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ABSTRACT Leucyl‐tRNA synthetase (LeuRS) is clinically validated molecular target for antibiotic development. Recently, we have reported several classes of small‐molecular inhibitors targeting aminoacyl‐adenylate binding site of Mycobacterium tuberculosis LeuRS with antibacterial activity. In this work, we performed in silico site‐directed mutagenesis of M. tuberculosis LeuRS synthetic site in order to identify the most critical amino acid residues for the interaction with substrate and prove binding modes of inhibitors. We carried out 20‐ns molecular dynamics (MD) simulations and used umbrella sampling (US) method for the calculation of the binding free energy (ΔGb) of leucyl‐adenylate with wild‐type and mutated forms of LeuRS. According to molecular modeling results, it was found that His89, Tyr93, and Glu660 are essential amino acid residues both for aminoacyl‐adenylate affinity and hydrogen bond formation. We have selected His89 for experimental site‐directed mutagenesis since according to our previous molecular docking results this amino acid residue was predicted to be important for inhibitor interaction in adenine‐binding region. We obtained recombinant mutant M. tuberculosis LeuRS H89A. Using aminoacylation assay we have found that the mutation of His89 to Ala in the active site of M. tuberculosis LeuRS results in significant decrease of inhibitory activity for compounds belonging to three different chemical classes—3‐phenyl‐5‐(1‐phenyl‐1H‐[1,2,3]triazol‐4‐yl)‐[1,2,4]oxadiazoles, N‐benzylidene‐N′‐thiazol‐2‐yl‐hydrazines, and 1‐oxo‐1H‐isothiochromene‐3‐carboxylic acid (4‐phenyl‐thiazol‐2‐yl)‐amide derivatives. Therefore, the interaction with His89 should be taken into account during further M. tuberculosis LeuRS inhibitors development and optimization. [ABSTRACT FROM AUTHOR]

Aspergillus montevidensis is an important domesticated fungus that has been applied to produce many traditional fermented foods under high osmotic conditions. However, the detailed mechanisms of tolerance to osmotic stress remain largely unknown. Here, we construct a target-deleted strain (ΔLeuRS) of A. montevidensis and found that the ΔLeuRS mutants grew slowly and suppressed the development of the cleistothecium compared to the wide-type strains (WT) under salt-stressed and non-stressed conditions. Furthermore, differentially expressed genes (p < 0.001) governed by LeuRS were involved in salt tolerance, ABC transporter, amino acid metabolism, sugar metabolism, and the reproduction process. The ΔLeuRS strains compared to WT strains under short- and long-term salinity stress especially altered accumulation levels of metabolites, such as amino acids and derivatives, carbohydrates, organic acids, and fatty acids. This study provides new insights into the underlying mechanisms of salinity tolerance and lays a foundation for flavor improvement of foods fermented with A. montevidensis. [ABSTRACT FROM AUTHOR]

Isoleucyl-tRNA synthetase (IleRS), leucyl-tRNA synthetase (LeuRS), and valyl-tRNA synthetase (ValRS) are enzymes that have potential for the determination of l -isoleucine, l -leucine, and l -valine in food products and plasma. However, the disadvantages of these enzymes are their specificity and sensitivity. Here, we examined the substrate specificity of IleRS, LeuRS, and ValRS under various conditions of pyrophosphate amplification to improve their specificity and sensitivity. The amount of pyrophosphate produced in IleRS, LeuRS, and ValRS reactions was amplified after the addition of excess adenosine-5′-triphosphate and magnesium ions, and was approximately 9-, 8-, and 7-fold higher, respectively, for each of the initial l -amino acid substrates (50 μM). However, in addition to their target amino acids, IleRS, LeuRS, and ValRS also reacted with l -valine, l -lysine, and l -threonine, respectively. This substrate misrecognition was overcome by making the reaction pH more acidic and by increasing the magnesium ion concentration. The pyrophosphate amplification in IleRS, LeuRS, and ValRS reactions resulted in the production of p1, p4-di (adenosine) 5′-tetraphosphate. We also observed a strong positive correlation (R = 0.99) between the amount of pyrophosphate produced and the initial concentration of l -amino acid with 5 and 50 μM l -isoleucine, l -leucine, and l -valine. Our results suggest that amino acid assays using IleRS, LeuRS, and ValRS are promising methods to accurately measure l -valine, l -isoleucine, and l -leucine in food products and plasma. [ABSTRACT FROM AUTHOR]

