scholarly journals Selective Inhibitors of Methionyl-tRNA Synthetase Have Potent Activity against Trypanosoma brucei Infection in Mice

2011 ◽  
Vol 55 (5) ◽  
pp. 1982-1989 ◽  
Author(s):  
Sayaka Shibata ◽  
J. Robert Gillespie ◽  
Angela M. Kelley ◽  
Alberto J. Napuli ◽  
Zhongsheng Zhang ◽  
...  
Keyword(s):  
Cell Growth ◽  
Trypanosoma Brucei ◽  
Drug Targets ◽  
Treatment Options ◽  
Therapeutic Index ◽  
Comparative Sequence Analysis ◽  
Trna Synthetase ◽  
Sub Saharan Africa ◽  
Content Type ◽  
Trna Synthetases

ABSTRACTHuman African trypanosomiasis continues to be an important public health threat in extensive regions of sub-Saharan Africa. Treatment options for infected patients are unsatisfactory due to toxicity, difficult administration regimes, and poor efficacy of available drugs. The aminoacyl-tRNA synthetases were selected as attractive drug targets due to their essential roles in protein synthesis and cell survival. Comparative sequence analysis disclosed differences between the trypanosome and mammalian methionyl-tRNA synthetases (MetRSs) that suggested opportunities for selective inhibition using drug-like molecules. Experiments using RNA interference on the single MetRS ofTrypanosoma bruceidemonstrated that this gene product was essential for normal cell growth. Small molecules (diaryl diamines) similar to those shown to have potent activity on prokaryotic MetRS enzymes were synthesized and observed to have inhibitory activity on theT. bruceiMetRS (50% inhibitory concentration, <50 nM) and on bloodstream forms ofT. bruceicultures (50% effective concentration, as low as 4 nM). Twenty-one compounds had a close correlation between enzyme binding/inhibition andT. bruceigrowth inhibition, indicating that they were likely to be acting on the intended target. The compounds had minimal effects on mammalian cell growth at 20 μM, demonstrating a wide therapeutic index. The most potent compound was tested in the murine model of trypanosomiasis and demonstrated profound parasite suppression and delayed mortality. A homology model of theT. bruceiMetRS based on other MetRS structures was used to model binding of the lead diaryl diamine compounds. Future studies will focus on improving the pharmacological properties of the MetRS inhibitors.

scholarly journals Genetic Validation of Aminoacyl-tRNA Synthetases as Drug Targets in Trypanosoma brucei

2014 ◽  
Vol 13 (4) ◽  
pp. 504-516 ◽  
Author(s):  
Savitha Kalidas ◽  
Igor Cestari ◽  
Severine Monnerat ◽  
Qiong Li ◽  
Sandesh Regmi ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Trypanosoma Brucei ◽  
Drug Targets ◽  
Trna Synthetase ◽  
New Drugs ◽  
Sub Saharan Africa ◽  
Content Type ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases

ABSTRACT Human African trypanosomiasis (HAT) is an important public health threat in sub-Saharan Africa. Current drugs are unsatisfactory, and new drugs are being sought. Few validated enzyme targets are available to support drug discovery efforts, so our goal was to obtain essentiality data on genes with proven utility as drug targets. Aminoacyl-tRNA synthetases (aaRSs) are known drug targets for bacterial and fungal pathogens and are required for protein synthesis. Here we survey the essentiality of eight Trypanosoma brucei aaRSs by RNA interference (RNAi) gene expression knockdown, covering an enzyme from each major aaRS class: valyl-tRNA synthetase (ValRS) (class Ia), tryptophanyl-tRNA synthetase (TrpRS-1) (class Ib), arginyl-tRNA synthetase (ArgRS) (class Ic), glutamyl-tRNA synthetase (GluRS) (class 1c), threonyl-tRNA synthetase (ThrRS) (class IIa), asparaginyl-tRNA synthetase (AsnRS) (class IIb), and phenylalanyl-tRNA synthetase (α and β) (PheRS) (class IIc). Knockdown of mRNA encoding these enzymes in T. brucei mammalian stage parasites showed that all were essential for parasite growth and survival in vitro . The reduced expression resulted in growth, morphological, cell cycle, and DNA content abnormalities. ThrRS was characterized in greater detail, showing that the purified recombinant enzyme displayed ThrRS activity and that the protein localized to both the cytosol and mitochondrion. Borrelidin, a known inhibitor of ThrRS, was an inhibitor of T. brucei ThrRS and showed antitrypanosomal activity. The data show that aaRSs are essential for T. brucei survival and are likely to be excellent targets for drug discovery efforts.

