scholarly journals Efficacy of a Binuclear Cyclopalladated Compound Therapy for Cutaneous Leishmaniasis in the Murine Model of Infection with Leishmania amazonensis and Its Inhibitory Effect on Topoisomerase 1B

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Angela Maria Arenas Velásquez ◽  
Willian Campos Ribeiro ◽  
Vutey Venn ◽  
Silvia Castelli ◽  
Mariana Santoro de Camargo ◽  
...  
Keyword(s):  
Leishmania Donovani ◽  
Parasite Load ◽  
Inhibitory Effect ◽  
Peritoneal Macrophages ◽  
Leishmania Amazonensis ◽  
Content Type ◽  
Intracellular Amastigotes ◽  
Therapeutic Alternatives

ABSTRACT Leishmaniasis is a disease found throughout the (sub)tropical parts of the world caused by protozoan parasites of the Leishmania genus. Despite the numerous problems associated with existing treatments, pharmaceutical companies continue to neglect the development of better ones. The high toxicity of current drugs combined with emerging resistance makes the discovery of new therapeutic alternatives urgent. We report here the evaluation of a binuclear cyclopalladated complex containing Pd(II) and N,N′-dimethylbenzylamine (Hdmba) against Leishmania amazonensis. The compound [Pd(dmba)(μ-N3)]2 (CP2) inhibits promastigote growth (50% inhibitory concentration [IC50] = 13.2 ± 0.7 μM) and decreases the proliferation of intracellular amastigotes in in vitro incubated macrophages (IC50 = 10.2 ± 2.2 μM) without a cytotoxic effect when tested against peritoneal macrophages (50% cytotoxic concentration = 506.0 ± 10.7 μM). In addition, CP2 was also active against T. cruzi intracellular amastigotes (IC50 = 2.3 ± 0.5 μM, selective index = 225), an indication of its potential for use in Chagas disease therapy. In vivo assays using L. amazonensis-infected BALB/c showed an 80% reduction in parasite load compared to infected and nontreated animals. Also, compared to amphotericin B treatment, CP2 did not show any side effects, which was corroborated by the analysis of plasma levels of different hepatic and renal biomarkers. Furthermore, CP2 was able to inhibit Leishmania donovani topoisomerase 1B (Ldtopo1B), a potentially important target in this parasite. (This study has been registered at ClinicalTrials.gov under identifier NCT02169141.)

scholarly journals In Vitro and In Vivo Evaluation of an Adamantyl-Based Phenyl Sulfonyl Acetamide against Cutaneous Leishmaniasis Models of Leishmania amazonensis

2020 ◽  
Vol 64 (12) ◽  
Author(s):  
Camila C. Santos ◽  
Huaisheng Zhang ◽  
Marcos M. Batista ◽  
Gabriel M. de Oliveira ◽  
Kelly C. Demarque ◽  
...  
Keyword(s):  
Cutaneous Leishmaniasis ◽  
Parasite Load ◽  
Leishmania Amazonensis ◽  
Host Cells ◽  
In Vivo Evaluation ◽  
Content Type ◽  
Suboptimal Dose ◽  
Low Toxicity

ABSTRACT Phenotypic assay against Leishmania amazonensis in vitro and in vivo led to identification of an adamantyl-based phenyl sulfonyl acetamide (compound 1) as a promising antileishmanial agent. Compound 1 inhibited the growth of intracellular forms of L. amazonensis (50% inhibitory concentration [IC50] = 4 μM) and exhibited low toxicity to host cells, with a selectivity index (SI) of >125. However, in a cutaneous leishmaniasis (CL) mouse model, compound 1 did not reduce lesions and parasite load when administered as monotherapy or when given simultaneously with a suboptimal dose of miltefosine.

scholarly journals Activity of the Pore-Forming Virulence Factor Listeriolysin O Is Reversibly Inhibited by Naturally Occurring S-Glutathionylation

2017 ◽  
Vol 85 (4) ◽  
Author(s):  
Jonathan L. Portman ◽  
Qiongying Huang ◽  
Michelle L. Reniere ◽  
Anthony T. Iavarone ◽  
Daniel A. Portnoy
Keyword(s):  
Protein Function ◽  
Inhibitory Effect ◽  
Cysteine Residue ◽  
Infection Model ◽  
Listeriolysin O ◽  
Content Type ◽  
Pore Forming Proteins

