes: Selectivity for Steroids and Chemical Validation in Bloodstream .

Molecules 2021 Jan 12;26(2). Epub 2021 Jan 12.

Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Mataojo 2020, Montevideo 11400, Uruguay.

Glucose 6-phosphate dehydrogenase (G6PDH) fulfills an essential role in cell physiology by catalyzing the production of NADPH and of a precursor for the de novo synthesis of ribose 5-phosphate. In trypanosomatids, G6PDH is essential for in vitro proliferation, antioxidant defense and, thereby, drug resistance mechanisms. So far, 16α-brominated epiandrosterone represents the most potent hit targeting trypanosomal G6PDH. Here, we extended the investigations on this important drug target and its inhibition by using a small subset of androstane derivatives. In , immunofluorescence revealed a cytoplasmic distribution of G6PDH and the absence of signal in major organelles. Cytochemical assays confirmed parasitic G6PDH as the molecular target of epiandrosterone. Structure-activity analysis for a set of new (dehydro)epiandrosterone derivatives revealed that bromination at position 16α of the cyclopentane moiety yielded more potent G6PDH inhibitors than the corresponding β-substituted analogues. For the 16α brominated compounds, the inclusion of an acetoxy group at position 3 either proved detrimental or enhanced the activity of the epiandrosterone or the dehydroepiandrosterone derivatives, respectively. Most derivatives presented single digit μM EC against infective and the killing mechanism involved an early thiol-redox unbalance. This data suggests that infective African trypanosomes lack efficient NADPH-synthesizing pathways, beyond the Pentose Phosphate, to maintain thiol-redox homeostasis.
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http://dx.doi.org/10.3390/molecules26020358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826790PMC
January 2021

Recombinant antibody against Trypanosoma cruzi from patients with chronic Chagas heart disease recognizes mammalian nervous system.

EBioMedicine 2021 Jan 9;63:103206. Epub 2021 Jan 9.

Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET), Buenos Aires, Argentina. Electronic address:

Background: To deeply understand the role of antibodies in the context of Trypanosoma cruzi infection, we decided to characterize A2R1, a parasite antibody selected from single-chain variable fragment (scFv) phage display libraries constructed from B cells of chronic Chagas heart disease patients.

Methods: Immunoblot, ELISA, cytometry, immunofluorescence and immunohistochemical assays were used to characterize A2R1 reactivity. To identify the antibody target, we performed an immunoprecipitation and two-dimensional electrophoresis coupled to mass spectrometry and confirmed A2R1 specific interaction by producing the antigen in different expression systems. Based on these data, we carried out a comparative in silico analysis of the protein target´s orthologues, focusing mainly on post-translational modifications.

Findings: A2R1 recognizes a parasite protein of ~50 kDa present in all life cycle stages of T. cruzi, as well as in other members of the kinetoplastid family, showing a defined immunofluorescence labeling pattern consistent with the cytoskeleton. A2R1 binds to tubulin, but this interaction relies on its post-translational modifications. Interestingly, this antibody also targets mammalian tubulin only present in brain, staining in and around cell bodies of the human peripheral and central nervous system.

Interpretation: Our findings demonstrate for the first time the existence of a human antibody against T. cruzi tubulin capable of cross-reacting with a human neural protein. This work re-emphasizes the role of molecular mimicry between host and parasitic antigens in the development of pathological manifestations of T. cruzi infection.
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http://dx.doi.org/10.1016/j.ebiom.2020.103206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7809174PMC
January 2021

Mechanistic and biological characterisation of novel -substituted paullones targeting the biosynthesis of trypanothione in .

J Enzyme Inhib Med Chem 2020 Dec;35(1):1345-1358

Laboratory Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Montevideo, Uruguay.

Trypanothione synthetase (TryS) produces -bis(glutathionyl)spermidine (or trypanothione) at the expense of ATP. Trypanothione is a metabolite unique and essential for survival and drug-resistance of trypanosomatid parasites. In this study, we report the mechanistic and biological characterisation of optimised -substituted paullone analogues with anti-TryS activity. Several of the new derivatives retained submicromolar IC against leishmanial TryS. The binding mode to TryS of the most potent paullones has been revealed by means of kinetic, biophysical and molecular modelling approaches. A subset of analogues showed an improved potency (EC 0.5-10 µM) and selectivity (20-35) against the clinically relevant stage of (mucocutaneous leishmaniasis) and (visceral leishmaniasis). For a selected derivative, the mode of action involved intracellular depletion of trypanothione. Our findings shed light on the molecular interaction of TryS with rationally designed inhibitors and disclose a new set of compounds with on-target activity against different species.
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http://dx.doi.org/10.1080/14756366.2020.1780227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7717452PMC
December 2020

The Redox Activity of Protein Disulfide Isomerase Inhibits ALS Phenotypes in Cellular and Zebrafish Models.

iScience 2020 May 25;23(5):101097. Epub 2020 Apr 25.

Centre for MND Research, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW 2109, Australia; Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC 3086, Australia.

Pathological forms of TAR DNA-binding protein 43 (TDP-43) are present in almost all cases of amyotrophic lateral sclerosis (ALS), and 20% of familial ALS cases are due to mutations in superoxide dismutase 1 (SOD1). Redox regulation is critical to maintain cellular homeostasis, although how this relates to ALS is unclear. Here, we demonstrate that the redox function of protein disulfide isomerase (PDI) is protective against protein misfolding, cytoplasmic mislocalization of TDP-43, ER stress, ER-Golgi transport dysfunction, and apoptosis in neuronal cells expressing mutant TDP-43 or SOD1, and motor impairment in zebrafish expressing mutant SOD1. Moreover, previously described PDI mutants present in patients with ALS (D292N, R300H) lack redox activity and were not protective against ALS phenotypes. Hence, these findings implicate the redox activity of PDI centrally in ALS, linking it to multiple cellular processes. They also imply that therapeutics based on PDI's redox activity will be beneficial in ALS.
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http://dx.doi.org/10.1016/j.isci.2020.101097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7240177PMC
May 2020

Novel distamycin analogues that block the cell cycle of African trypanosomes with high selectivity and potency.

Eur J Med Chem 2020 Mar 8;189:112043. Epub 2020 Jan 8.

