scholarly journals Evaluating 5-Nitrothiazoles as Trypanocidal Agents

2015 ◽  
Vol 60 (2) ◽  
pp. 1137-1140 ◽  
Author(s):  
Ivan P. O'Shea ◽  
Mohammed Shahed ◽  
Benjamín Aguilera-Venegas ◽  
Shane R. Wilkinson
Keyword(s):  
Trypanosoma Brucei ◽  
Mammalian Cells ◽  
Cytotoxic Effect ◽  
Human African Trypanosomiasis ◽  
Type I ◽  
Antiparasitic Activity ◽  
Content Type ◽  
Growth Inhibitory ◽  
Activity Dependent ◽  
Related Compounds

ABSTRACTThe growth-inhibitory properties of a 5-nitrothiazole series were evaluated againstTrypanosoma brucei. A subset of related compounds displayed the greatest potency toward the parasite while exhibiting little cytotoxic effect on mammalian cells, with this antiparasitic activity dependent on expression of a type I nitroreductase by the trypanosome. We conclude that the 5-nitrothiazole class of nitroheterocyclic drugs may represent a new lead in the treatment of human African trypanosomiasis.

scholarly journals Targeting the Substrate Preference of a Type I Nitroreductase To Develop Antitrypanosomal Quinone-Based Prodrugs

2012 ◽  
Vol 56 (11) ◽  
pp. 5821-5830 ◽  
Author(s):  
Belinda S. Hall ◽  
Emma Louise Meredith ◽  
Shane R. Wilkinson
Keyword(s):  
Trypanosoma Brucei ◽  
Type I ◽  
Antiparasitic Activity ◽  
Content Type ◽  
Biochemical Characteristics ◽  
Trypanocidal Activity ◽  
Donor And Acceptor ◽  
Enzyme Substrates ◽  
Electron Donor And Acceptor ◽  
Using Data

ABSTRACTNitroheterocyclic prodrugs are used to treat infections caused byTrypanosoma cruziandTrypanosoma brucei. A key component in selectivity involves a specific activation step mediated by a protein homologous with type I nitroreductases, enzymes found predominantly in prokaryotes. Using data from determinations based on flavin cofactor, oxygen-insensitive activity, substrate range, and inhibition profiles, we demonstrate that NTRs fromT. cruziandT. bruceidisplay many characteristics of their bacterial counterparts. Intriguingly, both enzymes preferentially use NADH and quinones as the electron donor and acceptor, respectively, suggesting that they may function as NADH:ubiquinone oxidoreductases in the parasite mitochondrion. We exploited this preference to determine the trypanocidal activity of a library of aziridinyl benzoquinones against bloodstream-formT. brucei. Biochemical screens using recombinant NTR demonstrated that several quinones were effective substrates for the parasite enzyme, havingKcat/Kmvalues 2 orders of magnitude greater than those of nifurtimox and benznidazole. In tests againstT. brucei, antiparasitic activity mirrored the biochemical data, with the most potent compounds generally being preferred enzyme substrates. Trypanocidal activity was shown to be NTR dependent, as parasites with elevated levels of this enzyme were hypersensitive to the aziridinyl agent. By unraveling the biochemical characteristics exhibited by the trypanosomal NTRs, we have shown that quinone-based compounds represent a class of trypanocidal compound.

scholarly journals Evaluating 5-Nitrofurans as Trypanocidal Agents

2013 ◽  
Vol 57 (4) ◽  
pp. 1638-1647 ◽  
Author(s):  
Christopher Bot ◽  
Belinda S. Hall ◽  
Guzmán Álvarez ◽  
Rossanna Di Maio ◽  
Mercedes González ◽  
...  
Keyword(s):  
Human African Trypanosomiasis ◽  
Bloodstream Form ◽  
Type I ◽  
Biochemical Screening ◽  
African Trypanosomiasis ◽  
Open Chain ◽  
Content Type ◽  
Growth Inhibitory ◽  
Initial Activation

