Polyethylene Glycol Impacts Conformation and Dynamics of Escherichia coli Prolyl-tRNA Synthetase Via Crowding and Confinement Effects.

Polyethylene glycol (PEG) is a flexible, nontoxic polymer commonly used in biological and medical research, and it is generally regarded as biologically inert. PEG molecules of variable sizes are also used as crowding agents to mimic intracellular environments. A recent study with PEG crowders revealed decreased catalytic activity of Escherichia coli prolyl-tRNA synthetase (Ec ProRS), where the smaller molecular weight PEGs had the maximum impact. The molecular mechanism of the crowding effects of PEGs is not clearly understood. PEG may impact protein conformation and dynamics, thus its function. In the present study, the effects of PEG molecules of various molecular weights and concentrations on the conformation and dynamics of Ec ProRS were investigated using a combined experimental and computational approach including intrinsic tryptophan fluorescence spectroscopy, atomic force microscopy, and atomistic molecular dynamic simulations. Results of the present study suggest that lower molecular weight PEGs in the dilute regime have modest effects on the conformational dynamics of Ec ProRS but impact the catalytic function primarily via the excluded volume effect; they form large clusters blocking the active site pocket. In contrast, the larger molecular weight PEGs in dilute to semidilute regimes have a significant impact on the protein's conformational dynamics; they wrap on the protein surface through noncovalent interactions. Thus, lower-molecular-weight PEG molecules impact protein dynamics and function via crowding effects, whereas larger PEGs induce confinement effects. These results have implications for the development of inhibitors for protein targets in a crowded cellular environment.

Exploration of aminoacyl-tRNA synthetases from eukaryotic parasites for drug development.

Parasitic diseases result in considerable human morbidity and mortality. The continuous emergence and spread of new drug-resistant parasite strains is an obstacle to controlling and eliminating many parasitic diseases. Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous enzymes essential for protein synthesis. The design and development of diverse small molecule, drug-like inhibitors against parasite-encoded and expressed aaRSs have validated this enzyme family as druggable. In this work, we have compiled the progress to date towards establishing the druggability of aaRSs in terms of their biochemical characterization, validation as targets, inhibitor development, and structural interpretation from parasites responsible for malaria (Plasmodium), lymphatic filariasis (Brugia,Wuchereria bancrofti), giardiasis (Giardia), toxoplasmosis (Toxoplasma gondii), leishmaniasis (Leishmania), cryptosporidiosis (Cryptosporidium), and trypanosomiasis (Trypanosoma). This work thus provides a robust framework for the systematic dissection of aaRSs from these pathogens and will facilitate the cross-usage of potential inhibitors to jump-start anti-parasite drug development.

A hit expansion of 3-benzamidopyrazine-2-carboxamide: Toward inhibitors of prolyl-tRNA synthetase with antimycobacterial activity.

This study presents an exploration of the chemical space around derivatives of 3-benzamidopyrazine-2-carboxamides, previously identified as potent antimycobacterial compounds with predicted binding to mycobacterial prolyl-transfer RNA synthetase. New urea derivatives (Series-1) were generally inactive, probably due to their preference for cis-trans conformation (confirmed by density functional theory calculations and experimentally by nuclear overhauser effect spectroscopy NMR). Series-2 (3-benzamidopyrazine-2-carboxamides with disubstituted benzene ring) demonstrated that substituents larger than fluorine are not tolerated in the ortho position of the benzene ring. This series brought two new compounds (21: R = 2-F, 4-Cl and 22: R = 2-F, 4-Br) with in vitro activity against Mycobacterium tuberculosis H37Rv as well as multidrug-resistant clinical isolates, with minimum inhibitory concentration ranging from 6.25 to 25 µg/mL. The lactone-type derivatives 4H-pyrazino[2,3-d][1,3]oxazin-4-ones (Series-3) were inactive, but solvent stability studies of compound 29 indicated that they might be developed to usable lactone prodrugs of inhibitors of mycobacterial aspartate decarboxylase (PanD).

Aminoacyl-tRNA synthetase inhibition activates a pathway that branches from the canonical amino acid response in mammalian cells.