Group Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Montevideo, Uruguay. Electronic address:

Polyamides-based compounds related to the Streptomycetal distamycin and netropsin are potent cytostatic molecules that bind to AT-rich regions of the minor groove of the DNA, hence interfering with DNA replication and transcription. Recently, derivatives belonging to this scaffold have been reported to halt the proliferation of deadly African trypanosomes by different and unrelated mechanisms. Here we describe the synthesis and preliminary characterization of the anti-trypanosomal mode of action of new potent and selective distamycin analogues. Two tri-heterocyclic derivatives containing a central N-methyl pyrrole ring (16 and 17) displayed high activity (EC < 20 nM) and selectivity (selectivity index >5000 with respect to mammalian macrophages) against the infective form of T. brucei. Both compounds caused cell cycle arrest by blocking the replication of the mitochondrial DNA but without affecting its integrity. This mode of action clearly differs from that reported for classical minor groove binder (MGB) drugs, which induce the degradation of the mitochondrial DNA. In line with this, in vitro assays suggest that 16 and 17 have a comparatively lower affinity for different template DNAs than the MGB drug diminazene. Therapeutic efficacy studies and stability assays suggest that the pharmacological properties of the hits should be optimized. The compounds can be rated as excellent scaffolds for the design of highly potent and selective anti-T. brucei agents.
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http://dx.doi.org/10.1016/j.ejmech.2020.112043DOI Listing
March 2020

A simple, robust, and affordable bioluminescent assay for drug discovery against infective African trypanosomes.

Drug Dev Res 2020 Jan 20. Epub 2020 Jan 20.

Group Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Montevideo, Uruguay.

African trypanosomiasis is a major problem for human and animal health in endemic countries, where it threatens millions of people and affects economic development. New drugs are needed to overcome the toxicity, administration, low efficacy, and resistance issues of the current chemotherapy. Robust, simple, and economical high-throughput, whole-cell-based assays are required to accelerate the identification of novel chemical entities. With this aim, we generated a bioluminescent cell line of the bloodstream stage of Trypanosoma brucei brucei and established a screening assay. Trypanosomes were stably transfected to constitutively express a thermostable red-shifted luciferase. The growth phenotype and drug sensitivity of the reporter cell line were essentially identical to that of the parental cell line. The endogenous luciferase activity, measured by a simple bioluminescence assay, proved to be proportional to parasite number and metabolic status. The assay, optimized to detect highly potent compounds in a 96-well-plate format, was validated by screening a small compound library (inter-assay values for Z' factor and coefficient variation were 0.77 and 5.8%, respectively). With a hit-confirmation ratio of ~97%, the assay was potent enough to identify several hits with EC  ≤ 10 μM. Preliminary tests indicated that the assay can be scaled up to a 384-well-plate format without compromising its robustness. In summary, we have generated reporter trypanosomes and a simple, robust, and affordable bioluminescence screening assay with great potential to speed up the early-phase drug discovery against African trypanosomes.
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http://dx.doi.org/10.1002/ddr.21634DOI Listing
January 2020

Synthesis of bicyclic 1,4-thiazepines as novel anti- agents.

Medchemcomm 2019 Aug 11;10(8):1481-1487. Epub 2019 Jun 11.

Laboratorio de Química Farmacéutica , Departamento de Química Orgánica , Facultad de Química , Universidad de la República , Montevideo , Uruguay . Email: ; Email:

1,4-Thiazepines derivatives are pharmacologically important heterocycles with different applications in medicinal chemistry. In the present work, we describe the preparation of new bicyclic thiazolidinyl-1,4-thiazepines by reaction between azadithiane compounds and Michael acceptors. The reaction scope was explored and the yields were optimized. The activity of the new compounds was evaluated against and as anthelmintic models and The most active compound was , showing an EC = 2.8 ± 0.7 μM against and a selectivity index >71.
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http://dx.doi.org/10.1039/c9md00064jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6786244PMC
August 2019

An essential thioredoxin-type protein of Trypanosoma brucei acts as redox-regulated mitochondrial chaperone.

PLoS Pathog 2019 09 26;15(9):e1008065. Epub 2019 Sep 26.

Biochemie-Zentrum der Universität Heidelberg (BZH), Heidelberg, Germany.

Most known thioredoxin-type proteins (Trx) participate in redox pathways, using two highly conserved cysteine residues to catalyze thiol-disulfide exchange reactions. Here we demonstrate that the so far unexplored Trx2 from African trypanosomes (Trypanosoma brucei) lacks protein disulfide reductase activity but functions as an effective temperature-activated and redox-regulated chaperone. Immunofluorescence microscopy and fractionated cell lysis revealed that Trx2 is located in the mitochondrion of the parasite. RNA-interference and gene knock-out approaches showed that depletion of Trx2 impairs growth of both mammalian bloodstream and insect stage procyclic parasites. Procyclic cells lacking Trx2 stop proliferation under standard culture conditions at 27°C and are unable to survive prolonged exposure to 37°C, indicating that Trx2 plays a vital role that becomes augmented under heat stress. Moreover, we found that Trx2 contributes to the in vivo infectivity of T. brucei. Remarkably, a Trx2 version, in which all five cysteines were replaced by serine residues, complements for the wildtype protein in conditional knock-out cells and confers parasite infectivity in the mouse model. Characterization of the recombinant protein revealed that Trx2 can coordinate an iron sulfur cluster and is highly sensitive towards spontaneous oxidation. Moreover, we discovered that both wildtype and mutant Trx2 protect other proteins against thermal aggregation and preserve their ability to refold upon return to non-stress conditions. Activation of the chaperone function of Trx2 appears to be triggered by temperature-mediated structural changes and inhibited by oxidative disulfide bond formation. Our studies indicate that Trx2 acts as a novel chaperone in the unique single mitochondrion of T. brucei and reveal a new perspective regarding the physiological function of thioredoxin-type proteins in trypanosomes.
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http://dx.doi.org/10.1371/journal.ppat.1008065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6783113PMC
September 2019

Glucose-6-Phosphate Dehydrogenase from the Human Pathogen Trypanosoma cruzi Evolved Unique Structural Features to Support Efficient Product Formation.