ABSTRACT Cholesterol-dependent cytolysins (CDCs) represent a family of homologous pore-forming proteins secreted by many Gram-positive bacterial pathogens. CDCs mediate membrane binding partly through a conserved C-terminal undecapeptide, which contains a single cysteine residue. While mutational changes to other residues in the undecapeptide typically have severe effects, mutation of the cysteine residue to alanine has minor effects on overall protein function. Thus, the role of this highly conserved reactive cysteine residue remains largely unknown. We report here that the CDC listeriolysin O (LLO), secreted by the facultative intracellular pathogen Listeria monocytogenes, was posttranslationally modified by S-glutathionylation at this conserved cysteine residue and that either endogenously synthesized or exogenously added glutathione was sufficient to form this modification. When recapitulated with purified protein in vitro, this modification completely ablated the activity of LLO, and this inhibitory effect was fully reversible by treatment with reducing agents. A cysteine-to-alanine mutation in LLO rendered the protein completely resistant to inactivation by S-glutathionylation, and a mutant expressing this mutation retained full hemolytic activity. A mutant strain of L. monocytogenes expressing the cysteine-to-alanine variant of LLO was able to infect and replicate within bone marrow-derived macrophages indistinguishably from the wild type in vitro, yet it was attenuated 4- to 6-fold in a competitive murine infection model in vivo. This study suggests that S-glutathionylation may represent a mechanism by which CDC-family proteins are posttranslationally modified and regulated and help explain an evolutionary pressure to retain the highly conserved undecapeptide cysteine.

scholarly journals TCP1γ Subunit Is Indispensable for Growth and Infectivity of Leishmania donovani

2020 ◽  
Vol 64 (8) ◽  
Author(s):  
Shailendra Yadav ◽  
Jitendra Kuldeep ◽  
Mohammad I. Siddiqi ◽  
Neena Goyal
Keyword(s):  
Cell Cycle Progression ◽  
Leishmania Donovani ◽  
Gene Replacement ◽  
Titration Calorimetry ◽  
Content Type ◽  
T Complex ◽  
Complex Protein ◽  
Oligomeric Complex

ABSTRACT T-complex protein-1 (TCP1) is a ubiquitous group II chaperonin and is known to fold various proteins, such as actin and tubulin. In Leishmania donovani, the γ subunit of TCP1 (LdTCP1γ) has been cloned and characterized. It forms a high-molecular-weight homo-oligomeric complex that performs ATP-dependent protein folding. In the present study, we evaluated the essentiality of the LdTCP1γ gene. Gene replacement studies indicated that LdTCP1γ is essential for parasite survival. The LdTCP1γ single-allele-replacement mutants exhibited slowed growth and decreased infectivity in mouse macrophages compared to the growth and infectivity of the wild-type parasites. Modulation of LdTCP1γ expression in promastigotes also modulated cell cycle progression. Suramin, an antitrypanosomal drug, not only inhibited the luciferase refolding activity of the recombinant LdTCP1γ (rLdTCP1γ) homo-oligomeric complex but also exhibited potential antileishmanial efficacy both in vitro and in vivo. The interaction of suramin and LdTCP1γ was further validated by isothermal titration calorimetry. The study suggests LdTCP1γ as a potential drug target and also provides a framework for the development of a new class of drugs.

scholarly journals A Redox-Sensitive Thiol in Wis1 Modulates the Fission Yeast Mitogen-Activated Protein Kinase Response to H2O2 and Is the Target of a Small Molecule

2020 ◽  
Vol 40 (7) ◽  
Author(s):  
Johanna J. Sjölander ◽  
Agata Tarczykowska ◽  
Cecilia Picazo ◽  
Itziar Cossio ◽  
Itedale Namro Redwan ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Fission Yeast ◽  
Inhibitory Effect ◽  
Mitogen Activated Protein Kinase ◽  
Activation Loop ◽  
Content Type ◽  
Kinase Activation ◽  
Mitogen Activated Protein

ABSTRACT Oxidation of a highly conserved cysteine (Cys) residue located in the kinase activation loop of mitogen-activated protein kinase kinases (MAPKK) inactivates mammalian MKK6. This residue is conserved in the fission yeast Schizosaccharomyces pombe MAPKK Wis1, which belongs to the H2O2-responsive MAPK Sty1 pathway. Here, we show that H2O2 reversibly inactivates Wis1 through this residue (C458) in vitro. We found that C458 is oxidized in vivo and that serine replacement of this residue significantly enhances Wis1 activation upon addition of H2O2. The allosteric MAPKK inhibitor INR119, which binds in a pocket next to the activation loop and C458, prevented the inhibition of Wis1 by H2O2 in vitro and significantly increased Wis1 activation by low levels of H2O2 in vivo. We propose that oxidation of C458 inhibits Wis1 and that INR119 cancels out this inhibitory effect by binding close to this residue. Kinase inhibition through the oxidation of a conserved Cys residue in MKK6 (C196) is thus conserved in the S. pombe MAPKK Wis1.

scholarly journals Synergistic Activity between Statins and Bisphosphonates against Acute Experimental Toxoplasmosis