Signaling pathways that sense amino acid abundance are integral to tissue homeostasis and cellular defense. Our laboratory has previously shown that halofuginone (HF) inhibits the prolyl-tRNA synthetase catalytic activity of glutamyl-prolyl-tRNA synthetase (EPRS), thereby activating the amino acid response (AAR). We now show that HF treatment selectively inhibits inflammatory responses in diverse cell types and that these therapeutic benefits occur in cells that lack GCN2, the signature effector of the AAR. Depletion of arginine, histidine, or lysine from cultured fibroblast-like synoviocytes recapitulates key aspects of HF treatment, without utilizing GCN2 or mammalian target of rapamycin complex 1 pathway signaling. Like HF, the threonyl-tRNA synthetase inhibitor borrelidin suppresses the induction of tissue remodeling and inflammatory mediators in cytokine-stimulated fibroblast-like synoviocytes without GCN2, but both aminoacyl-tRNA synthetase (aaRS) inhibitors are sensitive to the removal of GCN1. GCN1, an upstream component of the AAR pathway, binds to ribosomes and is required for GCN2 activation. These observations indicate that aaRS inhibitors, like HF, can modulate inflammatory response without the AAR/GCN2 signaling cassette, and that GCN1 has a role that is distinct from its activation of GCN2. We propose that GCN1 participates in a previously unrecognized amino acid sensor pathway that branches from the canonical AAR.

Glutamyl-Prolyl-tRNA Synthetase Regulates Proline-Rich Pro-Fibrotic Protein Synthesis During Cardiac Fibrosis.

RATIONALE: Increased protein synthesis of profibrotic genes is a common feature in cardiac fibrosis and heart failure. Despite this observation, critical factors and molecular mechanisms for translational control of profibrotic genes during cardiac fibrosis remain unclear. OBJECTIVE: To investigate the role of a bifunctional ARS (aminoacyl-tRNA synthetase), EPRS (glutamyl-prolyl-tRNA synthetase) in translational control of cardiac fibrosis. METHODS AND RESULTS: Results from reanalyses of multiple publicly available data sets of human and mouse heart failure, demonstrated that EPRS acted as an integrated node among the ARSs in various cardiac pathogenic processes. We confirmed that EPRS was induced at mRNA and protein levels (≈1.5-2.5-fold increase) in failing hearts compared with nonfailing hearts using our cohort of human and mouse heart samples. Genetic knockout of one allele of Eprs globally (Eprs+/-) using CRISPR-Cas9 technology or in a Postn-Cre-dependent manner (Eprsflox/+; PostnMCM/+) strongly reduces cardiac fibrosis (≈50% reduction) in isoproterenol-, transverse aortic constriction-, and myocardial infarction (MI)-induced heart failure mouse models. Inhibition of EPRS using a PRS (prolyl-tRNA synthetase)-specific inhibitor, halofuginone, significantly decreases translation efficiency (TE) of proline-rich collagens in cardiac fibroblasts as well as TGF-ß (transforming growth factor-ß)-activated myofibroblasts. Overexpression of EPRS increases collagen protein expression in primary cardiac fibroblasts under TGF-ß stimulation. Using transcriptome-wide RNA-Seq and polysome profiling-Seq in halofuginone-treated fibroblasts, we identified multiple novel Pro-rich genes in addition to collagens, such as Ltbp2 (latent TGF-ß-binding protein 2) and Sulf1 (sulfatase 1), which are translationally regulated by EPRS. SULF1 is highly enriched in human and mouse myofibroblasts. In the primary cardiac fibroblast culture system, siRNA-mediated knockdown of SULF1 attenuates cardiac myofibroblast activation and collagen deposition. Overexpression of SULF1 promotes TGF-ß-induced myofibroblast activation and partially antagonizes anti-fibrotic effects of halofuginone treatment. CONCLUSIONS: Our results indicate that EPRS preferentially controls translational activation of proline codon rich profibrotic genes in cardiac fibroblasts and augments pathological cardiac remodeling. Graphical Abstract: A graphical abstract is available for this article.