J Mol Biol 2019 05 28;431(11):2143-2162. Epub 2019 Mar 28.

Laboratory Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Mataojo 2020, 11400 Montevideo, Uruguay. Electronic address:

Glucose-6-phosphate dehydrogenase (G6PDH) is the key enzyme supplying reducing power (NADPH) to the cells, by oxidation of glucose-6-phosphate (G6P), and in the process providing a precursor of ribose-5-phosphate. G6PDH is also a virulence factor of pathogenic trypanosomatid parasites. To uncover the biochemical and structural features that distinguish TcG6PDH from its human homolog, we have solved and analyzed the crystal structures of the G6PDH from Trypanosoma cruzi (TcG6PDH), alone and in complex with G6P. TcG6PDH crystallized as a tetramer and enzymatic assays further indicated that the tetramer is the active form in the parasite, in contrast to human G6PDH, which displays higher activity as a dimer. This quaternary structure was shown to be particularly stable. The molecular reasons behind this disparity were unveiled by structural analyses: a TcG6PDH-specific residue, R323, is located at the dimer-dimer interface, critically contributing with two salt bridges per subunit that are absent in the human enzyme. This explains why TcG6PDH dimerization impaired enzyme activity. The parasite protein is also distinct in displaying a 37-amino-acid extension at the N-terminus, which comprises the non-conserved C8 and C34 involved in the covalent linkage of two neighboring protomers. In addition, a cysteine triad (C53, C94 and C135) specific of Kinetoplastid G6PDHs proved critical for stabilization of TcG6PDH active site. Based on the structural and biochemical data, we posit that the N-terminal region and the catalytic site are highly dynamic. The unique structural features of TcG6PDH pave the way toward the design of efficacious and highly specific anti-trypanosomal drugs.
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http://dx.doi.org/10.1016/j.jmb.2019.03.023DOI Listing
May 2019

Kinetic studies reveal a key role of a redox-active glutaredoxin in the evolution of the thiol-redox metabolism of trypanosomatid parasites.

J Biol Chem 2019 03 28;294(9):3235-3248. Epub 2018 Dec 28.

the Laboratorio de Fisicoquímica Biológica and

Trypanosomes are flagellated protozoan parasites (kinetoplastids) that have a unique redox metabolism based on the small dithiol trypanothione (T(SH)). Although GSH may still play a biological role in trypanosomatid parasites beyond being a building block of T(SH), most of its functions are replaced by T(SH) in these organisms. Consequently, trypanosomes have several enzymes adapted to using T(SH) instead of GSH, including the glutaredoxins (Grxs). However, the mechanistic basis of Grx specificity for T(SH) is unknown. Here, we combined fast-kinetic and biophysical approaches, including NMR, MS, and fluorescent tagging, to study the redox function of Grx1, the only cytosolic redox-active Grx in trypanosomes. We observed that Grx1 reduces GSH-containing disulfides (including oxidized trypanothione) in very fast reactions ( > 5 × 10 m s). We also noted that disulfides without a GSH are much slower oxidants, suggesting a strongly selective binding of the GSH molecule. Not surprisingly, oxidized Grx1 was also reduced very fast by T(SH) (4.8 × 10 m s); however, GSH-mediated reduction was extremely slow (39 m s). This kinetic selectivity in the reduction step of the catalytic cycle suggests that Grx1 uses preferentially a dithiol mechanism, forming a disulfide on the active site during the oxidative half of the catalytic cycle and then being rapidly reduced by T(SH) in the reductive half. Thus, the reduction of glutathionylated substrates avoids GSSG accumulation in an organism lacking GSH reductase. These findings suggest that Grx1 has played an important adaptive role during the rewiring of the thiol-redox metabolism of kinetoplastids.
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http://dx.doi.org/10.1074/jbc.RA118.006366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398122PMC
March 2019

Trypanothione synthetase confers growth, survival advantage and resistance to anti-protozoal drugs in Trypanosoma cruzi.

Free Radic Biol Med 2019 01 23;130:23-34. Epub 2018 Oct 23.

Instituto de Patología Experimental, Universidad Nacional de Salta - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Salta, Argentina. Electronic address:

Background: Chagas cardiomyopathy, caused by Trypanosoma cruzi infection, continues to be a neglected illness, and has a major impact on global health. The parasite undergoes several stages of morphological and biochemical changes during its life cycle, and utilizes an elaborated antioxidant network to overcome the oxidants barrier and establish infection in vector and mammalian hosts. Trypanothione synthetase (TryS) catalyzes the biosynthesis of glutathione-spermidine adduct trypanothione (T(SH)) that is the principal intracellular thiol-redox metabolite in trypanosomatids.

Methods And Results: We utilized genetic overexpression (TryS) and pharmacological inhibition approaches to examine the role of TryS in T. cruzi proliferation, tolerance to oxidative stress and resistance to anti-protozoal drugs. Our data showed the expression and activity of TryS was increased in all morphological stages of TryS (vs. control) parasites. In comparison to controls, the TryS epimastigotes (insect stage) recorded shorter doubling time, and both epimastigotes and infective trypomastigotes of TryS exhibited 36-71% higher resistance to HO (50-1000 μM) and heavy metal (1-500 μM) toxicity. Treatment with TryS inhibitors (5-30 μM) abolished the proliferation and survival advantages against HO pressure in a dose-dependent manner in both TryS and control parasites. Further, epimastigote and trypomastigote forms of TryS (vs. control) T. cruzi tolerated higher doses of benznidazole and nifurtimox, the drugs currently administered for acute Chagas disease treatment.

Conclusions: TryS is essential for proliferation and survival of T. cruzi under normal and oxidant stress conditions, and provides an advantage to the parasite to develop resistance against currently used anti-trypanosomal drugs. TryS indispensability has been chemically validated with inhibitors that may be useful for drug combination therapy against Chagas disease.
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http://dx.doi.org/10.1016/j.freeradbiomed.2018.10.436DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331241PMC
January 2019

The lineage-specific, intrinsically disordered N-terminal extension of monothiol glutaredoxin 1 from trypanosomes contains a regulatory region.

Sci Rep 2018 09 12;8(1):13716. Epub 2018 Sep 12.

