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Cristina M Nonato - One of the best experts on this subject based on the ideXlab platform.
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natural products as inhibitors of Leishmania Major dihydroorotate dehydrogenase
European Journal of Medicinal Chemistry, 2018Co-Authors: Lucas A Chibli, Cristina M Nonato, Thomas J Schmidt, Felipe A Calil, Fernando B Da CostaAbstract:Abstract The flavoenzyme dihydroorotate dehydrogenase (DHODH) catalyzes the fourth reaction of the de novo pyrimidine biosynthetic pathway, which exerts vital functions in the cells, especially within DNA and RNA biosynthesis. Thus, this enzyme stands out as a new key molecular target for parasites causing Neglected Diseases (NDs). Focused on contributing to the development of new therapeutic alternatives for NDs, in this study, for the first time, a screening of 57 natural products for in vitro inhibition of Leishmania Major DHODH (LmDHODH) was carried out, including cross validation against the human DHODH (HsDHODH). A subset of natural products consisting of 21 sesquiterpene lactones (STLs) was submitted to QSAR studies. Additionally, thermostability studies by differential scanning fluorimetry (DSF) were performed to determine whether the STLs are effectively or not binding to the enzyme. The IC50 values against LmDHODH varied from 27 to 1200 μM; only irrelevant inhibition was obtained on HsDHODH. DSF assays confirmed binding of STLs to LmDHODH; moreover, it is suggested that such inhibitors might act in a different site other than the active site. A reliable QSAR model based on molecular descriptors was obtained (R2: 0.83; Q2CV: 0.69 and Q2EXT/F2: 0.66) indicating that stronger inhibition requires a balanced distribution of the hydrophobic regions across the molecular surface, as well as higher width and lower hydrophobicity of the molecules. A pharmacophore-based 3D-QSAR approach also afforded a useful model (R2: 0.72; Q2CV: 0.50 and Q2EXT/F2: 0.62), which confirmed the importance of proper orientation of the ligands, molecular surface features and shape for stronger inhibition, reflecting properties of a putative common binding site. These data indicated for the first time that natural products can actually inhibit LmDHODH and highlighted some metabolites as potentially interesting starting points for the discovery of more potent LmDHODH inhibitors, ultimately aiming at new effective therapeutic alternatives for Leishmaniasis and, possibly, other NDs caused by trypanosomatids.
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crystal structure of dihydroorotate dehydrogenase from Leishmania Major
Biochimie, 2012Co-Authors: Artur T Cordeiro, Patricia R Feliciano, Matheus Pinto Pinheiro, Cristina M NonatoAbstract:Dihydroorotate dehydrogenase (DHODH) is the fourth enzyme in the de novo pyrimidine biosynthetic pathway and has been exploited as the target for therapy against proliferative and parasitic diseases. In this study, we report the crystal structures of DHODH from Leishmania Major, the species of Leishmania associated with zoonotic cutaneous Leishmaniasis, in its apo form and in complex with orotate and fumarate molecules. Both orotate and fumarate were found to bind to the same active site and exploit similar interactions, consistent with a ping-pong mechanism described for class 1A DHODHs. Analysis of LmDHODH structures reveals that rearrangements in the conformation of the catalytic loop have direct influence on the dimeric interface. This is the first structural evidence of a relationship between the dimeric form and the catalytic mechanism. According to our analysis, the high sequence and structural similarity observed among trypanosomatid DHODH suggest that a single strategy of structure-based inhibitor design can be used to validate DHODH as a druggable target against multiple neglected tropical diseases such as Leishmaniasis, Sleeping sickness and Chagas' diseases.