Interspecies analysis of MYC targets identifies tRNA synthetases as mediators of growth and survival in MYC-overexpressing cells.

Aberrant MYC oncogene activation is one of the most prevalent characteristics of cancer. By overlapping datasets of Drosophila genes that are insulin-responsive and also regulate nucleolus size, we enriched for Myc target genes required for cellular biosynthesis. Among these, we identified the aminoacyl tRNA synthetases (aaRSs) as essential mediators of Myc growth control in Drosophila and found that their pharmacologic inhibition is sufficient to kill MYC-overexpressing human cells, indicating that aaRS inhibitors might be used to selectively target MYC-driven cancers. We suggest a general principle in which oncogenic increases in cellular biosynthesis sensitize cells to disruption of protein homeostasis.

Inhibitors of aminoacyl-tRNA synthetases as antimycobacterial compounds: An up-to-date review.

Aminoacyl-tRNA synthetases (aaRSs) are crucial for the correct assembly of amino acids to cognate tRNA to maintain the fidelity of proteosynthesis. AaRSs have become a hot target in antimicrobial research. Three aaRS inhibitors are already in clinical practice; antibacterial mupirocin inhibits the synthetic site of isoleucyl-tRNA synthetase, antifungal tavaborole inhibits the editing site of leucyl-tRNA synthetase, and antiprotozoal halofuginone inhibits proline-tRNA synthetase. According to the World Health Organization, tuberculosis globally remains the leading cause of death from a single infectious agent. The rising incidence of multidrug-resistant tuberculosis is alarming and urges the search for new antimycobacterial compounds, preferably with yet unexploited mechanism of action. In this literature review, we have covered the up-to-date state in the field of inhibitors of mycobacterial aaRSs. The most studied aaRS in mycobacteria is LeuRS with at least four structural types of inhibitors, followed by TyrRS and AspRS. Inhibitors of MetRS, LysRS, and PheRS were addressed in a single significant study each. In many cases, the enzyme inhibition activity translated into micromolar or submicromolar inhibition of growth of mycobacteria. The most promising aaRS inhibitor as an antimycobacterial compound is GSK656 (compound 8), the only aaRS inhibitor in clinical trials (Phase IIa) for systemic use against tuberculosis. GSK656 is orally available and shares the oxaborole tRNA-trapping mechanism of action with antifungal tavaborole.

Towards Novel 3-Aminopyrazinamide-Based Prolyl-tRNA Synthetase Inhibitors: In Silico Modelling, Thermal Shift Assay and Structural Studies.

Human cytosolic prolyl-tRNA synthetase (HcProRS) catalyses the formation of the prolyl-tRNAPro, playing an important role in protein synthesis. Inhibition of HcProRS activity has been shown to have potential benefits in the treatment of fibrosis, autoimmune diseases and cancer. Recently, potent pyrazinamide-based inhibitors were identified by a high-throughput screening (HTS) method, but no further elaboration was reported. The pyrazinamide core is a bioactive fragment found in numerous clinically validated drugs and has been subjected to various modifications. Therefore, we applied a virtual screening protocol to our in-house library of pyrazinamide-containing small molecules, searching for potential novel HcProRS inhibitors. We identified a series of 3-benzylaminopyrazine-2-carboxamide derivatives as positive hits. Five of them were confirmed by a thermal shift assay (TSA) with the best compounds 3b and 3c showing EC50 values of 3.77 and 7.34 µM, respectively, in the presence of 1 mM of proline (Pro) and 3.45 µM enzyme concentration. Co-crystal structures of HcProRS in complex with these compounds and Pro confirmed the initial docking studies and show how the Pro facilitates binding of the ligands that compete with ATP substrate. Modelling 3b into other human class II aminoacyl-tRNA synthetases (aaRSs) indicated that the subtle differences in the ATP binding site of these enzymes likely contribute to its potential selective binding of HcProRS. Taken together, this study successfully identified novel HcProRS binders from our anti-tuberculosis in-house compound library, displaying opportunities for repurposing old drug candidates for new applications such as therapeutics in HcProRS-related diseases.

Aminoacyl-tRNA Synthetases as Valuable Targets for Antimicrobial Drug Discovery.