Department of Chemical Sciences, University of Padova, via Marzolo 1, 35131, Padova, Italy.

Glutaredoxins (Grx) are small proteins conserved throughout all the kingdoms of life that are engaged in a wide variety of biological processes and share a common thioredoxin-fold. Among them, class II Grx are redox-inactive proteins involved in iron-sulfur (FeS) metabolism. They contain a single thiol group in their active site and use low molecular mass thiols such as glutathione as ligand for binding FeS-clusters. In this study, we investigated molecular aspects of 1CGrx1 from the pathogenic parasite Trypanosoma brucei brucei, a mitochondrial class II Grx that fulfills an indispensable role in vivo. Mitochondrial 1CGrx1 from trypanosomes differs from orthologues in several features including the presence of a parasite-specific N-terminal extension (NTE) whose role has yet to be elucidated. Previously we have solved the structure of a truncated form of 1CGrx1 containing only the conserved glutaredoxin domain but lacking the NTE. Our aim here is to investigate the effect of the NTE on the conformation of the protein. We therefore solved the NMR structure of the full-length protein, which reveals subtle but significant differences with the structure of the NTE-less form. By means of different experimental approaches, the NTE proved to be intrinsically disordered and not involved in the non-redox dependent protein dimerization, as previously suggested. Interestingly, the portion comprising residues 65-76 of the NTE modulates the conformational dynamics of the glutathione-binding pocket, which may play a role in iron-sulfur cluster assembly and delivery. Furthermore, we disclosed that the class II-strictly conserved loop that precedes the active site is critical for stabilizing the protein structure. So far, this represents the first communication of a Grx containing an intrinsically disordered region that defines a new protein subgroup within class II Grx.
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http://dx.doi.org/10.1038/s41598-018-31817-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6135854PMC
September 2018

A glutaredoxin in the mitochondrial intermembrane space has stage-specific functions in the thermo-tolerance and proliferation of African trypanosomes.

Redox Biol 2018 05 31;15:532-547. Epub 2018 Jan 31.

Biochemie-Zentrum der Universität Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany. Electronic address:

Trypanosoma brucei glutaredoxin 2 (Grx2) is a dithiol glutaredoxin that is specifically located in the mitochondrial intermembrane space. Bloodstream form parasites lacking Grx2 or both, Grx2 and the cytosolic Grx1, are viable in vitro and infectious to mice suggesting that neither oxidoreductase is needed for survival or infectivity to mammals. A 37 °C to 39 °C shift changes the cellular redox milieu of bloodstream cells to more oxidizing conditions and induces a significantly stronger growth arrest in wildtype parasites compared to the mutant cells. Grx2-deficient cells ectopically expressing the wildtype form of Grx2 with its C31QFC34 active site, but not the C34S mutant, regain the sensitivity of the parental strain, indicating that the physiological role of Grx2 requires both active site cysteines. In the procyclic insect stage of the parasite, Grx2 is essential. Both alleles can be replaced if procyclic cells ectopically express authentic or C34S, but not C31S/C34S Grx2, pointing to a redox role that relies on a monothiol mechanism. RNA-interference against Grx2 causes a virtually irreversible proliferation defect. The cells adopt an elongated morphology but do not show any significant alteration in the cell cycle. The growth retardation is attenuated by high glucose concentrations. Under these conditions, procyclic cells obtain ATP by substrate level phosphorylation suggesting that Grx2 might regulate a respiratory chain component.
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http://dx.doi.org/10.1016/j.redox.2018.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5975080PMC
May 2018

Alternative Thiol-Based Redox Systems.

Antioxid Redox Signal 2018 02 5;28(6):407-409. Epub 2018 Jan 5.

3 Redox Biology of Trypanosomes Lab, Institut Pasteur de Montevideo , Montevideo, Uruguay .

The maintenance of thiol-redox homeostasis is vital to the survival of living organisms. Sulfur-based low-molecular weight compounds and proteins synthesized by cells provide efficient and specific ways to counteract oxidative stress and regulate cellular processes. For these tasks, most organisms share the glutathione and thioredoxin NADPH-dependent redox systems. However, in certain lineages, evolution has taken different paths that led to the emergence of novel cysteine-based low-molecular weight redox cofactors, around which new redox systems evolved. These include the sugar-based cysteinyl derivatives mycothiol and bacillithiol, and ergothioneine (EGT), which are present in different phyla from bacteria. Within Eukarya, some fungi contain EGT, whereas trypanothione is unique to species from the Euglenozoa family. This Forum compiles the state-of-the-art knowledge about these noncanonical redox systems of pathogenic organisms. The functions in physiology and pathogenicity, as well as structural and biochemical specializations that these system components evolved, are thoroughly discussed. Antioxid. Redox Signal. 28, 407-409.
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http://dx.doi.org/10.1089/ars.2017.7464DOI Listing
February 2018

Polyamine-Based Thiols in Trypanosomatids: Evolution, Protein Structural Adaptations, and Biological Functions.

Antioxid Redox Signal 2018 02 27;28(6):463-486. Epub 2017 Nov 27.

1 Laboratory Redox Biology of Trypanosomes, Institut Pasteur de Montevideo , Montevideo, Uruguay .

Significance: Major pathogenic enterobacteria and protozoan parasites from the phylum Euglenozoa, such as trypanosomatids, are endowed with glutathione (GSH)-spermidine (Sp) derivatives that play important roles in signaling and metal and thiol-redox homeostasis. For some Euglenozoa lineages, the GSH-Sp conjugates represent the main redox cosubstrates around which entire new redox systems have evolved. Several proteins underwent molecular adaptations to synthesize and utilize the new polyamine-based thiols. Recent Advances: The genomes of closely related organisms have recently been sequenced, which allows mining and analysis of gene sequences that belong to these peculiar redox systems. Similarly, the three-dimensional structures of several of these proteins have been solved, which allows for comparison with their counterparts in classical redox systems that rely on GSH/glutaredoxin and thioredoxin.

Critical Issues: The evolutionary and structural aspects related to the emergence and use of GSH-Sp conjugates in Euglenozoa are reviewed focusing on unique structural specializations that proteins developed to use N,N-bisglutathionylspermidine (trypanothione) as redox cosubstrate. An updated overview on the biochemical and biological significance of the major enzymatic activities is also provided.