ABSTRACTThe nitroheterocycle nifurtimox, as part of a nifurtimox-eflornithine combination therapy, represents one of a limited number of treatments targetingTrypanosoma brucei, the causative agent of human African trypanosomiasis. The mode of action of this prodrug involves an initial activation reaction catalyzed by a type I nitroreductase (NTR), an enzyme found predominantly in prokaryotes, leading to the formation of a cytotoxic unsaturated open-chain nitrile metabolite. Here, we evaluate the trypanocidal activities of a library of other 5-nitrofurans against the bloodstream form ofT. bruceias a preliminary step in the identification of additional nitroaromatic compounds that can potentially partner with eflornithine. Biochemical screening against the purified enzyme revealed that all 5-nitrofurans were effective substrates forT. bruceiNTR (TbNTR), with the preferred compounds having apparentkcat/Kmvalues approximately 50-fold greater than those of nifurtimox. For several compounds,in vitroreduction by this nitroreductase yielded products characterized by mass spectrometry as either unsaturated or saturated open-chain nitriles. When tested against the bloodstream form ofT. brucei, many of the derivatives displayed significant growth-inhibitory properties, with the most potent compounds generating 50% inhibitory concentrations (IC50s) around 200 nM. The antiparasitic activities of the most potent agents were demonstrated to be NTR dependent, as parasites having reduced levels of the enzyme displayed resistance to the compounds, while parasites overexpressing TbNTR showed hypersensitivity. We conclude that other members of the 5-nitrofuran class of nitroheterocycles have the potential to treat human African trypanosomiasis, perhaps as an alternative partner prodrug to nifurtimox, in the next generation of eflornithine-based combinational therapies.

scholarly journals Evaluating Aziridinyl Nitrobenzamide Compounds as Leishmanicidal Prodrugs

2013 ◽  
Vol 58 (1) ◽  
pp. 370-377 ◽  
Author(s):  
Andrew A. Voak ◽  
Karin Seifert ◽  
Nuala A. Helsby ◽  
Shane R. Wilkinson
Keyword(s):  
Mammalian Cells ◽  
Leishmania Major ◽  
Inhibitory Effect ◽  
Luciferase Reporter ◽  
Flavin Mononucleotide ◽  
Infection Model ◽  
Type I ◽  
Luciferase Reporter Gene ◽  
Content Type ◽  
Growth Inhibitory

ABSTRACTMany of the nitroaromatic agents used in medicine function as prodrugs and must undergo activation before exerting their toxic effects. In most cases, this is catalyzed by flavin mononucleotide (FMN)-dependent type I nitroreductases (NTRs), a class of enzyme absent from higher eukaryotes but expressed by bacteria and several eukaryotic microbes, including trypanosomes andLeishmania. Here, we utilize this difference to evaluate whether members of a library of aziridinyl nitrobenzamides have activity againstLeishmania major. Biochemical screens using purifiedL. majorNTR (LmNTR) revealed that compounds containing an aziridinyl-2,4-dinitrobenzyl core were effective substrates for the enzyme and showed that the 4-nitro group was important for this activity. To facilitate drug screening against intracellular amastigote parasites, we generated leishmanial cells that expressed the luciferase reporter gene and optimized a mammalian infection model in a 96-well plate format. A subset of aziridinyl-2,4-dinitrobenzyl compounds possessing a 5-amide substituent displayed significant growth-inhibitory properties against the parasite, with the most potent agents generating 50% inhibitory concentrations of <100 nM for the intracellular form. This antimicrobial activity was shown to be LmNTR specific sinceL. major NTR+/−heterozygote parasites were slightly resistance to most aziridinyl dinitrobenzyl agents tested. When the most potent leishmanicidal agents were screened against the mammalian cells in which the amastigote parasites were propagated, no growth-inhibitory effect was observed at concentrations of up to 100 μM. We conclude that the aziridinyl nitrobenzamides represent a new lead structure that may have the potential to treat leishmanial infections.

scholarly journals A Tale of Three Species: Adaptation ofSodalis glossinidiusto Tsetse Biology,WigglesworthiaMetabolism, and Host Diet

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Rebecca J. Hall ◽  
Lindsey A. Flanagan ◽  
Michael J. Bottery ◽  
Vicki Springthorpe ◽  
Stephen Thorpe ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Human African Trypanosomiasis ◽  
Strong Dependence ◽  
Tsetse Fly ◽  
Disease Spread ◽  
Growth Media ◽  
Insect Vector ◽  
African Trypanosomiasis ◽  
Content Type ◽  
Sodalis Glossinidius