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Zhu-Hong Li ◽  
Catherine Li ◽  
Sergio H. Szajnman ◽  
Juan B. Rodriguez ◽  
Silvia N. J. Moreno
Keyword(s):  
Lethal Dose ◽  
Inhibitory Effect ◽  
Etiologic Agent ◽  
Farnesyl Diphosphate ◽  
Synergistic Activity ◽  
Content Type ◽  
Coa Reductase ◽  
Modest Effect

ABSTRACT Bisphosphonates are widely used for the treatment of bone disorders. These drugs also inhibit the growth of a variety of protozoan parasites, such as Toxoplasma gondii, the etiologic agent of toxoplasmosis. The target of the most potent bisphosphonates is the isoprenoid biosynthesis pathway enzyme farnesyl diphosphate synthase (FPPS). Based on our previous work on the inhibitory effect of sulfur-containing linear bisphosphonates against T. gondii, we investigated the potential synergistic interaction between one of these derivatives, 1-[(n-heptylthio)ethyl]-1,1-bisphosphonate (C7S), and statins, which are potent inhibitors of the host 3-hydroxy-3-methyl glutaryl-coenzyme A reductase (3-HMG-CoA reductase). C7S showed high activity against the T. gondii bifunctional farnesyl diphosphate (FPP)/geranylgeranyl diphosphate (GGPP) synthase (TgFPPS), which catalyzes the formation of FPP and GGPP (50% inhibitory concentration [IC50] = 31 ± 0.01 nM [mean ± standard deviation]), and modest effect against the human FPPS (IC50 = 1.3 ± 0.5 μM). We tested combinations of C7S with statins against the in vitro replication of T. gondii. We also treated mice infected with a lethal dose of T. gondii with similar combinations. We found strong synergistic activities when using low doses of C7S, which were stronger in vivo than when tested in vitro. We also investigated the synergism of several commercially available bisphosphonates with statins both in vitro and in vivo. Our results provide evidence that it is possible to develop drug combinations that act synergistically by inhibiting host and parasite enzymes in vitro and in vivo.

scholarly journals Role of F1C Fimbriae, Flagella, and Secreted Bacterial Components in the Inhibitory Effect of Probiotic Escherichia coli Nissle 1917 on Atypical Enteropathogenic E. coli Infection

2014 ◽  
Vol 82 (5) ◽  
pp. 1801-1812 ◽  
Author(s):  
Sylvia Kleta ◽  
Marcel Nordhoff ◽  
Karsten Tedin ◽  
Lothar H. Wieler ◽  
Rafal Kolenda ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Inhibitory Effect ◽  
Host Cells ◽  
Single Infection ◽  
Content Type ◽  
E Coli ◽  
Initial Adhesion

ABSTRACTEnteropathogenicEscherichia coli(EPEC) is recognized as an important intestinal pathogen that frequently causes acute and persistent diarrhea in humans and animals. The use of probiotic bacteria to prevent diarrhea is gaining increasing interest. The probioticE. colistrain Nissle 1917 (EcN) is known to be effective in the treatment of several gastrointestinal disorders. While bothin vitroandin vivostudies have described strong inhibitory effects of EcN on enteropathogenic bacteria, including pathogenicE. coli, the underlying molecular mechanisms remain largely unknown. In this study, we examined the inhibitory effect of EcN on infections of porcine intestinal epithelial cells with atypical enteropathogenicE. coli(aEPEC) with respect to single infection steps, including adhesion, microcolony formation, and the attaching and effacing phenotype. We show that EcN drastically reduced the infection efficiencies of aEPEC by inhibiting bacterial adhesion and growth of microcolonies, but not the attaching and effacing of adherent bacteria. The inhibitory effect correlated with EcN adhesion capacities and was predominantly mediated by F1C fimbriae, but also by H1 flagella, which served as bridges between EcN cells. Furthermore, EcN seemed to interfere with the initial adhesion of aEPEC to host cells by secretion of inhibitory components. These components do not appear to be specific to EcN, but we propose that the strong adhesion capacities enable EcN to secrete sufficient local concentrations of the inhibitory factors. The results of this study are consistent with a mode of action whereby EcN inhibits secretion of virulence-associated proteins of EPEC, but not their expression.

scholarly journals Novel Role of Calmodulin in Regulating Protein Transport to Mitochondria in a Unicellular Eukaryote

2013 ◽  
Vol 33 (22) ◽  
pp. 4579-4593 ◽  
Author(s):  
Abhishek Aich ◽  
Chandrima Shaha
Keyword(s):  
Leishmania Donovani ◽  
Protein Transport ◽  
Mitochondrial Protein ◽  
Unicellular Eukaryote ◽  
Mitochondrial Targeting ◽  
Content Type ◽  
Mitochondrial Translocation ◽  
Tryparedoxin Peroxidase