Future Directions: A thiol-redox system strictly dependent on trypanothione is a feature unique to trypanosomatids. The physicochemical properties of the polyamine-GSH conjugates were a major driving force for structural adaptation of proteins that use these thiols as ligand and redox cofactor. In fact, the structural differences of indispensable components of this system can be exploited toward selective drug development. Future research should clarify whether additional cellular processes are regulated by the trypanothione system. Antioxid. Redox Signal. 28, 463-486.
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http://dx.doi.org/10.1089/ars.2017.7133DOI Listing
February 2018

Diglycosyl diselenides alter redox homeostasis and glucose consumption of infective African trypanosomes.

Int J Parasitol Drugs Drug Resist 2017 12 12;7(3):303-313. Epub 2017 Aug 12.

Group Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Mataojo 2020, 11400 Montevideo, Uruguay. Electronic address:

With the aim to develop compounds able to target multiple metabolic pathways and, thus, to lower the chances of drug resistance, we investigated the anti-trypanosomal activity and selectivity of a series of symmetric diglycosyl diselenides and disulfides. Of 18 compounds tested the fully acetylated forms of di-β-D-glucopyranosyl and di-β-D-galactopyranosyl diselenides (13 and 15, respectively) displayed strong growth inhibition against the bloodstream stage of African trypanosomes (EC 0.54 μM for 13 and 1.49 μM for 15) although with rather low selectivity (SI < 10 assayed with murine macrophages). Nonacetylated versions of the same sugar diselenides proved to be, however, much less efficient or completely inactive to suppress trypanosome growth. Significantly, the galactosyl (15), and to a minor extent the glucosyl (13), derivative inhibited glucose catabolism but not its uptake. Both compounds induced redox unbalance in the pathogen. In vitro NMR analysis indicated that diglycosyl diselenides react with glutathione, under physiological conditions, via formation of selenenylsulfide bonds. Our results suggest that non-specific cellular targets as well as actors of the glucose and the redox metabolism of the parasite may be affected. These molecules are therefore promising leads for the development of novel multitarget antitrypanosomal agents.
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http://dx.doi.org/10.1016/j.ijpddr.2017.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5565762PMC
December 2017

Ornithine decarboxylase or gamma-glutamylcysteine synthetase overexpression protects Leishmania (Vianna) guyanensis against antimony.

Exp Parasitol 2017 Apr 3;175:36-43. Epub 2017 Feb 3.

Laboratório de Parasitologia Celular e Molecular, Centro de Pesquisas René Rachou CPqRR/Fiocruz, Av. Augusto de Lima 1715 301190-002, Belo Horizonte, MG, Brazil. Electronic address:

Trypanosomatids present a unique mechanism for detoxification of peroxides that is dependent on trypanothione (bisglutathionylspermidine). Ornithine decarboxylase (ODC) and γ-glutamylcysteine synthetase (GSH1) produce molecules that are direct precursors of trypanothione. In this study, Leishmania guyanensis odc and gsh1 overexpressor cell lines were generated to investigate the contribution of these genes to the trivalent antimony (Sb)-resistance phenotype. The ODC- or GSH1-overexpressors parasites presented an increase of two and four-fold in Sb-resistance index, respectively, when compared with the wild-type line. Pharmacological inhibition of ODC and GSH1 with the specific inhibitors α-difluoromethylornithine (DFMO) and buthionine sulfoximine (BSO), respectively, increased the antileishmanial effect of Sb in all cell lines. However, the ODC- and GSH1-overexpressor were still more resistant to Sb than the parental cell line. Together, our data shows that modulation of ODC and GSH1 levels and activity is sufficient to affect L. guyanensis susceptibility to Sb, and confirms a role of these genes in the Sb-resistance phenotype.
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http://dx.doi.org/10.1016/j.exppara.2017.02.001DOI Listing
April 2017

In vitro activity and mode of action of distamycin analogues against African trypanosomes.

Eur J Med Chem 2017 Jan 2;126:776-788. Epub 2016 Dec 2.

Laboratorio de Química Farmacéutica, Departamento de Química Orgánica, Facultad de Química, Universidad de la República, Montevideo, Uruguay. Electronic address:

Distamycin, a natural polyamide containing three heterocycle rings with a polar end, has inspired several groups to prepare synthetic analogues, which proved to have anti-trypanosomal and anti-tumoral activity. We describe the synthesis of bi and tri thiazoles amides that harbor different substitutions at their ends and the evaluation of their anti-Trypanosoma brucei activity. The most active compound 10b showed better biological activity (EC 310 nM and selectivity index 16) than the control drug nifurtimox (EC 15 μM and selectivity index 10). Studies on the mode of action show that the parasiticidal activity of 10b originates from disruption of lysosomal homeostasis, which is followed by release of redox active iron, an increase in oxidizing species and collapse of cell membrane integrity. In this respect, our study suggests that non-charged lipophylic distamycins destabilize cell membranes.
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http://dx.doi.org/10.1016/j.ejmech.2016.12.002DOI Listing
January 2017

5-Substituted 3-chlorokenpaullone derivatives are potent inhibitors of Trypanosoma brucei bloodstream forms.

Bioorg Med Chem 2016 08 13;24(16):3790-800. Epub 2016 Jun 13.

Technische Universität Braunschweig, Institut für Medizinische und Pharmazeutische Chemie, Beethovenstraße 55, D-38106 Braunschweig, Germany; Technische Universität Braunschweig, Center of Pharmaceutical Engineering (PVZ), Franz-Liszt-Straße 35A, D-38106 Braunschweig, Germany. Electronic address:

Trypanothione synthetase is an essential enzyme for kinetoplastid parasites which cause highly disabling and fatal diseases in humans and animals. Inspired by the observation that N(5)-substituted paullones inhibit the trypanothione synthetase from the related parasite Leishmania infantum, we designed and synthesized a series of new derivatives. Although none of the new compounds displayed strong inhibition of Trypanosoma brucei trypanothione synthetase, several of them caused a remarkable growth inhibition of cultivated Trypanosoma brucei bloodstream forms. The most potent congener 3a showed antitrypanosomal activity in double digit nanomolar concentrations and a selectivity index of three orders of magnitude versus murine macrophage cells.
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http://dx.doi.org/10.1016/j.bmc.2016.06.023DOI Listing
August 2016

Identification of Novel Chemical Scaffolds Inhibiting Trypanothione Synthetase from Pathogenic Trypanosomatids.

