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 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 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 Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

Utp22p acts in concert with Utp8p to channel aminoacyl-tRNA from the nucleolus to the nuclear tRNA export receptor Los1p but not Msn5p

2012 ◽  
Vol 90 (6) ◽  
pp. 731-749 ◽  
Author(s):  
Manoja B.K. Eswara ◽  
Ashley Clayton ◽  
Dev Mangroo
Keyword(s):  
Protein Complex ◽  
Trna Synthetase ◽  
Nuclear Pore ◽  
Independent Manner ◽  
Nuclear Retention ◽  
Trna Synthetases ◽  
U3 Snorna ◽  
Aminoacyl Trna Synthetases ◽  
Export Receptors

Utp8p is an essential nucleolar protein that channels aminoacyl-tRNAs from aminoacyl-tRNA synthetases in the nucleolus to the nuclear tRNA export receptors located in the nucleoplasm and nuclear pore complex in Saccharomyces cerevisiae . Utp8p is also part of the U3 snoRNA-associated protein complex involved in 18S rRNA biogenesis in the nucleolus. We report that Utp22p, which is another member of the U3 snoRNA-associated protein complex, is also an intranuclear component of the nuclear tRNA export machinery. Depletion of Utp22p results in nuclear retention of mature tRNAs derived from intron-containing and intronless precursors. Moreover, Utp22p copurifies with the nuclear tRNA export receptor Los1p, the aminoacyl-tRNA synthetase Tys1p and Utp8p, but not with the RanGTPase Gsp1p and the nuclear tRNA export receptor Msn5p. Utp22p interacts directly with Utp8p and Los1p in a tRNA-independent manner in vitro. Utp22p also interacts directly with Tys1p, but this binding is stimulated when Tys1p is bound to tRNA. However, Utp22p, unlike Utp8p, does not bind tRNA saturably. These data suggest that Utp22p recruits Utp8p to aminoacyl-tRNA synthetases in the nucleolus to collect aminoacyl-tRNA and then accompanies the Utp8p–tRNA complex to deliver the aminoacyl-tRNAs to Los1p but not Msn5p. It is possible that Nrap/Nol6, the mammalian orthologue of Utp22p, plays a role in channelling aminoacyl-tRNA to the nuclear tRNA export receptor exportin-t.

scholarly journals Multiple Mutations in Mycobacterium tuberculosis MmpL3 Increase Resistance to MmpL3 Inhibitors

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Matthew B. McNeil ◽  
Theresa O’Malley ◽  
Devon Dennison ◽  
Catherine D. Shelton ◽  
Bjorn Sunde ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
New Drugs ◽  
Content Type ◽  
Mechanism Of Resistance ◽  
Primary Mechanism ◽  
Resistant Mutants ◽  
Mycobacterial Cell Wall

ABSTRACT The Mycobacterium tuberculosis protein MmpL3 performs an essential role in cell wall synthesis, since it effects the transport of trehalose monomycolates across the inner membrane. Numerous structurally diverse pharmacophores have been identified as inhibitors of MmpL3 largely based on the identification of resistant isolates with mutations in MmpL3. For some compounds, it is possible there are different primary or secondary targets. Here, we have investigated resistance to the spiral amine class of compounds. Isolation and sequencing of resistant mutants demonstrated that all had mutations in MmpL3. We hypothesized that if additional targets of this pharmacophore existed, then successive rounds to generate resistant isolates might reveal mutations in other loci. Since compounds were still active against resistant isolates, albeit with reduced potency, we isolated resistant mutants in this background at higher concentrations. After a second round of isolation with the spiral amine, we found additional mutations in MmpL3. To increase our chance of finding alternative targets, we ran a third round of isolation using a different molecule scaffold (AU1235, an adamantyl urea). Surprisingly, we obtained further mutations in MmpL3. Multiple mutations in MmpL3 increased the level and spectrum of resistance to different pharmacophores but did not incur a fitness cost in vitro. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores. IMPORTANCE Mycobacterium tuberculosis is a major global human pathogen, and new drugs and new drug targets are urgently required. Cell wall biosynthesis is a major target of current tuberculosis drugs and of new agents under development. Several new classes of molecules appear to have the same target, MmpL3, which is involved in the export and synthesis of the mycobacterial cell wall. However, there is still debate over whether MmpL3 is the primary or only target for these classes. We wanted to confirm the mechanism of resistance for one series. We identified mutations in MmpL3 which led to resistance to the spiral amine series. High-level resistance to these compounds and two other series was conferred by multiple mutations in the same protein (MmpL3). These mutations did not reduce growth rate in culture. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores.