ABSTRACTThe tsetse fly is the insect vector for theTrypanosoma bruceiparasite, the causative agent of human African trypanosomiasis. The colonization and spread of the trypanosome correlate positively with the presence of a secondary symbiotic bacterium,Sodalis glossinidius. The metabolic requirements and interactions of the bacterium with its host are poorly understood, and herein we describe a metabolic model ofS. glossinidiusmetabolism. The model enabled the design and experimental verification of a defined medium that supportsS. glossinidiusgrowthex vivo. This has been used subsequently to analyzein vitroaspects ofS. glossinidiusmetabolism, revealing multiple unique adaptations of the symbiont to its environment. Continued dependence on a sugar, and the importance of the chitin monomerN-acetyl-d-glucosamine as a carbon and energy source, suggests adaptation to host-derived molecules. Adaptation to the amino acid-rich blood diet is revealed by a strong dependence onl-glutamate as a source of carbon and nitrogen and by the ability to rescue a predictedl-arginine auxotrophy. Finally, the selective loss of thiamine biosynthesis, a vitamin provided to the host by the primary symbiontWigglesworthia glossinidia, reveals an intersymbiont dependence. The reductive evolution ofS. glossinidiusto exploit environmentally derived metabolites has resulted in multiple weaknesses in the metabolic network. These weaknesses may become targets for reagents that inhibitS. glossinidiusgrowth and aid the reduction of trypanosomal transmission.IMPORTANCEHuman African trypanosomiasis is caused by theTrypanosoma bruceiparasite. The tsetse fly vector is of interest for its potential to prevent disease spread, as it is essential forT. bruceilife cycle progression and transmission. The tsetse’s mutualistic endosymbiontSodalis glossinidiushas a link to trypanosome establishment, providing a disease control target. Here, we describe a new, experimentally verified model ofS. glossinidiusmetabolism. This model has enabled the development of a defined growth medium that was used successfully to test aspects ofS. glossinidiusmetabolism. We presentS. glossinidiusas uniquely adapted to life in the tsetse, through its reliance on the blood diet and host-derived sugars. Additionally,S. glossinidiushas adapted to the tsetse’s obligate symbiontWigglesworthia glossinidiaby scavenging a vitamin it produces for the insect. This work highlights the use of metabolic modeling to design defined growth media for symbiotic bacteria and may provide novel inhibitory targets to block trypanosome transmission.

scholarly journals Mammalian Casein Kinase 1α and Its Leishmanial Ortholog Regulate Stability of IFNAR1 and Type I Interferon Signaling

2009 ◽  
Vol 29 (24) ◽  
pp. 6401-6412 ◽  
Author(s):  
Jianghuai Liu ◽  
Lucas P. Carvalho ◽  
Sabyasachi Bhattacharya ◽  
Christopher J. Carbone ◽  
K. G. Suresh Kumar ◽  
...  
Keyword(s):  
Er Stress ◽  
Mammalian Cells ◽  
Leishmania Major ◽  
Type I Interferon ◽  
Casein Kinase ◽  
Type I ◽  
Bona Fide ◽  
Activity Dependent

ABSTRACT Phosphorylation of the degron of the IFNAR1 chain of the type I interferon (IFN) receptor triggers ubiquitination and degradation of this receptor and, therefore, plays a crucial role in negative regulation of IFN-α/β signaling. Besides the IFN-stimulated and Jak activity-dependent pathways, a basal ligand-independent phosphorylation of IFNAR1 has been described and implicated in downregulating IFNAR1 in response to virus-induced endoplasmic reticulum (ER) stress. Here we report purification and characterization of casein kinase 1α (CK1α) as a bona fide major IFNAR1 kinase that confers basal turnover of IFNAR1 and cooperates with ER stress stimuli to mediate phosphorylation-dependent degradation of IFNAR1. Activity of CK1α was required for phosphorylation and downregulation of IFNAR1 in response to ER stress and viral infection. While many forms of CK1 were capable of phosphorylating IFNAR1 in vitro, human CK1α and L-CK1 produced by the protozoan Leishmania major were also capable of increasing IFNAR1 degron phosphorylation in cells. Expression of leishmania CK1 in mammalian cells stimulated the phosphorylation-dependent downregulation of IFNAR1 and attenuated its signaling. Infection of mammalian cells with L. major modestly decreased IFNAR1 levels and attenuated cellular responses to IFN-α in vitro. We propose a role for mammalian and parasite CK1 enzymes in regulating IFNAR1 stability and type I IFN signaling.