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fumarate hydratase isoforms of Leishmania Major subcellular localization structural and kinetic properties
International Journal of Biological Macromolecules, 2012Co-Authors: Patricia R Feliciano, Antonio J Costafilho, Shreedhara Gupta, Fabio H Dyszy, Marcelo Diasbaruffi, Paul A M Michels, Cristina M NonatoAbstract:Fumarate hydratases (FHs; EC 4.2.1.2) are enzymes that catalyze the reversible hydration of fumarate to S-malate. Parasitic protists that belong to the genus Leishmania and are responsible for a complex of vector-borne diseases named Leishmaniases possess two genes that encode distinct putative FH enzymes. Genome sequence analysis of Leishmania Major Friedlin reveals the existence of genes LmjF24.0320 and LmjF29.1960 encoding the putative enzymes LmFH-1 and LmFH-2, respectively. In the present work, the FH activity of both L. Major enzymes has been confirmed. Circular dichroism studies suggest important differences in terms of secondary structure content when comparing LmFH isoforms and even larger differences when comparing them to the homologous human enzyme. CD melting experiments revealed that both LmFH isoforms are thermolabile enzymes. The catalytic efficiency under aerobic and anaerobic environments suggests that they are both highly sensitive to oxidation and damaged by oxygen. Intracellular localization studies located LmFH-1 in the mitochondrion, whereas LmFH-2 was found predominantly in the cytosol with possibly also some in glycosomes. The high degree of sequence conservation in different Leishmania species, together with the relevance of FH activity for the energy metabolism in these parasites suggest that FHs might be exploited as targets for broad-spectrum antiLeishmanial drugs.
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cloning expression purification and characterization of Leishmania Major dihydroorotate dehydrogenase
Protein Expression and Purification, 2006Co-Authors: Patricia R Feliciano, Artur T Cordeiro, Antonio J Costafilho, Cristina M NonatoAbstract:Abstract Leishmania Major Friedlin (LmjF) is a protozoan parasite whose genomic sequence has been recently elucidated. Here we have cloned, overexpressed, purified, and characterized the product of the gene from LmjF chromosome 16: LmjF16.0530, which encodes a protein with putative dihydroorotate dehydrogenase activity. Dihydroorotate dehydrogenase (DHODH) is a flavoprotein that catalyses the oxidation of l -dihydroorotate to orotate, the fourth sequential step in the de novo pyrimidine nucleotide synthesis pathway. The predicted enzyme from L. Major was cloned and expressed in Escherichia coli strain BL21(DE3) as a histidine-tag fusion protein and purified to homogeneity using affinity chromatography. The final product was homogeneous in SDS–PAGE gel electrophoresis. The dihydroorotate oxidase activity has been assayed and the steady-state kinetic mechanism has been determined using fumarate as the oxidizing substrate. The catalysis by LmDHODH enzyme proceeds by a Ping-Pong Bi–Bi mechanism and the kinetic parameters K m were calculated to be 90 and 418 μM for dihydroorotate and fumarate, respectively, and V max was calculated to be 11 μmol min −1 mg −1 . Our results confirmed that the product of the gene LmjF16.0530, whose function has previously been predicted based on homology to known proteins, can therefore be positively assigned as L. Major DHODH.
Charehdar Sorour - One of the best experts on this subject based on the ideXlab platform.
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nanogold for the treatment of zoonotic cutaneous Leishmaniasis caused by Leishmania Major mrho ir 75 er an animal trial with methanol extract of eucalyptus camaldulensis
Journal of Pharmaceutical & Health Sciences, 2011Co-Authors: Torabi Negin, Mohebali Mehdi, Shahverdi Ahmad Reza, Rezayat Seyed Mehdi, Edrissian Gholam Hossein, Esmaeili Jamileh, Charehdar SorourAbstract:This experimental study was conducted to assess of effectiveness of two nanogold concentrations against cutaneous Leishmaniasis (CL) caused by Iranian strain of Leishmania Major in BALB/c mice model. Cutaneous lesions were experimentally induced by inoculation with Iranian Leishmania Major promastigotes (MRHO/IR/75/ER) in 61 BALB/c mice. The infected mice were enrolled to four groups: interventional groups which were treated with two different concentrations of nanogold and two control groups. Nanogold solutions applied topically twice daily for 28 days (N=30) and the results compared with control groups (N=31).The results showed that amastigote number into the lesions, were significantly decreased in interventional groups compared with control groups (p=0.001). Nanogold solutions were also decreased mortality rate in the mice (p=0.003). No statistically difference was found between 40 μg/ml and 0.4μg/ml of nanogold concentrations in amastigote reduction (p=0.648).