scholarly journals Structure-guided enhancement of selectivity of chemical probe inhibitors targeting bacterial seryl-tRNA synthetase

2019 ◽  
Author(s):  
Ricky Cain ◽  
Ramya Salimraj ◽  
Avinash S. Punekar ◽  
Dom Bellini ◽  
Colin W. G. Fishwick ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Drug Discovery ◽  
Bacterial Species ◽  
Trna Synthetase ◽  
Human Homologue ◽  
X Ray ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Chemical Inhibition ◽  
Selective Chemical

AbstractAminoacyl-tRNA synthetases are ubiquitous and essential enzymes for protein synthesis and also a variety of other metabolic processes, especially in bacterial species. Bacterial aminoacyl-tRNA synthetases represent attractive and validated targets for antimicrobial drug discovery if issues of prokaryotic versus eukaryotic selectivity and antibiotic resistance generation can be addressed. We have determined high resolution X-ray crystal structures of the Escherichia coli and Staphylococcus aureus seryl-tRNA synthetases in complex with aminoacyl adenylate analogues and applied a structure-based drug discovery approach to explore and identify a series of small molecule inhibitors that selectively inhibit bacterial seryl-tRNA synthetases with greater than two orders of magnitude compared to their human homologue, demonstrating a route to selective chemical inhibition of these bacterial targets.

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 Relaxed Substrate Specificity Leads to Extensive tRNA Mischarging by Streptococcus pneumoniae Class I and Class II Aminoacyl-tRNA Synthetases

mBio ◽  
2014 ◽  
Vol 5 (5) ◽  
Author(s):  
Jennifer Shepherd ◽  
Michael Ibba
Keyword(s):  
Quality Control ◽  
Cell Wall ◽  
Protein Synthesis ◽  
Streptococcus Pneumoniae ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Trna Synthetase ◽  
Content Type ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases

ABSTRACTAminoacyl-tRNA synthetases provide the first step in protein synthesis quality control by discriminating cognate from noncognate amino acid and tRNA substrates. While substrate specificity is enhanced in many instances bycis-andtrans-editing pathways, it has been revealed that in organisms such asStreptococcus pneumoniaesome aminoacyl-tRNA synthetases display significant tRNA mischarging activity. To investigate the extent of tRNA mischarging in this pathogen, the aminoacylation profiles of class I isoleucyl-tRNA synthetase (IleRS) and class II lysyl-tRNA synthetase (LysRS) were determined. Pneumococcal IleRS mischarged tRNAIlewith both Val, as demonstrated in other bacteria, and Leu in a tRNA sequence-dependent manner. IleRS substrate specificity was achieved in an editing-independent manner, indicating that tRNA mischarging would only be significant under growth conditions where Ile is depleted. Pneumococcal LysRS was found to misaminoacylate tRNALyswith Ala and to a lesser extent Thr and Ser, with mischarging efficiency modulated by the presence of an unusual U4:G69 wobble pair in the acceptor stems of both pneumococcal tRNALysisoacceptors. Addition of thetrans-editing factor MurM, which also functions in peptidoglycan synthesis, reduced Ala-tRNALysproduction by LysRS, providing evidence for cross talk between the protein synthesis and cell wall biogenesis pathways. Mischarging of tRNALysby AlaRS was also observed, and this would provide additional potential MurM substrates. More broadly, the extensive mischarging activities now described for a number ofStreptococcus pneumoniaeaminoacyl-tRNA synthetases suggest that adaptive misaminoacylation may contribute significantly to the viability of this pathogen during amino acid starvation.IMPORTANCEStreptococcus pneumoniaeis a common causative agent of several debilitating and potentially life-threatening infections, such as pneumonia, meningitis, and infectious endocarditis. Such infections are increasingly difficult to treat due to widespread development of penicillin resistance. High-level penicillin resistance is known to depend in part upon MurM, a protein involved in both aminoacyl-tRNA-dependent synthesis of indirect amino acid cross-linkages within cell wall peptidoglycan and in translation quality control. The involvement of MurM in both protein synthesis and antibiotic resistance identify it as a potential target for the development of new and potent antibiotics for pneumococcal infections. The goals of this work were to identify and characterizeS. pneumoniaepathways that can synthesize mischarged tRNAs and to relate these activities to expected changes in protein and peptidoglycan biosynthesis during antibiotic and nutritional stress.