PLoS Negl Trop Dis 2016 Apr 12;10(4):e0004617. Epub 2016 Apr 12.

Laboratory Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Montevideo, Uruguay.

Background: The search for novel chemical entities targeting essential and parasite-specific pathways is considered a priority for neglected diseases such as trypanosomiasis and leishmaniasis. The thiol-dependent redox metabolism of trypanosomatids relies on bis-glutathionylspermidine [trypanothione, T(SH)2], a low molecular mass cosubstrate absent in the host. In pathogenic trypanosomatids, a single enzyme, trypanothione synthetase (TryS), catalyzes trypanothione biosynthesis, which is indispensable for parasite survival. Thus, TryS qualifies as an attractive drug target candidate.

Methodology/principal Finding: A library composed of 144 compounds from 7 different families and several singletons was screened against TryS from three major pathogen species (Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum). The screening conditions were adjusted to the TryS´ kinetic parameters and intracellular concentration of substrates corresponding to each trypanosomatid species, and/or to avoid assay interference. The screening assay yielded suitable Z' and signal to noise values (≥0.85 and ~3.5, respectively), and high intra-assay reproducibility. Several novel chemical scaffolds were identified as low μM and selective tri-tryp TryS inhibitors. Compounds displaying multi-TryS inhibition (N,N'-bis(3,4-substituted-benzyl) diamine derivatives) and an N5-substituted paullone (MOL2008) halted the proliferation of infective Trypanosoma brucei (EC50 in the nM range) and Leishmania infantum promastigotes (EC50 = 12 μM), respectively. A bis-benzyl diamine derivative and MOL2008 depleted intracellular trypanothione in treated parasites, which confirmed the on-target activity of these compounds.

Conclusions/significance: Novel molecular scaffolds with on-target mode of action were identified as hit candidates for TryS inhibition. Due to the remarkable species-specificity exhibited by tri-tryp TryS towards the compounds, future optimization and screening campaigns should aim at designing and detecting, respectively, more potent and broad-range TryS inhibitors.
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http://dx.doi.org/10.1371/journal.pntd.0004617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4829233PMC
April 2016

Binding Mode and Selectivity of Steroids towards Glucose-6-phosphate Dehydrogenase from the Pathogen Trypanosoma cruzi.

Molecules 2016 Mar 17;21(3):368. Epub 2016 Mar 17.

Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Mataojo 2020, Montevideo 11400, Uruguay.

Glucose-6-phosphate dehydrogenase (G6PDH) plays a housekeeping role in cell metabolism by generating reducing power (NADPH) and fueling the production of nucleotide precursors (ribose-5-phosphate). Based on its indispensability for pathogenic parasites from the genus Trypanosoma, G6PDH is considered a drug target candidate. Several steroid-like scaffolds were previously reported to target the activity of G6PDH. Epiandrosterone (EA) is an uncompetitive inhibitor of trypanosomal G6PDH for which its binding site to the enzyme remains unknown. Molecular simulation studies with the structure of Trypanosoma cruzi G6PDH revealed that EA binds in a pocket close to the G6P binding-site and protrudes into the active site blocking the interaction between substrates and hence catalysis. Site directed mutagenesis revealed the important steroid-stabilizing effect of residues (L80, K83 and K84) located on helix α-1 of T. cruzi G6PDH. The higher affinity and potency of 16α-Br EA by T. cruzi G6PDH is explained by the formation of a halogen bond with the hydrogen from the terminal amide of the NADP+-nicotinamide. At variance with the human enzyme, the inclusion of a 21-hydroxypregnane-20-one moiety to a 3β-substituted steroid is detrimental for T. cruzi G6PDH inhibition. The species-specificity of certain steroid derivatives towards the parasite G6PDH and the corresponding biochemically validated binding models disclosed in this work may prove valuable for the development of selective inhibitors against the pathogen's enzyme.
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http://dx.doi.org/10.3390/molecules21030368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6273692PMC
March 2016

Selenoproteins of African trypanosomes are dispensable for parasite survival in a mammalian host.

Mol Biochem Parasitol 2016 Mar-Apr;206(1-2):13-9. Epub 2016 Mar 11.

Redox Biology of Trypanosomes Laboratory, Institut Pasteur de Montevideo, Uruguay. Electronic address:

The trace element selenium is found in polypeptides as selenocysteine, the 21(st) amino acid that is co-translationally inserted into proteins at a UGA codon. In proteins, selenocysteine usually plays a role as an efficient redox catalyst. Trypanosomatids previously examined harbor a full set of genes encoding the machinery needed for selenocysteine biosynthesis and incorporation into three selenoproteins: SelK, SelT and, the parasite-specific, Seltryp. We investigated the selenoproteome of kinetoplastid species in recently sequenced genomes and assessed the in vivo relevance of selenoproteins for African trypanosomes. Database mining revealed that SelK, SelT and Seltryp genes are present in most kinetoplastids, including the free-living species Bodo saltans, and Seltryp was lost in the subgenus Viannia from the New World Leishmania. Homology and sinteny with bacterial sulfur dioxygenases and sulfur transferases suggest a putative role for Seltryp in sulfur metabolism. A Trypanosoma brucei selenocysteine synthase (SepSecS) null-mutant, in which selenoprotein synthesis is abolished, displayed similar sensitivity to oxidative stress induced by a short-term exposure to high concentrations of methylglyoxal or H2O2 to that of the parental wild-type cell line. Importantly, the infectivity of the SepSecS knockout cell line was not impaired when tested in a mouse infection model and compensatory effects via up-regulation of proteins involved in thiol-redox metabolism were not observed. Collectively, our data show that selenoproteins are not required for survival of African trypanosomes in a mammalian host and exclude a role for selenoproteins in parasite antioxidant defense and/or virulence. On this basis, selenoproteins can be disregarded as drug target candidates.
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http://dx.doi.org/10.1016/j.molbiopara.2016.03.002DOI Listing
July 2017

Glutaredoxin-deficiency confers bloodstream Trypanosoma brucei with improved thermotolerance.