scholarly journals TbKAP6, a Mitochondrial HMG Box-Containing Protein in Trypanosoma brucei, Is the First Trypanosomatid Kinetoplast-Associated Protein Essential for Kinetoplast DNA Replication and Maintenance

2014 ◽  
Vol 13 (7) ◽  
pp. 919-932 ◽  
Author(s):  
Jianyang Wang ◽  
Valeria Pappas-Brown ◽  
Paul T. Englund ◽  
Robert E. Jensen
Keyword(s):  
Trypanosoma Brucei ◽  
Mammalian Cells ◽  
High Mobility ◽  
Kinetoplast Dna ◽  
Content Type ◽  
Hmg Box ◽  
Mitochondrial Nucleoids ◽  
Topo Ii

ABSTRACT Kinetoplast DNA (kDNA), the mitochondrial genome of trypanosomatids, is a giant planar network of catenated minicircles and maxicircles. In vivo kDNA is organized as a highly condensed nucleoprotein disk. So far, in Trypanosoma brucei , proteins involved in the maintenance of the kDNA condensed structure remain poorly characterized. In Crithidia fasciculata , some small basic histone H1-like k inetoplast- a ssociated p roteins (CfKAP) have been shown to condense isolated kDNA networks in vitro . High-mobility group (HMG) box-containing proteins, such as mitochondrial transcription factor A (TFAM) in mammalian cells and Abf2 in the budding yeast, have been shown essential for the packaging of mitochondrial DNA (mtDNA) into mitochondrial nucleoids, remodeling of mitochondrial nucleoids, gene expression, and maintenance of mtDNA. Here, we report that TbKAP6, a mitochondrial HMG box-containing protein, is essential for parasite cell viability and involved in kDNA replication and maintenance. The RNA interference (RNAi) depletion of TbKAP6 stopped cell growth. Replication of both minicircles and maxicircles was inhibited. RNAi or overexpression of TbKAP6 resulted in the disorganization, shrinkage, and loss of kDNA. Minicircle release, the first step in kDNA replication, was inhibited immediately after induction of RNAi, but it quickly increased 3-fold upon overexpression of TbKAP6. Since the release of covalently closed minicircles is mediated by a type II topoisomerase (topo II), we examined the potential interactions between TbKAP6 and topo II. Recombinant TbKAP6 (rTbKAP6) promotes the topo II-mediated decatenation of kDNA. rTbKAP6 can condense isolated kDNA networks in vitro . These results indicate that TbKAP6 is involved in the replication and maintenance of kDNA.

scholarly journals New Chemical Scaffolds for Human African Trypanosomiasis Lead Discovery from a Screen of Tyrosine Kinase Inhibitor Drugs

2014 ◽  
Vol 58 (4) ◽  
pp. 2202-2210 ◽  
Author(s):  
Ranjan Behera ◽  
Sarah M. Thomas ◽  
Kojo Mensa-Wilmot
Keyword(s):  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Human African Trypanosomiasis ◽  
Kinase Inhibitor ◽  
New Drugs ◽  
Proof Of Concept ◽  
African Trypanosomiasis ◽  
Lead Discovery ◽  
Content Type ◽  
Growth Inhibitory