Background. The most serious challenge in the treatment of tuberculosis is the multidrug resistance of Mycobacterium tuberculosis to available antibiotics. This provides the stimulus for the search of novel molecular targets and approaches to overcome resistance emergence. Nowadays, M. tuberculosis leucyl-tRNA synthetase (LeuRS) is a clinically validated molecular target for antituberculosis drug discovery. The aim of our study was to develop small-molecular inhibitors of M. tuberculosis LeuRS with antibacterial activity. Methods. Molecular docking, pharmacophore screening and machine learning approaches were used for in silico design of M. tuberculosis LeuRS inhibitors. Molecular docking was performed with DOCK 4.0 software. Ligand-oriented pharmacophore models were built using web-server PharmaGist. Pharmacophore screening was conducted using PharmDeveloper program. The artificial neural networks were built using module net from R 3.6.1. The inhibitory activity of compounds toward recombinant aminoacyl-tRNA synthetase was investigated in aminoacylation assays. The antibacterial activity of compounds was studied toward pathogenic strain M. tuberculosis H37Rv in microdilution assay. Results. Using molecular docking approach we have found inhibitors of M. tuberculosis LeuRS among N-Benzylidene-N’-thiazol2-yl-hydrazines with IC50 values in the range from 2.27 mM to 19.3 mM. The most promising compound among studied derivatives inhibits growth of pathogenic bacteria M. tuberculosis H37Rv with IC50 = 10 mM. Then, as a strategy to overcome resistance emergence in M. tuberculosis we used the idea of multitarget inhibition, since the probability of mutations simultaneously in both enzymes is negligible. As molecular targets we have selected M. tuberculosis LeuRS and methionyl-tRNA synthetase (MetRS). Using molecular docking approach we identified dual-targeted inhibitors among N-benzylidene-N’-thiazol2-yl-hydrazines. The most promising compound among studied derivatives inhibits growth of M. tuberculosis H37Rv with IC50 = 1.56 mM. Applying a ligand-based pharmacophore approach we found five novel dual-targeted hit compounds from different chemical classes. The most active compound inhibits LeuRS and MetRS with IC50 values of 13 mM and 13.8 mM, correspondingly. Using the single-hidden-layer neural network models we identified dual-targeted inhibitors among 2-(quinolin2-ylsulfanyl)-acetamides. The most active compound among studied derivatives inhibits MetRS and LeuRS with IC50 values of 33 mM and 23.9 mM, respectively. Conclusions. Using in silico approaches we identified several chemical classes of inhibitors targeting M. tuberculosis LeuRS and MetRS with IC50 values in micromolar concentration range. It was found that the most promising compounds which possess antibacterial activity belong to N-benzylidene-N’-thiazol-2-yl-hydrazine derivatives. Therefore, the compounds in this class can be valuable for further biological research and optimization. [ABSTRACT FROM AUTHOR]

The current study is focused on the combination of pyrazinamide with 6-aminobenzo[c][1,2]oxaborol-1(3H)-ol, which is a crucial pharmacophore of several antimicrobial agents. The use of benzoxaborole moiety could afford the formation of a spiro adduct between benzoxaborole moiety and 3'-terminal adenosine nucleotide Ade76 of tRNALeu. In the form of this spiro adduct, it may potentially inhibit the enzyme leucyl-tRNA synthetase (LeuRS). A large heterocyclic substitution in position 6 of the benzoxaborole moiety could lead to the enhanced selectivity of the intended compounds to the bacterial enzyme due to steric clashes with eukaryotic types of LeuRS. The target compounds were synthesized by condensation of 6-aminobenzo[c][1,2]oxaborol-1(3H)-ol with variously substituted heteroaromatic acids that underwent previous activation. The synthetic products and the isolated condensation intermediates were subjected to biological in vitro screening against fungi and bacteria, including mycobacteria and in vitro cytotoxicity screening against the HepG2 cancer cell line. Some of the compounds showed moderate antimycobacterial activity with persisted low toxicity.