Aminoacyl-tRNA synthetases (aaRSs) catalyze the esterification of tRNA with a cognate amino acid and are essential enzymes in all three kingdoms of life. Due to their important role in the translation of the genetic code, aaRSs have been recognized as suitable targets for the development of small molecule anti-infectives. In this review, following a concise discussion of aaRS catalytic and proof-reading activities, the various inhibitory mechanisms of reported natural and synthetic aaRS inhibitors are discussed. Using the expanding repository of ligand-bound X-ray crystal structures, we classified these compounds based on their binding sites, focusing on their ability to compete with the association of one, or more of the canonical aaRS substrates. In parallel, we examined the determinants of species-selectivity and discuss potential resistance mechanisms of some of the inhibitor classes. Combined, this structural perspective highlights the opportunities for further exploration of the aaRS enzyme family as antimicrobial targets.

Progress and challenges in aminoacyl-tRNA synthetase-based therapeutics.

Aminoacyl-tRNA synthetases (ARSs) are universal enzymes that catalyze the attachment of amino acids to the 3' ends of their cognate tRNAs. The resulting aminoacylated tRNAs are escorted to the ribosome where they enter protein synthesis. By specifically matching amino acids to defined anticodon sequences in tRNAs, ARSs are essential to the physical interpretation of the genetic code. In addition to their canonical role in protein synthesis, ARSs are also involved in RNA splicing, transcriptional regulation, translation, and other aspects of cellular homeostasis. Likewise, aminoacylated tRNAs serve as amino acid donors for biosynthetic processes distinct from protein synthesis, including lipid modification and antibiotic biosynthesis. Thanks to the wealth of details on ARS structures and functions and the growing appreciation of their additional roles regulating cellular homeostasis, opportunities for the development of clinically useful ARS inhibitors are emerging to manage microbial and parasite infections. Exploitation of these opportunities has been stimulated by the discovery of new inhibitor frameworks, the use of semi-synthetic approaches combining chemistry and genome engineering, and more powerful techniques for identifying leads from the screening of large chemical libraries. Here, we review the inhibition of ARSs by small molecules, including the various families of natural products, as well as inhibitors developed by either rational design or high-throughput screening as antibiotics and anti-parasitic therapeutics.

Inhibition of tRNA Synthetases Induces Persistence in Chlamydia.

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections, and Chlamydia pneumoniae causes community-acquired respiratory infections. In vivo, the host immune system will release gamma interferon (IFN-γ) to combat infection. IFN-γ activates human cells to produce the tryptophan (Trp)-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO). Consequently, there is a reduction in cytosolic Trp in IFN-γ-activated host cells. In evolving to obligate intracellular dependence, Chlamydia has significantly reduced its genome size and content, as it relies on the host cell for various nutrients. Importantly, C. trachomatis and C. pneumoniae are Trp auxotrophs and are starved for this essential nutrient when the human host cell is exposed to IFN-γ. To survive this, chlamydiae enter an alternative developmental state referred to as persistence. Chlamydial persistence is characterized by a halt in the division cycle, aberrant morphology, and, in the case of IFN-γ-induced persistence, Trp codon-dependent changes in transcription. We hypothesize that these changes in transcription are dependent on the particular amino acid starvation state. To investigate the chlamydial response mechanisms acting when other amino acids become limiting, we tested the efficacy of prokaryote-specific tRNA synthetase inhibitors, indolmycin and AN3365, to mimic starvation of Trp and leucine, respectively. We show that these drugs block chlamydial growth and induce changes in morphology and transcription consistent with persistence. Importantly, growth inhibition was reversed when the compounds were removed from the medium. With these data, we find that indolmycin and AN3365 are valid tools that can be used to mimic the persistent state independently of IFN-γ.

Identification of Selective Novel Hits against Plasmodium falciparum Prolyl tRNA Synthetase Active Site and a Predicted Allosteric Site Using in silico Approaches.