ABSTRACTHuman African trypanosomiasis (HAT) is caused by the protozoanTrypanosoma brucei. New drugs are needed to treat HAT because of undesirable side effects and difficulties in the administration of the antiquated drugs that are currently used. In human proliferative diseases, protein tyrosine kinase (PTK) inhibitors (PTKIs) have been developed into drugs (e.g., lapatinib and erlotinib) by optimization of a 4-anilinoquinazoline scaffold. Two sets of facts raise a possibility that drugs targeted against human PTKs could be “hits” for antitrypanosomal lead discoveries. First, trypanosome protein kinases bind some drugs, namely, lapatinib, CI-1033, and AEE788. Second, the pan-PTK inhibitor tyrphostin A47 blocks the endocytosis of transferrin and inhibits trypanosome replication. Following up on these concepts, we performed a focused screen of various PTKI drugs as possible antitrypanosomal hits. Lapatinib, CI-1033, erlotinib, axitinib, sunitinib, PKI-166, and AEE788 inhibited the replication of bloodstreamT. brucei, with a 50% growth inhibitory concentration (GI50) between 1.3 μM and 2.5 μM. Imatinib had no effect (i.e., GI50> 10 μM). To discover leads among the drugs, a mouse model of HAT was used in a proof-of-concept study. Orally administered lapatinib reduced parasitemia, extended the survival of all treated mice, and cured the trypanosomal infection in 25% of the mice. CI-1033 and AEE788 reduced parasitemia and extended the survival of the infected mice. On the strength of these data and noting their oral bioavailabilities, we propose that the 4-anilinoquinazoline and pyrrolopyrimidine scaffolds of lapatinib, CI-1033, and AEE788 are worth optimizing againstT. bruceiin medicinal chemistry campaigns (i.e., scaffold repurposing) to discover new drugs against HAT.

scholarly journals In VitroandIn VivoInvestigation of the Inhibition of Trypanosoma brucei Cell Growth by Lipophilic Bisphosphonates

2015 ◽  
Vol 59 (12) ◽  
pp. 7530-7539 ◽  
Author(s):  
Gyongseon Yang ◽  
Wei Zhu ◽  
Kuglae Kim ◽  
Soo Young Byun ◽  
Gahee Choi ◽  
...  
Keyword(s):  
Cell Growth ◽  
Bone Resorption ◽  
Mouse Model ◽  
Trypanosoma Brucei ◽  
Human African Trypanosomiasis ◽  
Farnesyl Diphosphate ◽  
Farnesyl Diphosphate Synthase ◽  
Titration Calorimetry ◽  
Content Type ◽  
Mouse Model Of Infection

ABSTRACTWe report the results of a screen of a library of 925 potential prenyl synthase inhibitors againstTrypanosoma bruceifarnesyl diphosphate synthase (TbFPPS) and againstT. brucei, the causative agent of human African trypanosomiasis. The most potent compounds were lipophilic analogs of the bone resorption drug zoledronate, some of which had submicromolar to low micromolar activity against bloodstream formT. bruceiand selectivity indices of up to ∼300. We evaluated the effects of two such inhibitors on survival and parasitemia in aT. bruceimouse model of infection and found that survival increased by up to 16 days. We also investigated the binding of three lipophilic bisphosphonates to an expressed TbFPPS using crystallography and investigated the thermodynamics of binding using isothermal titration calorimetry.

scholarly journals Entry and Killing of Tetrahymena thermophila by Bacterially Produced Shiga Toxin

mBio ◽  
2012 ◽  
Vol 4 (1) ◽  
Author(s):  
Gino Stolfa ◽  
Gerald B. Koudelka
Keyword(s):  
Protein Synthesis ◽  
Shiga Toxin ◽  
Mammalian Cells ◽  
Cytotoxic Effect ◽  
Tetrahymena Thermophila ◽  
Vesicular Trafficking ◽  
Carbohydrate Binding ◽  
Antipredator Defense ◽  
Content Type ◽  
Genes Encoding