Peter J Myler - One of the best experts on this subject based on the ideXlab platform.
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kinetoplastid specific histone variant functions are conserved in Leishmania Major
Molecular and Biochemical Parasitology, 2013Co-Authors: Britta A Anderson, Peter J Myler, Iris L K Wong, Loren Baugh, Gowthaman Ramasamy, Stephen M BeverleyAbstract:Regions of transcription initiation and termination in kinetoplastid protists lack known eukaryotic promoter and terminator elements, although epigenetic marks such as histone variants and the modified DNA base J have been localized to these regions in Trypanosoma brucei, Trypanosoma cruzi, and/or Leishmania Major. Phenotypes of base J mutants vary significantly across trypanosomatids, implying divergence in the epigenetic networks governing transcription during evolution. Here, we demonstrate that the histone variants H2A.Z and H2B.V are essential in L. Major using a powerful quantitative plasmid segregation-based test. In contrast, H3.V is not essential for viability or normal growth in Leishmania. Steady-state transcript levels and the efficiency of transcription termination at convergent strand switch regions (SSRs) in H3V-null parasites were comparable to WT parasites. Our genetic tests show a conservation of histone variant phenotypes between L. Major and T. brucei, unlike the diversity of phenotypes associated with genetic manipulation of the DNA base J modification.
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histone acetylations mark origins of polycistronic transcription in Leishmania Major
BMC Genomics, 2009Co-Authors: Sean Thomas, Peter J Myler, Amanda Green, Nancy R Sturm, David A CampbellAbstract:Many components of the RNA polymerase II transcription machinery have been identified in kinetoplastid protozoa, but they diverge substantially from other eukaryotes. Furthermore, protein-coding genes in these organisms lack individual transcriptional regulation, since they are transcribed as long polycistronic units. The transcription initiation sites are assumed to lie within the 'divergent strand-switch' regions at the junction between opposing polycistronic gene clusters. However, the mechanism by which Kinetoplastidae initiate transcription is unclear, and promoter sequences are undefined. The chromosomal location of TATA-binding protein (TBP or TRF4), Small Nuclear Activating Protein complex (SNAP50), and H3 histones were assessed in Leishmania Major using microarrays hybridized with DNA obtained through chromatin immunoprecipitation (ChIP-chip). The TBP and SNAP50 binding patterns were almost identical and high intensity peaks were associated with tRNAs and snRNAs. Only 184 peaks of acetylated H3 histone were found in the entire genome, with substantially higher intensity in rapidly-dividing cells than stationary-phase. The Majority of the acetylated H3 peaks were found at divergent strand-switch regions, but some occurred at chromosome ends and within polycistronic gene clusters. Almost all these peaks were associated with lower intensity peaks of TBP/SNAP50 binding a few kilobases upstream, evidence that they represent transcription initiation sites. The first genome-wide maps of DNA-binding protein occupancy in a kinetoplastid organism suggest that H3 histones at the origins of polycistronic transcription of protein-coding genes are acetylated. Global regulation of transcription initiation may be achieved by modifying the acetylation state of these origins.