Mol Biochem Parasitol 2015 Dec 6;204(2):93-105. Epub 2016 Feb 6.

Biochemie-Zentrum der Universität Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany. Electronic address:

As constituents of their unusual trypanothione-based thiol metabolism, African trypanosomes express two dithiol glutaredoxins (Grxs), a cytosolic Grx1 and a mitochondrial Grx2, with so far unknown biological functions. As revealed by gel shift assays, in the mammalian bloodstream form of Trypanosoma brucei, Grx1 is in the fully reduced state. Upon diamide treatment of the cells, Grx1 forms an active site disulfide bridge that is rapidly re-reduced after stress removal; Cys76, a conserved non-active site Cys remains in the thiol state. Deletion of both grx1 alleles does not result in any proliferation defect of neither the procyclic insect form nor the bloodstream form, even not under various stress conditions. In addition, the Grx1-deficient parasites are fully infectious in the mouse model. A functional compensation by Grx2 is unlikely as identical levels of Grx2 were found in wildtype and Grx1-deficient cells. In the classical hydroxyethyl disulfide assay, Grx1-deficient bloodstream cells display 50-60% of the activity of wildtype cells indicating that the cytosolic oxidoreductase accounts for a major part of the total deglutathionylation capacity of the parasite. Intriguingly, at elevated temperature, proliferation of the Grx1-deficient bloodstream parasites is significantly less affected compared to wildtype cells. When cultured for three days at 39°C, only 51% of the cells in the wildtype population retained normal morphology with single mitochondrial and nuclear DNA (1K1N), whereas 27% of the cells displayed ≥2K2N. In comparison, 64% of the Grx1-deficient cells kept the 1K1N phenotype and only 18% had ≥2K2N. The data suggest that Grx1 plays a role in the regulation of the thermotolerance of the parasites by (in)directly interfering with the progression of the cell cycle, a process that may comprise protein (de)glutathionylation step(s).
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http://dx.doi.org/10.1016/j.molbiopara.2016.02.001DOI Listing
December 2015

Measurement and meaning of cellular thiol:disufhide redox status.

Authors:
Marcelo A Comini

Free Radic Res 2016 ;50(2):246-71

a Laboratory Redox Biology of Trypanosomes , Institut Pasteur de Montevideo , Mataojo 2020 , Montevideo , Uruguay.

The functional group of cysteine is a thiol group (SH) that, due to its chemical reactivity, is able to undergo a wide array of modifications each with the potential to confer a different property or function to the molecule harboring this residue. Most of these modifications involve the reversible oxidation of the thiol to sulfenic acid (SOH), and disulfide, including intra- and intermolecular disulfides between polypeptides and glutathione (glutathionylation). The reversibility of these oxidations allows thiol groups to serve as versatile chemical and structural transducing elements in several low molecular mass metabolites and proteins. A plethora of cellular functions such as DNA and protein synthesis, protein secretion, cytoskeleton architecture, differentiation, apoptosis, and anti-oxidant defense, are recognized to be modulated, at certain stage, by thiol-disulfide exchange mechanisms of redox active thiol groups. All organisms are equipped with enzymatic systems composed by NADPH-dependent reductases, redoxins, and peroxidases that provide kinetic control of global thiol-redox homeostasis as well as target selectivity. These redox systems are distributed in different subcellular compartments and are not in equilibrium with each other. In consequence, measuring cellular thiol-disulfide status represents a challenge for studies aimed to obtain dynamic and spatio-temporal resolution. This review provides a summary of the methods and tools available to quantify the thiol redox status of cells.
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http://dx.doi.org/10.3109/10715762.2015.1110241DOI Listing
October 2016

A New Class of Thioredoxin-Related Protein Able to Bind Iron-Sulfur Clusters.

Antioxid Redox Signal 2016 01 27;24(4):205-216. Epub 2015 Oct 27.

1 Worm Biology Laboratory, Institut Pasteur de Montevideo, Montevideo, Uruguay .

Aims: Members of the thioredoxin (Trx) protein family participate mainly in redox pathways and have not been associated with Fe/S binding, in contrast to some closely related glutaredoxins (Grxs). Cestode parasites possess an unusual diversity of Trxs and Trx-related proteins with unexplored functions. In this study, we addressed the biochemical characterization of a new class of Trx-related protein (IsTRP) and a classical monothiol Grx (EgGrx5) from the human pathogen Echinococcus granulosus.

Results: The dimeric form of IsTRP coordinates FeS in a glutathione-independent manner; instead, Fe/S binding relies on the CXXC motif conserved among Trxs. This novel binding mechanism allows holo-IsTRP to be highly resistant to oxidation. IsTRP lacks canonical reductase activities. Mitochondrially targeted IsTRP aids growth of a Grx5 null yeast strain. Similar complementation assays performed with EgGrx5 revealed functional conservation for class II Grxs, despite the presence of nonconserved structural elements. IsTRP is a cestode lineage-specific protein highly expressed in the gravid adult worm, which releases the infective stage critical for dissemination.

Innovation: IsTRP is the first member from the Trx family to be reported to bind Fe/S. We disclose a novel mechanism of Fe/S coordination within the Trx folding unit, which renders the cluster highly resistant to oxidation-mediated disassembly.

Conclusion: We demonstrate that IsTRP defines a new protein family within the Trx superfamily, confirm the conservation of function for class II Grx from nonphylogenetically related species, and highlight the versatility of the Trx folding unit to acquire Fe/S binding as a recurrent emergent function. Antioxid. Redox Signal. 00, 000-000.
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http://dx.doi.org/10.1089/ars.2015.6377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6913166PMC
January 2016

Antitumor and antiparasitic activity of novel ruthenium compounds with polycyclic aromatic ligands.

J Inorg Biochem 2015 Sep 9;150:38-47. Epub 2015 Jun 9.