scholarly journals An aminoacylation independent activity of PheRS/FARS promotes growth and proliferation

2020 ◽  
Author(s):  
Manh Tin Ho ◽  
Jiongming Lu ◽  
Beat Suter
Keyword(s):  
Cell Growth ◽  
Trna Synthetase ◽  
Tumor Formation ◽  
Follicle Cells ◽  
Strong Increase ◽  
Trna Synthetases ◽  
Elevated Expression ◽  
Wing Discs ◽  
Cell Growth And Proliferation ◽  
Normal Twin

Summary / AbstractAminoacyl-tRNA synthetases (aaRSs) not only load the appropriate amino acid onto their cognate tRNA, but many of them perform additional functions that are not necessarily related to their canonical activities. Phenylalanyl-tRNA synthetase (PheRS/FARS) levels are elevated in various cancer cells compared to their normal cell counterparts. However, whether and how these levels might contribute to tumor formation was not clear. Here, we show that PheRS is required for cell growth and proliferation. Interestingly, elevated expression of the α-PheRS subunit alone stimulates cell growth and proliferation. In the wing discs system, this leads to a strong increase of mitotic cells. Clonal analysis of twin spots in dividing follicle cells revealed that elevated expression of the α-PheRS subunit causes cells to grow and proliferate about 25% faster than their normal twin cells. Importantly, this stimulation of growth and proliferation neither required the β-PheRS subunit nor the aminoacylation activity, and it did not visibly stimulate translation. These results, therefore, revealed a non-canonical function of an ancient housekeeping enzyme, providing novel insight into its roles in health and diseases.

scholarly journals Serine-Threonine Kinases Encoded by SplithipAHomologs Inhibit Tryptophanyl-tRNA Synthetase

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Stine Vang Nielsen ◽  
Kathryn Jane Turnbull ◽  
Mohammad Roghanian ◽  
Rene Bærentsen ◽  
Maja Semanjski ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Gene Family ◽  
Sequence Similarity ◽  
Trna Synthetase ◽  
Bacterial Toxin ◽  
Content Type ◽  
Multidrug Tolerance ◽  
K 12

ABSTRACTType II toxin-antitoxin (TA) modules encode a stable toxin that inhibits cell growth and an unstable protein antitoxin that neutralizes the toxin by direct protein-protein contact.hipBAofEscherichia colistrain K-12 codes for HipA, a serine-threonine kinase that phosphorylates and inhibits glutamyl-tRNA synthetase. Induction ofhipAinhibits charging of glutamyl-tRNA that, in turn, inhibits translation and induces RelA-dependent (p)ppGpp synthesis and multidrug tolerance. Here, we describe the discovery of a three-component TA gene family that encodes toxin HipT, which exhibits sequence similarity with the C-terminal part of HipA. A genetic screening revealed thattrpSin high copy numbers suppresses HipT-mediated growth inhibition. We show that HipT ofE. coliO127 is a kinase that phosphorylates tryptophanyl-tRNA synthetasein vitroat a conserved serine residue. Consistently, induction ofhipTinhibits cell growth and stimulates production of (p)ppGpp. The gene immediately upstream fromhipT, calledhipS, encodes a small protein that exhibits sequence similarity with the N terminus of HipA. HipT kinase was neutralized by cognate HipSin vivo, whereas the third component, HipB, encoded by the first gene of the operon, did not counteract HipT kinase activity. However, HipB augmented the ability of HipS to neutralize HipT. Analysis of two additionalhipBST-homologous modules showed that, indeed, HipS functions as an antitoxin in these cases also. Thus,hipBSTconstitutes a novel family of tricomponent TA modules wherehipAhas been split into two genes,hipSandhipT, that function as a novel type of TA pair.IMPORTANCEBacterial toxin-antitoxin (TA) modules confer multidrug tolerance (persistence) that may contribute to the recalcitrance of chronic and recurrent infections. The first high-persister gene identified washipAofEscherichia colistrain K-12, which encodes a kinase that inhibits glutamyl-tRNA synthetase. ThehipAgene encodes the toxin of thehipBATA module, whilehipBencodes an antitoxin that counteracts HipA. Here, we describe a novel, widespread TA gene family,hipBST, that encodes HipT, which exhibits sequence similarity with the C terminus of HipA. HipT is a kinase that phosphorylates tryptophanyl-tRNA synthetase and thereby inhibits translation and induces the stringent response. Thus, this new TA gene family may contribute to the survival and spread of bacterial pathogens.