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transcription initiation and termination on Leishmania Major chromosome 3
Eukaryotic Cell, 2004Co-Authors: Santiago Martinezcalvillo, Dan Nguyen, Kenneth Stuart, Peter J MylerAbstract:Genome projects involving Leishmania and other trypanosomatids have revealed that most genes in these organisms are organized into large clusters of genes on the same DNA strand. We have previously shown that transcription of the entire Leishmania Major Friedlin (LmjF) chromosome 1 (chr1) initiates bidirectionally between two divergent gene clusters. Here, we analyze transcription of LmjF chr3, which contains two convergent clusters of 67 and 30 genes, separated by a tRNA gene, with a single divergent protein-coding gene located close to the “left” telomere. Nuclear run-on analyses indicate that specific transcription of chr3 initiates bidirectionally between the single subtelomeric gene and the adjacent 67-gene cluster, close to the “right” telomere upstream of the 30-gene cluster, and upstream of the tRNA gene. Transcription on both strands terminates within the tRNA-gene region. Transient-transfection studies support the role of the tRNA-gene region as a transcription terminator for RNA polymerase II (Pol II) and Pol III, and also for Pol I.
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transcription of Leishmania Major friedlin chromosome 1 initiates in both directions within a single region
Molecular Cell, 2003Co-Authors: Santiago Martinezcalvillo, Dan Nguyen, Kenneth Stuart, Peter J MylerAbstract:Abstract Almost nothing is known about the sequences involved in transcription initiation of protein-coding genes in the parasite Leishmania . We describe here the transcriptional analysis of chromosome 1 (chr1) from Leishmania Major Friedlin (LmjF) which encodes the first 29 genes on one DNA strand, and the remaining 50 on the opposite strand. Strand-specific nuclear run-on assays showed that a low level of nonspecific transcription probably takes place over the entire chromosome, but an ∼10-fold higher level of coding strand-specific RNA polymerase II (Pol II)-mediated transcription initiates within the strand-switch region. 5′ RACE studies localized the initiation sites to a
Uta Schurigt - One of the best experts on this subject based on the ideXlab platform.
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inhibitory effect of phenothiazine and phenoxazine derived chloroacetamides on Leishmania Major growth and trypanosoma brucei trypanothione reductase
European Journal of Medicinal Chemistry, 2016Co-Authors: Ana Marcu, Uta Schurigt, Klaus Muller, Heidrun Moll, Luise R Krauthsiegel, Helge PrinzAbstract:Abstract A number of phenothiazine-, phenoxazine- and related tricyclics-derived chloroacetamides were synthesized and evaluated in vitro for antiprotozoal activities against Leishmania Major ( L. Major ) promastigotes. Several analogs were remarkably potent inhibitors, with antiLeishmanial activities being comparable or superior to those of the reference antiprotozoal drugs. Furthermore, we explored the structure–activity relationships of N-10 haloacetamides that influence the potency of such analogs toward inhibition of L. Major promastigote growth in vitro . With respect to the mechanism of action, selected compounds were evaluated for time-dependent inactivation of Trypanosoma brucei trypanothione reductase. Our results are indicative of a covalent interaction which could account for potent antiprotozoal activities.
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cinnamic acid bornyl ester derivatives from valeriana wallichii exhibit antiLeishmanial in vivo activity in Leishmania Major infected balb c mice
PLOS ONE, 2015Co-Authors: Anita Masic, Ana Maria Valencia Hernandez, Sudipta Hazra, Jan Glaser, Ulrike Holzgrabe, Banasri Hazra, Uta SchurigtAbstract:Human Leishmaniasis covers a broad spectrum of clinical manifestations ranging from self-healing cutaneous Leishmaniasis to severe and lethal visceral Leishmaniasis caused among other species by Leishmania Major or Leishmania donovani, respectively. Some drug candidates are in clinical trials to substitute current therapies, which are facing emerging drug-resistance accompanied with serious side effects. Here, two cinnamic acid bornyl ester derivatives (1 and 2) were assessed for their antiLeishmanial activity. Good selectivity and antiLeishmanial activity of bornyl 3-phenylpropanoate (2) in vitro prompted the antiLeishmanial assessment in vivo. For this purpose, BALB/c mice were infected with Leishmania Major promastigotes and treated with three doses of 50 mg/kg/day of compound 2. The treatment prevented the characteristic swelling at the site of infection and correlated with reduced parasite burden. Transmitted light microscopy and transmission electron microscopy of Leishmania Major promastigotes revealed that compounds 1 and 2 induce mitochondrial swelling. Subsequent studies on Leishmania Major promastigotes showed the loss of mitochondrial transmembrane potential (ΔΨm) as a putative mode of action. As the cinnamic acid bornyl ester derivatives 1 and 2 had exhibited antiLeishmanial activity in vitro, and compound 2 in Leishmania Major-infected BALB/c mice in vivo, they can be regarded as possible lead structures for the development of new antiLeishmanial therapeutic approaches.