Departament de Química Inorgànica, Universitat de Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain. Electronic address:

Five novel ruthenium(II)-arene complexes with polycyclic aromatic ligands were synthesized, comprising three compounds of the formula [RuCl(η(6)-p-cym)(L)][PF6], where p-cym = 1-isopropyl-4-methylbenzene and L are the bidentate aromatic ligands 1,10-phenanthroline-5,6-dione, 1, 5-amine-1,10-phenanthroline, 4, or 5,6-epoxy-5,6-dihydro-phenanthroline, 5. In the other two complexes [RuCl2(η(6)-p-cym)(L')], the metal is coordinated to a monodentate ligand L', where L' is phenanthridine, 2, or 9-carbonylanthracene, 3. All compounds were fully characterized by mass spectrometry and elemental analysis, as well as NMR and IR spectroscopic techniques. Obtained ruthenium compounds as well as their respective ligands were tested for their antiparasitic and antitumoral activities. Even though all compounds showed lower Trypanosoma brucei activity than the free ligands, they also resulted less toxic on mammalian cells. Cytotoxicity assays on HL60 cells showed a moderate antitumoral activity for all ruthenium compounds. Compound 1 was the most potent antitumoral (IC50 = 1.26±0.78 μM) and antiparasitic (IC50 = 0.19 ± 0.05 μM) agent, showing high selectivity towards the parasites (selectivity index >100). As complex 1 was the most promising antitumoral compound, its interaction with ubiquitin as potential target was also studied. In addition, obtained ruthenium compounds were found to bind DNA, and they are thought to interact with this macromolecule mainly through intercalation of the aromatic ligand.
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http://dx.doi.org/10.1016/j.jinorgbio.2015.06.007DOI Listing
September 2015

9- and 11-Substituted 4-azapaullones are potent and selective inhibitors of African trypanosoma.

Eur J Med Chem 2014 Aug 11;83:274-83. Epub 2014 Jun 11.

Technische Universität Braunschweig, Institut für Medizinische und Pharmazeutische Chemie, Beethovenstraße 55, D-38106 Braunschweig, Germany. Electronic address:

Trypanosomes from the "brucei" complex are pathogenic parasites endemic in sub-Saharan Africa and causative agents of severe diseases in humans and livestock. In order to identify new antitrypanosomal chemotypes against African trypanosomes, 4-azapaullones carrying α,β-unsaturated carbonyl chains in 9- or 11-position were synthesized employing a procedure with a Heck reaction as key step. Among the so prepared compounds, 5a and 5e proved to be potent antiparasitic agents with antitrypanosomal activity in the submicromolar range.
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http://dx.doi.org/10.1016/j.ejmech.2014.06.020DOI Listing
August 2014

Genetic and chemical analyses reveal that trypanothione synthetase but not glutathionylspermidine synthetase is essential for Leishmania infantum.

Free Radic Biol Med 2014 Aug 20;73:229-38. Epub 2014 May 20.

IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, 4150-180 Porto, Portugal. Electronic address:

Trypanothione is a unique and essential redox metabolite of trypanosomatid parasites, the biosynthetic pathway of which is regarded as a promising target for antiparasitic drugs. Synthesis of trypanothione occurs by the consecutive conjugation of two glutathione molecules to spermidine. Both reaction steps are catalyzed by trypanothione synthetase (TRYS), a molecule known to be essential in Trypanosoma brucei. However, other trypanosomatids (including some Leishmania species and Trypanosoma cruzi) potentially express one additional enzyme, glutathionylspermidine synthetase (GSPS), capable of driving the first step of trypanothione synthesis yielding glutathionylspermidine. Because this monothiol can substitute for trypanothione in some reactions, the possibility existed that TRYS was redundant in parasites harboring GSPS. To clarify this issue, the functional relevance of both GSPS and TRYS was investigated in Leishmania infantum (Li). Employing a gene-targeting approach, we generated a gsps(-/-) knockout line, which was viable and capable of replicating in both life cycle stages of the parasite, thus demonstrating the superfluous role of LiGSPS. In contrast, elimination of both LiTRYS alleles was not possible unless parasites were previously complemented with an episomal copy of the gene. Retention of extrachromosomal LiTRYS in the trys(-/-)/+TRYS line after several passages in culture further supported the essentiality of this gene for survival of L. infantum (including its clinically relevant stage), hence ruling out the hypothesis of functional complementation by LiGSPS. Chemical targeting of LiTRYS with a drug-like compound was shown to also lead to parasite death. Overall, this study disqualifies GSPS as a target for drug development campaigns and, by genetic and chemical evidence, validates TRYS as a chemotherapeutic target in a parasite endowed with GSPS and, thus, probably along the entire trypanosomatid lineage.
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http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.007DOI Listing
August 2014

(1)H, (13)C and (15)N resonance assignment of the mature form of monothiol glutaredoxin 1 from the pathogen Trypanosoma brucei.

Biomol NMR Assign 2015 Apr 16;9(1):143-6. Epub 2014 May 16.

Department of Chemical Sciences, University of Padova, Via Marzolo 1, 35131, Padova, Italy.

Glutaredoxins (Grx) are small proteins, conserved throughout all the kingdoms of life, which are engaged in a wide variety of biological processes. According to the number of cysteines in their active site, Grx are classified as dithiolic or monothiolic (1-C-Grx). In most organisms, 1-C-Grx are implicated in iron-sulfur cluster (FeS) metabolism and utilize glutathione as cofactor. Trypanosomatids are parasitic protozoa of the order Kinetoplastida, which cause severe diseases in humans and domestic animals. These parasites exploit a unique thiol-dependent redox system based on bis(glutathionyl)spermidine (trypanothione) rather than on glutathione. Mitochondrial 1-C-Grx1 from trypanosomes differs from orthologues in several features including the use of trypanothione as ligand for FeS binding and the presence of a parasite-specific N-terminal extension. We have recently shown that 1-C-Grx1 from Trypanosoma brucei is indispensable for parasite survival in mouse, making this protein a potential drug target candidate against trypanosomiasis. However, structural information for the full-length form of 1-C-Grx1 is still lacking. Here, we report the NMR resonance assignment of the mature form of Tb1-C-Grx1 including an N-terminal tail, paving the way to disclose the role of this intrinsically disordered region in the protein function.
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http://dx.doi.org/10.1007/s12104-014-9561-3DOI Listing
April 2015