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Genetic validation of aminoacyl-tRNA synthetases as drug targets in Trypanosoma brucei.

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.

SUBMITTER: Kalidas S

PROVIDER: S-EPMC4000095 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundTreatment of parasitic diseases has been challenging due to evolution of drug resistant parasites, and thus there is need to identify new class of drugs and drug targets. Protein translation is important for survival of malarial parasite, Plasmodium, and the pathway is present in all of its life cycle stages. Aminoacyl tRNA synthetases are primary enzymes in protein translation as they catalyse amino acid addition to the cognate tRNA. This study sought to understand differences between Plasmodium and human aminoacyl tRNA synthetases through bioinformatics analysis.MethodsPlasmodium berghei, Plasmodium falciparum, Plasmodium fragile, Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Plasmodium yoelii and human aminoacyl tRNA synthetase sequences were retrieved from UniProt database and grouped into 20 families based on amino acid specificity. These families were further divided into two classes. Both families and classes were analysed. Motif discovery was carried out using the MEME software, sequence identity calculation was done using an in-house Python script, multiple sequence alignments were performed using PROMALS3D and TCOFFEE tools, and phylogenetic tree calculations were performed using MEGA vs 7.0 tool. Possible alternative binding sites were predicted using FTMap webserver and SiteMap tool.ResultsMotif discovery revealed Plasmodium-specific motifs while phylogenetic tree calculations showed that Plasmodium proteins have different evolutionary history to the human homologues. Human aaRSs sequences showed low sequence identity (below 40%) compared to Plasmodium sequences. Prediction of alternative binding sites revealed potential druggable sites in PfArgRS, PfMetRS and PfProRS at regions that are weakly conserved when compared to the human homologues. Multiple sequence analysis, motif discovery, pairwise sequence identity calculations and phylogenetic tree analysis showed significant differences between parasite and human aaRSs proteins despite functional and structural conservation. These differences may provide a basis for further exploration of Plasmodium aminoacyl tRNA synthetases as potential drug targets.ConclusionThis study showed that, despite, functional and structural conservation, Plasmodium aaRSs have key differences from the human homologues. These differences in Plasmodium aaRSs can be targeted to develop anti-malarial drugs with less toxicity to the host.

Project description: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.

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Genetic validation of aminoacyl-tRNA synthetases as drug targets in Trypanosoma brucei.

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