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autophagic digestion of Leishmania Major by host macrophages is associated with differential expression of bnip3 ctse and the mirnas mir 101c mir 129 and mir 210
Parasites & Vectors, 2015Co-Authors: Benjamin Frank, Ana Marcu, Antonio Luis De Oliveira Almeida Petersen, Heike Weber, Christian Stigloher, Jeremy C Mottram, Claus Juergen Scholz, Uta SchurigtAbstract:Background Autophagy participates in innate immunity by eliminating intracellular pathogens. Consequently, numerous microorganisms have developed strategies to impair the autophagic machinery in phagocytes. In the current study, interactions between Leishmania Major (L. m.) and the autophagic machinery of bone marrow-derived macrophages (BMDM) were analyzed.
Thomas L. Poulos - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of the Leishmania Major peroxidase cytochrome c complex
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Victoria S Jasion, Tzanko Doukov, Stephanie Hai Pineda, Thomas L. PoulosAbstract:The causative agent of Leishmaniasis is the protozoan parasite Leishmania Major. Part of the host protective mechanism is the production of reactive oxygen species including hydrogen peroxide. In response, L. Major produces a peroxidase, L. Major peroxidase (LmP), that helps to protect the parasite from oxidative stress. LmP is a heme peroxidase that catalyzes the peroxidation of mitochondrial cytochrome c. We have determined the crystal structure of LmP in a complex with its substrate, L. Major cytochrome c (LmCytc) to 1.84 A, and compared the structure to its close homolog, the yeast cytochrome c peroxidase–cytochrome c complex. The binding interface between LmP and LmCytc has one strong and one weak ionic interaction that the yeast system lacks. The differences between the steady-state kinetics correlate well with the Lm redox pair being more dependent on ionic interactions, whereas the yeast redox pair depends more on nonpolar interactions. Mutagenesis studies confirm that the ion pairs at the intermolecular interface are important to both kcat and KM. Despite these differences, the electron transfer path, with respect to the distance between hemes, along the polypeptide chain is exactly the same in both redox systems. A potentially important difference, however, is the side chains involved. LmP has more polar groups (Asp and His) along the pathway compared with the nonpolar groups (Leu and Ala) in the yeast system, and as a result, the electrostatic environment along the presumed electron transfer path is substantially different.
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Leishmania Major peroxidase is a cytochrome c peroxidase
Biochemistry, 2012Co-Authors: Victoria S Jasion, Thomas L. PoulosAbstract:Leishmania Major peroxidase (LmP) exhibits both ascorbate and cytochrome c peroxidase activities. Our previous results illustrated that LmP has a much higher activity against horse heart cytochrome c than ascorbate, suggesting that cytochrome c may be the biologically important substrate. To elucidate the biological function of LmP, we have recombinantly expressed, purified, and determined the 2.08 A crystal structure of L. Major cytochrome c (LmCytc). Like other types of cytochrome c, LmCytc has an electropositive surface surrounding the exposed heme edge that serves as the site of docking with redox partners. Kinetic assays performed with LmCytc and LmP show that LmCytc is a much better substrate for LmP than horse heart cytochrome c. Furthermore, unlike the well-studied yeast system, the reaction follows classic Michaelis–Menten kinetics and is sensitive to an increasing ionic strength. Using the yeast cocrystal as a control, protein–protein docking was performed using Rosetta to develop a model for th...