Recently, there has been increased interest in aminoacyl tRNA synthetases (aaRSs) as potential malarial drug targets. These enzymes play a key role in protein translation by the addition of amino acids to their cognate tRNA. The aaRSs are present in all Plasmodium life cycle stages, and thus present an attractive malarial drug target. Prolyl tRNA synthetase is a class II aaRS that functions in charging tRNA with proline. Various inhibitors against Plasmodium falciparum ProRS (PfProRS) active site have been designed. However, none have gone through clinical trials as they have been found to be highly toxic to human cells. Recently, a possible allosteric site was reported in PfProRS with two possible allosteric modulators: glyburide and TCMDC-124506. In this study, we sought to identify novel selective inhibitors targeting PfProRS active site and possible novel allosteric modulators of this enzyme. To achieve this, virtual screening of South African natural compounds against PfProRS and the human homologue was carried out using AutoDock Vina. The modulation of protein motions by ligand binding was studied by molecular dynamics (MD) using the GROningen MAchine for Chemical Simulations (GROMACS) tool. To further analyse the protein global motions and energetic changes upon ligand binding, principal component analysis (PCA), and free energy landscape (FEL) calculations were performed. Further, to understand the effect of ligand binding on the protein communication, dynamic residue network (DRN) analysis of the MD trajectories was carried out using the MD-TASK tool. A total of ten potential natural hit compounds were identified with strong binding energy scores. Binding of ligands to the protein caused observable global and residue level changes. Dynamic residue network calculations showed increase in betweenness centrality (BC) metric of residues at the allosteric site implying these residues are important in protein communication. A loop region at the catalytic domain between residues 300 and 350 and the anticodon binding domain showed significant contributions to both PC1 and PC2. Large motions were observed at a loop in the Z-domain between residues 697 and 710 which was also in agreement with RMSF calculations that showed increase in flexibility of residues in this region. Residues in this loop region are implicated in ATP binding and thus a change in dynamics may affect ATP binding affinity. Free energy landscape (FEL) calculations showed that the holo protein (protein-ADN complex) and PfProRS-SANC184 complexes were stable, as shown by the low energy with very few intermediates and hardly distinguishable low energy barriers. In addition, FEL results agreed with backbone RMSD distribution plots where stable complexes showed a normal RMSD distribution while unstable complexes had multimodal RMSD distribution. The betweenness centrality metric showed a loss of functional importance of key ATP binding site residues upon allosteric ligand binding. The deep basins in average L observed at the allosteric region imply that there is high accessibility of residues at this region. To further analyse BC and average L metrics data, we calculated the ΔBC and ΔL values by taking each value in the holo protein BC or L matrix less the corresponding value in the ligand-bound complex BC or L matrix. Interestingly, in allosteric complexes, residues located in a loop region implicated in ATP binding had negative ΔL values while in orthosteric complexes these residues had positive ΔL values. An increase in contact frequency between residues Ser263, Thr267, Tyr285, and Leu707 at the allosteric site and residues Thr397, Pro398, Thr402, and Gln395 at the ATP binding TXE loop was observed. In summary, this study identified five potential orthosteric inhibitors and five allosteric modulators against PfProRS. Allosteric modulators changed ATP binding site dynamics, as shown by RMSF, PCA, and DRN calculations. Changes in dynamics of the ATP binding site and increased contact frequency between residues at the proposed allosteric site and the ATP binding site may explain how allosteric modulators distort the ATP binding site and thus might inhibit PfProRS. The scaffolds of the identified hits in the study can be used as a starting point for antimalarial inhibitor development with low human cytotoxicity.

Synthesis and Biological Evaluation of 1,3-Dideazapurine-Like 7-Amino-5-Hydroxymethyl-Benzimidazole Ribonucleoside Analogues as Aminoacyl-tRNA Synthetase Inhibitors.