scholarly journals Genetic Validation of Trypanosoma brucei Glutathione Synthetase as an Essential Enzyme

2014 ◽  
Vol 13 (5) ◽  
pp. 614-624 ◽  
Author(s):  
Chelsea Pratt ◽  
Suong Nguyen ◽  
Margaret A. Phillips
Keyword(s):  
Trypanosoma Brucei ◽  
Drug Targets ◽  
Glutathione Synthetase ◽  
Biosynthetic Enzymes ◽  
Polyamine Biosynthesis ◽  
Content Type ◽  
Stage Disease ◽  
Trypanosomatid Parasites ◽  
Vector Borne ◽  
Essential Enzyme

ABSTRACTHuman African trypanosomiasis (HAT) is a debilitating and fatal vector-borne disease. Polyamine biosynthesis is the target of one of the key drugs (eflornithine) used for the treatment of late-stage disease, suggesting that the pathway might be exploited for the identification of additional drug targets. The polyamine spermidine is required in trypanosomatid parasites for formation of a unique redox cofactor termed trypanothione, which is formed from the conjugation of glutathione to spermidine. Here we characterize recombinantTrypanosoma bruceiglutathione synthetase (TbGS) and show that depletion ofTbGS in blood-form parasites using a regulated knockout strategy leads to loss of trypanothione and to cell death as quantified by fluorescence-activated cell sorter (FACS) analysis. These data suggest that >97% depletion ofTbGS is required before trypanothione is depleted and cell growth arrest is observed. Exogenous glutathione was able to partially compensate for the loss ofTbGS, suggesting that parasites are able to transport intact glutathione. Finally, reduced expression ofTbGS leads to increased levels of upstream glutathione biosynthetic enzymes and decreased expression of polyamine biosynthetic enzymes, providing evidence that the cells cross regulate the two branches of the trypanothione biosynthetic pathway to maintain spermidine and trypanothione homeostasis.

scholarly journals Aminoacyl tRNA Synthetases as Malarial Drug Targets: A Comparative Bioinformatics Study

2018 ◽  
Author(s):  
Dorothy Wavinya Nyamai ◽  
Özlem Tastan Bishop
Keyword(s):  
Amino Acid ◽  
Drug Targets ◽  
Motif Discovery ◽  
Protein Translation ◽  
Trna Synthetase ◽  
Potential Drug ◽  
Multiple Sequence ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Potential Drug Targets

AbstractTreatment of parasitic diseases has been challenging due to the development of drug resistance by parasites, and thus there is need to identify new class of drugs and drug targets. Protein translation is important for survival of plasmodium and the pathway is present in all the life cycle stages of the plasmodium parasite. Aminoacyl tRNA synthetases are primary enzymes in protein translation as they catalyse the first reaction where an amino acid is added to the cognate tRNA. Currently, there is limited research on comparative studies of aminoacyl tRNA synthetases as potential drug targets. The aim of this study is to understand differences between plasmodium and human aminoacyl tRNA synthetases through bioinformatics analysis. Plasmodium falciparum, P. fragile, P. vivax, P. ovale, P. knowlesi, P. bergei, P. malariae and human aminoacyl tRNA synthetase sequences were retrieved from UniProt database and grouped into 20 families based on amino acid specificity. Despite functional and structural conservation, multiple sequence analysis, motif discovery, pairwise sequence identity calculations and molecular phylogenetic analysis showed striking differences between parasite and human proteins. Prediction of alternate binding sites revealed potential druggable sites in PfArgRS, PfMetRS and PfProRS at regions that were weakly conserved when compared to the human homologues. These differences provide a basis for further exploration of plasmodium aminoacyl tRNA synthetases as potential drug targets.