Aminoacyl-tRNA synthetases are crucial enzymes involved in protein synthesis and various cellular physiological reactions. Aside from their standard role in linking amino acids to the corresponding tRNAs, they also impact protein homeostasis by controlling the level of soluble amino acids within the cell. For instance, leucyl-tRNA synthetase (LARS1) acts as a leucine sensor for the mammalian target of rapamycin complex 1 (mTORC1), and may also function as a probable GTPase-activating protein (GAP) for the RagD subunit of the heteromeric activator of mTORC1. In turn, mTORC1 regulates cellular processes, such as protein synthesis, autophagy, and cell growth, and is implicated in various human diseases including cancer, obesity, diabetes, and neurodegeneration. Hence, inhibitors of mTORC1 or a deregulated mTORC1 pathway may offer potential cancer therapies. In this study, we investigated the structural requirements for preventing the sensing and signal transmission from LARS to mTORC1. Building upon recent studies on mTORC1 regulation activation by leucine, we lay the foundation for the development of chemotherapeutic agents against mTORC1 that can overcome resistance to rapamycin. Using a combination of in-silico approaches to develop and validate an alternative interaction model, discussing its benefits and advancements. Finally, we identified a set of compounds ready for testing to prevent LARS1/RagD protein-protein interactions. We establish a basis for creating chemotherapeutic drugs targeting mTORC1, which can conquer resistance to rapamycin. We utilize in-silico methods to generate and confirm an alternative interaction model, outlining its advantages and improvements, and pinpoint a group of novel substances that can prevent LARS1/RagD interactions.Communicated by Ramaswamy H. Sarma.

Post-fermented tea has multiple health-promoting efficacy; however, the molecular mechanisms of quality formation remain largely unknown. Here, we found that leucyl-tRNA synthetase (LeuRS) is essential for the formation of 'golden flowers' in dark teas fermented by A. montevidensis. Metabolomics based on UHPLC-Q-TOF/MS revealed 67, 130, and 98 differential metabolites in the compared groups of fermented teas by wide-type strains (WT)/by Δ leuRS mutants (MT) at 3, 9 and 15 days, respectively. These differential metabolites consisted mainly of flavonoids, sugars, organic acids, and fatty acids. Transcriptome analysis showed that hundreds of genes were significantly differentially expressed compared to WT with MT, which were mainly involved in KEGG pathways such as starch and sucrose metabolism, degradation of aromatic compounds, amino acid metabolism and lipid metabolism. These findings highlighted that LeuRS plays a crucial role in the development of the fungal cleistothecium and the flavor formation of the post-fermented teas with A. montevidensis. • LeuRS plays a key role in the formation of 'golden flowers' in fermented dark tea. • LeuRS influenced the metabolization of flavonoids of fermented dark tea. • LeuRS modulated the metabolization of sugars of fermented dark tea. • LeuRS affected the metabolism of organic acids and fatty acids of fermented dark tea. [ABSTRACT FROM AUTHOR]

Background: The most serious challenge in the treatment of tuberculosis is the multidrug resistance of Mycobacterium tuberculosis to existing antibiotics. As a strategy to overcome resistance we used a multitarget drug design approach. The purpose of the work was to discover dual-targeted inhibitors of mycobacterial LeuRS and MetRS with machine learning. Methods: The artificial neural networks were built using module nnet from R 3.6.1. The inhibitory activity of compounds toward LeuRS and MetRS was investigated in aminoacylation assays. Results: Using a machine-learning approach, we identified dual-targeted inhibitors of LeuRS and MetRS among 2-(quinolin-2-ylsulfanyl)-acetamide derivatives. The most active compound inhibits MetRS and LeuRS with IC 50 values of 33 μm and 23.9 μm, respectively. Conclusion: 2-(Quinolin-2-ylsulfanyl)-acetamide scaffold can be useful for further research.