Aminoacyl-tRNA synthetases (aaRSs) have become viable targets for the development of antimicrobial agents due to their crucial role in protein translation. A series of six amino acids were coupled to the purine-like 7-amino-5-hydroxymethylbenzimidazole nucleoside analogue following an optimized synthetic pathway. These compounds were designed as aaRS inhibitors and can be considered as 1,3-dideazaadenine analogues carrying a 2-hydroxymethyl substituent. Despite our intentions to obtain N1-glycosylated 4-aminobenzimidazole congeners, resembling the natural purine nucleosides glycosylated at the N9-position, we obtained the N3-glycosylated benzimidazole derivatives as the major products, resembling the respective purine N7-glycosylated nucleosides. A series of X-ray crystal structures of class I and II aaRSs in complex with newly synthesized compounds revealed interesting interactions of these "base-flipped" analogues with their targets. While the exocyclic amine of the flipped base mimics the reciprocal interaction of the N3-purine atom of aminoacyl-sulfamoyl adenosine (aaSA) congeners, the hydroxymethyl substituent of the flipped base apparently loses part of the standard interactions of the adenine N1 and the N6-amine as seen with aaSA analogues. Upon the evaluation of the inhibitory potency of the newly obtained analogues, nanomolar inhibitory activities were noted for the leucine and isoleucine analogues targeting class I aaRS enzymes, while rather weak inhibitory activity against the corresponding class II aaRSs was observed. This class bias could be further explained by detailed structural analysis.

Aminoacyl tRNA synthetases as malarial drug targets: a comparative bioinformatics study.

BACKGROUND: Treatment 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. METHODS: Plasmodium 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. RESULTS: Motif 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. CONCLUSION: This 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.

ATP-directed capture of bioactive herbal-based medicine on human tRNA synthetase.

Febrifugine is the active component of the Chinese herb Chang Shan (Dichroa febrifuga Lour.), which has been used for treating malaria-induced fever for about 2,000 years. Halofuginone (HF), the halogenated derivative of febrifugine, has been tested in clinical trials for potential therapeutic applications in cancer and fibrotic disease. Recently, HF was reported to inhibit T(H)17 cell differentiation by activating the amino acid response pathway, through inhibiting human prolyl-transfer RNA synthetase (ProRS) to cause intracellular accumulation of uncharged tRNA. Curiously, inhibition requires the presence of unhydrolysed ATP. Here we report an unusual 2.0 Å structure showing that ATP directly locks onto and orients two parts of HF onto human ProRS, so that one part of HF mimics bound proline and the other mimics the 3' end of bound tRNA. Thus, HF is a new type of ATP-dependent inhibitor that simultaneously occupies two different substrate binding sites on ProRS. Moreover, our structure indicates a possible similar mechanism of action for febrifugine in malaria treatment. Finally, the elucidation here of a two-site modular targeting activity of HF raises the possibility that substrate-directed capture of similar inhibitors might be a general mechanism that could be applied to other synthetases.

A new set of assays for the discovery of aminoacyl-tRNA synthetase inhibitors.

Current biochemical methods available to monitor the activity of aminoacyl-tRNA synthetases (ARS) are ill-suited to high-throughput screening approaches for the identification of small-molecule inhibitors of these enzymes. In an attempt to improve the limitations of current assays we have developed a suite of new methods designed to streamline the discovery of new ARS antagonists. This set of assays includes approaches to monitor ARS activity in vitro, in human cells, and in bacteria. They are applicable to several ARSs from any given organism, can be easily adapted to very high-throughput set-ups, and allow for a multi-factorial selection of drug candidates.

Leishmania donovani Encodes a Functional Selenocysteinyl-tRNA Synthase.

The synthesis of selenocysteine, the 21st amino acid, occurs on its transfer RNA (tRNA), tRNA(Sec). tRNA(Sec) is initially aminoacylated with serine by seryl-tRNA synthetase and the resulting seryl moiety is converted to phosphoserine by O-phosphoseryl-tRNA kinase (PSTK) in eukaryotes. The selenium donor, selenophosphate is synthesized from selenide and ATP by selenophosphate synthetase. Selenocysteinyl-tRNA synthase (SepSecS) then uses the O-phosphoseryl-tRNA(Sec) and selenophosphate to form Sec-tRNA(Sec) in eukaryotes. Here, we report the characterization of selenocysteinyl-tRNA synthase from Leishmania donovani. Kinetoplastid SepSecS enzymes are phylogenetically closer to worm SepSecS. LdSepSecS was found to exist as a tetramer. Leishmania SepSecS enzyme was found to be active and able to complement the ΔselA deletion in Escherichia coli JS1 strain only in the presence of archaeal PSTK, indicating the conserved nature of the PSTK-SepSecS pathway. LdSepSecS was found to localize in the cytoplasm of the parasite. Gene deletion studies indicate that Leishmania SepSecS is dispensable for the parasite survival. The parasite was found to encode three selenoproteins, which were only expressed in the presence of SepSecS. Selenoproteins of L. donovani are not required for the growth of the promastigotes. Auranofin, a known inhibitor of selenoprotein synthesis showed the same sensitivity toward the wild-type and null mutants suggesting its effect is not through binding to selenoproteins. The three-dimensional structural comparison indicates that human and Leishmania homologs are structurally highly similar but their association modes leading to tetramerization seem different.