scholarly journals Aminoacyl tRNA synthetases as potential drug targets of the Panthera pathogen Babesia

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Jyoti Chhibber-Goel ◽  
Sarthak Joshi ◽  
Amit Sharma
Keyword(s):  
Drug Targets ◽  
Bos Taurus ◽  
Homo Sapiens ◽  
Ixodes Scapularis ◽  
Protein Translation ◽  
Trna Synthetase ◽  
Panthera Tigris ◽  
Illegal Hunting ◽  
Trna Synthetases ◽  
Causative Agents

Abstract Background A century ago, pantheras were abundant across Asia. Illegal hunting and trading along with loss of habitat have resulted in the designation of Panthera as a genus of endangered species. In addition to the onslaught from humans, pantheras are also susceptible to outbreaks of several infectious diseases, including babesiosis. The latter is a hemoprotozoan disease whose causative agents are the eukaryotic parasites of the apicomplexan genus Babesia. Babesiosis affects a varied range of animals including humans (Homo sapiens), bovines (e.g. Bos taurus), pantheras (e.g. Panthera tigris, P. leo, P. pardus) and equines. Babesia spp. are transmitted by the tick vector Ixodes scapularis or ticks of domestic animals, namely Rhipicephalus (Boophilus) microplus and R. (B.) decoloratus. At the level of protein translation within these organisms, the conserved aminoacyl tRNA synthetase (aaRS) family offers an opportunity to identify the sequence and structural differences in the host (Panthera) and parasites (Babesia spp.) in order to exploit these for drug targeting Babesia spp. Methods Using computational tools we investigated the genomes of Babesia spp. and Panthera tigris so as to annotate their aaRSs. The sequences were analysed and their subcellular localizations were predicted using Target P1.1, SignalP 3.0, TMHMM v.2.0 and Deeploc 1.0 web servers. Structure-based analysis of the aaRSs from P. tigris and its protozoan pathogens Babesia spp. was performed using Phyre2 and chimera. Results We identified 33 (B. bovis), 34 (B. microti), 33 (B. bigemina) and 33 (P. tigris) aaRSs in these respective organisms. Poor sequence identity (~ 20–50%) between aaRSs from Babesia spp. and P. tigris was observed and this merits future experiments to validate new drug targets against Babesia spp. Conclusions Overall this work provides a foundation for experimental investigation of druggable aaRSs from Babesia sp. in an effort to control Babesiosis in Panthera.

scholarly journals Molecular basis of the multifaceted functions of human leucyl-tRNA synthetase in protein synthesis and beyond

2020 ◽  
Vol 48 (9) ◽  
pp. 4946-4959
Author(s):  
Ru-Juan Liu ◽  
Tao Long ◽  
Hao Li ◽  
JingHua Zhao ◽  
Jing Li ◽  
...  
Keyword(s):  
Drug Development ◽  
Molecular Basis ◽  
Drug Targets ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Assembly Mechanism ◽  
Mtorc1 Pathway ◽  
Pathogenic Microbes

Abstract Human cytosolic leucyl-tRNA synthetase (hcLRS) is an essential and multifunctional enzyme. Its canonical function is to catalyze the covalent ligation of leucine to tRNALeu, and it may also hydrolyze mischarged tRNAs through an editing mechanism. Together with eight other aminoacyl-tRNA synthetases (AaRSs) and three auxiliary proteins, it forms a large multi-synthetase complex (MSC). Beyond its role in translation, hcLRS has an important moonlight function as a leucine sensor in the rapamycin complex 1 (mTORC1) pathway. Since this pathway is active in cancer development, hcLRS is a potential target for anti-tumor drug development. Moreover, LRS from pathogenic microbes are proven drug targets for developing antibiotics, which however should not inhibit hcLRS. Here we present the crystal structure of hcLRS at a 2.5 Å resolution, the first complete structure of a eukaryotic LRS, and analyze the binding of various compounds that target different sites of hcLRS. We also deduce the assembly mechanism of hcLRS into the MSC through reconstitution of the entire mega complex in vitro. Overall, our study provides the molecular basis for understanding both the multifaceted functions of hcLRS and for drug development targeting these functions.