ABSTRACTPhage-encoded Shiga toxin (Stx) acts as a bacterial defense against the eukaryotic predatorTetrahymena thermophila. It is unknown how Stx entersTetrahymenaprotozoa or how it kills them.Tetrahymenaprotozoa are phagocytotic; hence, Stx could gain entry to the cytoplasm through the oral apparatus or via endocytosis. We find that Stx2 can killT. thermophilaprotozoa that lack an oral apparatus, indicating that Stx2 can enter these cells via endocytosis. As opposed to the lack of effect on mammalian phagocytes, Stx2 produced by bacteria encapsulated within phagocytotic vesicles is also capable of killingTetrahymena. Addition of an excess of the carbohydrate binding subunits of Stx2 (StxB) and/or ricin (ricin B) blocks Stx2 cytotoxicity. Thus, regardless of whether Stx2 enters the cytoplasm by endocytosis or from the phagocytotic vesicle, this transport is mediated by a putative glycoconjugate receptor. Bacteriophage-mediated lysis of Stx-encoding bacteria is necessary for Stx toxicity inTetrahymena; i.e., toxin released as a consequence of digestion of bacteria byTetrahymenais harmless to the cell. This finding provides a rationale as to why the genes encoding Stx are found almost exclusively on bacteriophages; Stx must be released from the bacteria prior to the digestion of the cell, or it will not be able to exert its cytotoxic effect. It also suggests a reason why other bacterial exotoxins are also found only on temperate bacteriophages. Incubation ofTetrahymenawith purified Stx2 decreases total protein synthesis. This finding indicates that, similar to mammalian cells, Stx2 killsTetrahymenaby inactivating its ribosomes.IMPORTANCETetrahymenais a bacterial predator and a model for mammalian phagocytosis and intracellular vesicular trafficking. Phage-encoded exotoxins apparently have evolved for the purpose of bacterial antipredator defense. These exotoxins kill mammalian cells by inactivating universally conserved factors and/or pathways.Tetrahymenaand susceptible mammalian cells are killed when exposed to bacteriophage-encoded Shiga toxin (Stx). Stx toxicity in mammalian cells requires Stx binding to the globotriaosyl ceramide (Gb3) receptor, followed by receptor-mediated endocytosis (RME). We show that, similar to mammalian cells, internalized Stx inhibits protein synthesis inTetrahymena. AlthoughTetrahymenalacks Gb3, our results suggest that the cytotoxic effect of Stx onTetrahymenais apparently mediated by a receptor, thereby arguing for the existence of RME inTetrahymena. As opposed to the case with mammalian phagocytes, Stx produced by bacteria insideTetrahymenais cytotoxic, suggesting that these cells may represent a “missing link” between unicellular eukaryotic bacterial predators and phagocytotic mammalian cells.

scholarly journals Pharmacokinetics, Trypanosoma brucei gambiense Efficacy, and Time of Drug Action of DB829, a Preclinical Candidate for Treatment of Second-Stage Human African Trypanosomiasis

2013 ◽  
Vol 57 (11) ◽  
pp. 5330-5343 ◽  
Author(s):  
Tanja Wenzler ◽  
Sihyung Yang ◽  
Olivier Braissant ◽  
David W. Boykin ◽  
Reto Brun ◽  
...  
Keyword(s):  
Single Dose ◽  
Trypanosoma Brucei ◽  
Human African Trypanosomiasis ◽  
Drug Action ◽  
African Trypanosomiasis ◽  
Content Type ◽  
Second Stage ◽  
Trypanosoma Brucei Gambiense

ABSTRACTHuman African trypanosomiasis (HAT, also called sleeping sickness), a neglected tropical disease endemic to sub-Saharan Africa, is caused by the parasitesTrypanosoma brucei gambienseandT. brucei rhodesiense. Current drugs against this disease have significant limitations, including toxicity, increasing resistance, and/or a complicated parenteral treatment regimen. DB829 is a novel aza-diamidine that demonstrated excellent efficacy in mice infected withT. b. rhodesienseorT. b. bruceiparasites. The current study examined the pharmacokinetics,in vitroandin vivoactivity againstT. b. gambiense, and time of drug action of DB829 in comparison to pentamidine. DB829 showed outstandingin vivoefficacy in mice infected with parasites ofT. b. gambiensestrains, despite having higherin vitro50% inhibitory concentrations (IC50s) than againstT. b. rhodesiensestrain STIB900. A single dose of DB829 administered intraperitoneally (5 mg/kg of body weight) cured all mice infected with differentT. b. gambiensestrains. No cross-resistance was observed between DB829 and pentamidine inT. b. gambiensestrains isolated from melarsoprol-refractory patients. Compared to pentamidine, DB829 showed a greater systemic exposure when administered intraperitoneally, partially contributing to its improved efficacy. Isothermal microcalorimetry andin vivotime-to-kill studies revealed that DB829 is a slower-acting trypanocidal compound than pentamidine. A single dose of DB829 (20 mg/kg) administered intraperitoneally clears parasites from mouse blood within 2 to 5 days. In summary, DB829 is a promising preclinical candidate for the treatment of first- and second-stage HAT caused by bothTrypanosoma bruceisubspecies.

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