Lower eukaryotes like the kinetoplastid parasites are good models to study evolution of cellular pathways during steps to eukaryogenesis. In this study, a kinetoplastid parasite,Leishmania donovani, was used to understand the process of mitochondrial translocation of a nucleus-encoded mitochondrial protein, the mitochondrial tryparedoxin peroxidase (mTXNPx). We report the presence of an N-terminal cleavable mitochondrial targeting signal (MTS) validated through deletion and grafting experiments. We also establish a novel finding of calmodulin (CaM) binding to the MTS of mTXNPx through specific residues. Mutation of CaM binding residues, keeping intact the residues involved in mitochondrial targeting and biochemical inhibition of CaM activity bothin vitroandin vivo, prevented mitochondrial translocation. Through reconstituted import assays, we demonstrate obstruction of mitochondrial translocation either in the absence of CaM or Ca2+or in the presence of CaM inhibitors. We also demonstrate the prevention of temperature-driven mTXNPx aggregation in the presence of CaM. These findings establish the idea that CaM is required for the transport of the protein to mitochondria through maintenance of translocation competence posttranslation.

scholarly journals Quinine Sulphate Microparticles as Treatment for Leishmaniasis

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Grace Lovia Allotey-Babington ◽  
Seth Kwabena Amponsah ◽  
Thomas Nettey ◽  
Clement Sasu ◽  
Henry Nettey
Keyword(s):  
Zeta Potential ◽  
Encapsulation Efficiency ◽  
Leishmania Donovani ◽  
Treatment Options ◽  
Parasite Load ◽  
Pharmacokinetic Profile ◽  
Drug Formulation ◽  
Quinine Sulphate

Background. Leishmaniasis is a neglected tropical disease caused by the Leishmania parasite and transmitted by the female phlebotomine sandfly. The disease can affect the skin (least fatal) or internal organs (most fatal). Current treatment options for leishmaniasis have a number of adverse effects, and there appears to be resistance by the protozoan parasite (Leishmania spp.). Reports suggest that quinine sulphate, not indicated for leishmaniasis, is effective in killing the Leishmania parasite. Indeed, the efficacy of any drug is dependent on the concentration at the target site, which is also almost dependent on drug formulation. The current study assessed the pharmacokinetic profile of the microparticulate formulation of quinine sulphate and its in vitro and in vivo efficacy against Leishmania donovani. Methods. Quinine sulphate was encapsulated in bovine serum albumin by the spray-drying method. Quinine sulphate microparticles were evaluated for size, zeta potential, drug content, encapsulation efficiency, and in vitro release properties. Afterwards, the pharmacokinetic characteristics of quinine sulphate microparticles were estimated and in vivo efficacy studies were also conducted. Results. The size range of the quinine sulphate microparticles was between 2.0 and 5.0 µm. Microparticles had an average zeta potential of −35.2 mV and an encapsulation efficiency of 94.5%. Also, Cmax, t1/2, and AUC were all significantly desirable for quinine sulphate microparticles compared to the drug powder. Quinine sulphate microparticles significantly reduced parasite load in rat organs than amphotericin B. Conclusion. Overall, quinine sulphate microparticles had better pharmacokinetic profile and showed higher efficacy against Leishmania donovani parasites in vivo. Thus, quinine sulphate microparticles have the potential, especially, in treating visceral leishmaniasis.

scholarly journals Snapshot Profiling of the Antileishmanial Potency of Lead Compounds and Drug Candidates against Intracellular Leishmania donovani Amastigotes, with a Focus on Human-Derived Host Cells

2017 ◽  
Vol 61 (3) ◽  
Author(s):  
Markela Koniordou ◽  
Stephen Patterson ◽  
Susan Wyllie ◽  
Karin Seifert
Keyword(s):  
Host Cell ◽  
Leishmania Donovani ◽  
Host Cells ◽  
Content Type ◽  
Human Macrophages ◽  
Drug Candidates ◽  
Lead Compounds ◽  
Intracellular Amastigotes ◽  
Peritoneal Exudate Macrophages

ABSTRACT This study characterized the in vitro potencies of antileishmanial agents against intracellular Leishmania donovani amastigotes in primary human macrophages, obtained with or without CD14-positive monocyte enrichment, phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 cells, and mouse peritoneal exudate macrophages (PEMs). Host cell-dependent potency was confirmed for pentavalent and trivalent antimony. Fexinidazole was inactive against intracellular amastigotes across the host cell panel. Fexinidazole sulfone, (R)-PA-824, (S)-PA-824, and VL-2098 displayed similar potency in all of the host cells tested.

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