scholarly journals MupB, a New High-Level Mupirocin Resistance Mechanism in Staphylococcus aureus

2012 ◽  
Vol 56 (4) ◽  
pp. 1916-1920 ◽  
Author(s):  
Christine Seah ◽  
David C. Alexander ◽  
Lisa Louie ◽  
Andrew Simor ◽  
Donald E. Low ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Shuttle Vector ◽  
Resistance Mechanism ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Content Type ◽  
Trna Synthetases ◽  
Mupirocin Resistance ◽  
High Level ◽  
Bacterial Protein Synthesis

ABSTRACTMupirocin is a topical antibiotic used for the treatment of skin infections and the eradication of methicillin-resistantStaphylococcus aureuscarriage. It inhibits bacterial protein synthesis by interfering with isoleucyl-tRNA synthetase activity. High-level mupirocin resistance (MIC of ≥512 μg/ml) is mediated by the expression ofmupA(ileS2), which encodes an alternate isoleucyl-tRNA synthetase. In this study, we describe high-level mupirocin resistance mediated by a novel locus,mupB. ThemupBgene (3,102 bp) shares 65.5% sequence identity withmupAbut only 45.5% identity withileS. The deduced MupB protein shares 58.1% identity (72.3% similarity) and 25.4% identity (41.8% similarity) with MupA and IleS, respectively. Despite this limited homology, MupB contains conserved motifs found in class I tRNA synthetases. Attempts to transfer high-level mupirocin resistance via conjugation or transformation (using plasmid extracts from anmupB-containing strain) were unsuccessful. However, by cloning themupBgene into a shuttle vector, it was possible to transfer the resistance phenotype to susceptibleS. aureusby electroporation, proving thatmupBwas responsible for the high-level mupirocin resistance. Further studies need to be done to determine the prevalence ofmupBand to understand risk factors and outcomes associated with resistance mediated by this gene.

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 Proteomic identification of novel cytoskeletal proteins associated with TbPLK, an essential regulator of cell morphogenesis in Trypanosoma brucei

2015 ◽  
Vol 26 (17) ◽  
pp. 3013-3029 ◽  
Author(s):  
Michael R. McAllaster ◽  
Kyojiro N. Ikeda ◽  
Ana Lozano-Núñez ◽  
Dorothea Anrather ◽  
Verena Unterwurzacher ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Molecular Mechanisms ◽  
Treatment Options ◽  
Cytoskeletal Proteins ◽  
Sub Saharan Africa ◽  
Molecular Networks ◽  
Cell Morphogenesis ◽  
Daughter Cells ◽  
Sub Saharan ◽  
Attachment Zone

Trypanosoma brucei is the causative agent of African sleeping sickness, a devastating disease endemic to sub-Saharan Africa with few effective treatment options. The parasite is highly polarized, including a single flagellum that is nucleated at the posterior of the cell and adhered along the cell surface. These features are essential and must be transmitted to the daughter cells during division. Recently we identified the T. brucei homologue of polo-like kinase (TbPLK) as an essential morphogenic regulator. In the present work, we conduct proteomic screens to identify potential TbPLK binding partners and substrates to better understand the molecular mechanisms of kinase function. These screens identify a cohort of proteins, most of which are completely uncharacterized, which localize to key cytoskeletal organelles involved in establishing cell morphology, including the flagella connector, flagellum attachment zone, and bilobe structure. Depletion of these proteins causes substantial changes in cell division, including mispositioning of the kinetoplast, loss of flagellar connection, and prevention of cytokinesis. The proteins identified in these screens provide the foundation for establishing the molecular networks through which TbPLK directs cell morphogenesis in T. brucei.

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