Discovery of a novel prolyl-tRNA synthetase inhibitor and elucidation of its binding mode to the ATP site in complex with l-proline.

Prolyl-tRNA synthetase (PRS) is a member of the aminoacyl-tRNA synthetase family of enzymes and catalyzes the synthesis of prolyl-tRNAPro using ATP, l-proline, and tRNAPro as substrates. An ATP-dependent PRS inhibitor, halofuginone, was shown to suppress autoimmune responses, suggesting that the inhibition of PRS is a potential therapeutic approach for inflammatory diseases. Although a few PRS inhibitors have been derivatized from natural sources or substrate mimetics, small-molecule human PRS inhibitors have not been reported. In this study, we discovered a novel series of pyrazinamide PRS inhibitors from a compound library using pre-transfer editing activity of human PRS enzyme. Steady-state biochemical analysis on the inhibitory mode revealed its distinctive characteristics of inhibition with proline uncompetition and ATP competition. The binding activity of a representative compound was time-dependently potentiated by the presence of l-proline with Kd of 0.76 nM. Thermal shift assays demonstrated the stabilization of PRS in complex with l-proline and pyrazinamide PRS inhibitors. The binding mode of the PRS inhibitor to the ATP site of PRS enzyme was elucidated using the ternary complex crystal structure with l-proline. The results demonstrated the different inhibitory and binding mode of pyrazinamide PRS inhibitors from preceding halofuginone. Furthermore, the PRS inhibitor inhibited intracellular protein synthesis via a different mode than halofuginone. In conclusion, we have identified a novel drug-like PRS inhibitor with a distinctive binding mode. This inhibitor was effective in a cellular context. Thus, the series of PRS inhibitors are considered to be applicable to further development with differentiation from preceding halofuginone.

Prolyl-tRNA synthetase inhibition promotes cell death in SK-MEL-2 cells through GCN2-ATF4 pathway activation.

Protein translation is highly activated in cancer tissues through oncogenic mutations and amplifications, and this can support survival and aberrant proliferation. Therefore, blocking translation could be a promising way to block cancer progression. The process of charging a cognate amino acid to tRNA, a crucial step in protein synthesis, is mediated by tRNA synthetases such as prolyl tRNA synthetase (PRS). Interestingly, unlike pan-translation inhibitors, we demonstrated that a novel small molecule PRS inhibitor (T-3861174) induced cell death in several tumor cell lines including SK-MEL-2 without complete suppression of translation. Additionally, our findings indicated that T-3861174-induced cell death was caused by activation of the GCN2-ATF4 pathway. Furthermore, the PRS inhibitor exhibited significant anti-tumor activity in several xenograft models without severe body weight losses. These results indicate that PRS is a druggable target, and suggest that T-3861174 is a potential therapeutic agent for cancer therapy.

Novel boronic acid derivatives of bis(indolyl) methane as anti-MRSA agents.

Towards the search for a new generation of antibiotics to control methicillin-resistant Staphylococcus aureus (MRSA), the design and synthesis of various bis indolyl methane (BIM) derivatives based on their different electron donor and acceptor properties of the substituents have been made, in which boronic acid derivatives of BIM are found to be active against MRSA. The observed evidence with the lead compound reveals their strong anti-MRSA activity, which paves the way of design and further development of a new generation antibiotics.