scholarly journals A Leucyl-tRNA Synthetase Inhibitor with Broad-Spectrum Antimycobacterial Activity

2021 ◽  
Vol 65 (5) ◽  
Author(s):  
Uday S. Ganapathy ◽  
Rubén González del Rio ◽  
Mónica Cacho-Izquierdo ◽  
Fátima Ortega ◽  
Joël Lelièvre ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
De Novo ◽  
Resistant Mutant ◽  
Trna Synthetase ◽  
New Drugs ◽  
Mouse Lung ◽  
Infection Model ◽  
Mutant Selection ◽  
Content Type

ABSTRACT Global infections by nontuberculous mycobacteria (NTM) are steadily rising. New drugs are needed to treat NTM infections, but the NTM drug pipeline remains poorly populated and focused on repurposing or reformulating approved antibiotics. We sought to accelerate de novo NTM drug discovery by testing advanced compounds with established activity against Mycobacterium tuberculosis. 3-Aminomethyl 4-halogen benzoxaboroles, a novel class of leucyl-tRNA synthetase inhibitors, were recently discovered as active against M. tuberculosis. Here, we report that the benzoxaborole EC/11770 is not only a potent antitubercular agent but is active against the M. abscessus and M. avium complexes. Focusing on M. abscessus, which causes the most-difficult-to-cure NTM disease, we show that EC/11770 retained potency against drug-tolerant biofilms in vitro and was effective in a mouse lung infection model. Resistant mutant selection experiments showed a low frequency of resistance and confirmed leucyl-tRNA synthetase as the target. This work establishes the benzoxaborole EC/11770 as a novel preclinical candidate for the treatment of NTM lung disease and tuberculosis and validates leucyl-tRNA synthetase as an attractive target for the development of broad-spectrum antimycobacterials.

scholarly journals Leishmania donovani Parasites Are Inhibited by the Benzoxaborole AN2690 Targeting Leucyl-tRNA Synthetase

2018 ◽  
Vol 62 (9) ◽  
Author(s):  
Reetika Manhas ◽  
Smriti Tandon ◽  
Shib Sankar Sen ◽  
Neha Tiwari ◽  
Manoj Munde ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Drug Targets ◽  
Fungal Pathogens ◽  
Leishmania Donovani ◽  
Protozoan Parasite ◽  
Gene Replacement ◽  
Trna Synthetase ◽  
Content Type

ABSTRACT Visceral leishmaniasis is an important public health threat in parts of India. It is caused by a protozoan parasite, Leishmania donovani. Currently available drugs manifest severe side effects. Hence, there is a need to identify new drug targets and drugs. Aminoacyl-tRNA synthetases, required for protein synthesis, are known drug targets for bacterial and fungal pathogens. The aim of the present study was to obtain essentiality data for Leishmania donovani leucyl-tRNA synthetase (LdLRS) by gene replacement. Gene replacement studies indicate that this enzyme plays an essential role in the viability of this pathogenic organism and appears to be indispensable for its survival in vitro. The heterozygous mutant parasites demonstrated a growth deficit and reduced infectivity in mouse macrophages compared to the wild-type cells. We also report that Leishmania donovani recombinant LRS displayed aminoacylation activity and that the protein localized to both the cytosol and the mitochondrion. A broad-spectrum antifungal, 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690), was found to inhibit parasite growth in both the promastigote and amastigote stages in vitro as well as in vivo in BALB/c mice. This compound exhibited low toxicity to mammalian cells. AN2690 was effective in inhibiting the aminoacylation activity of the recombinant LdLRS. We provide preliminary chemical validation of LdLRS as a drug target by showing that AN2690 is an inhibitor both of L. donovani LRS and of L. donovani cell growth.

Sign in / Sign up

Export Citation Format

Share Document