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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]

A protein synthesis enzyme, leucyl-tRNA synthetase (LRS), serves as a leucine sensor for the mechanistic target of rapamycin complex 1 (mTORC1), which is a central effector for protein synthesis, metabolism, autophagy, and cell growth. However, its significance in mTORC1 signaling and cancer growth and its functional relationship with other suggested leucine signal mediators are not well-understood. Here we show the kinetics of the Rag GTPase cycle during leucine signaling and that LRS serves as an initiating "ON" switch via GTP hydrolysis of RagD that drives the entire Rag GTPase cycle, whereas Sestrin2 functions as an "OFF" switch by controlling GTP hydrolysis of RagB in the Rag GTPase-mTORC1 axis. The LRS-RagD axis showed a positive correlation with mTORC1 activity in cancer tissues and cells. The GTP-GDP cycle of the RagD-RagB pair, rather than the RagC-RagA pair, is critical for leucine-induced mTORC1 activation. The active RagD-RagB pair can overcome the absence of the RagC-RagA pair, but the opposite is not the case. This work suggests that the GTPase cycle of RagD-RagB coordinated by LRS and Sestrin2 is critical for controlling mTORC1 activation, and thus will extend the current understanding of the amino acid-sensing mechanism.

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.

Leucyl-tRNA synthetase (LARS) is an enzyme that catalyses the ligation of leucine with leucine tRNA. LARS is also essential to sensitize the intracellular leucine concentration to the mammalian target of rapamycin complex 1 (mTORC1) activation. Biallelic mutation in the LARS gene causes infantile liver failure syndrome type 1 (ILFS1), which is characterized by acute liver failure, anaemia, and neurological disorders, including microcephaly and seizures. However, the molecular mechanism underlying ILFS1 under LARS deficiency has been elusive. Here, we generated Lars deficient (larsb−/−) zebrafish that showed progressive liver failure and anaemia, resulting in early lethality within 12 days post fertilization. The atg5-morpholino knockdown and bafilomycin treatment partially improved the size of the liver and survival rate in larsb−/− zebrafish. These findings indicate the involvement of autophagy in the pathogenesis of larsb−/− zebrafish. Indeed, excessive autophagy activation was observed in larsb−/− zebrafish. Therefore, our data clarify a mechanistic link between LARS and autophagy in vivo. Furthermore, autophagy regulation by LARS could lead to development of new therapeutics for IFLS1. [ABSTRACT FROM AUTHOR]

Leucyl-tRNA synthetases (LRS) catalyze the linkage of leucine with tRNALeu. A large insertion CP1 domain (Connective Polypeptide 1) in LRS is responsible for post-transfer editing of mis-charged aminoacyl-tRNAs. Here, we characterized the CP1 domain of Leishmania donovani, a protozoan parasite, and its role in editing activity and interaction with broad spectrum anti-fungal, AN2690. The deletion mutant of LRS, devoid of CP1 domain (LRS-CP1Δ) was constructed, followed by determination of its role in editing and aminoacylation. Binding of AN2690 and different amino acids with CP1 deletion mutant and full length LRS was evaluated using isothermal titration calorimetry (ITC) and molecular dynamics simulations. The recombinant LRS-CP1Δ protein did not catalyze the aminoacylation and the editing reaction when compared to full-length LRS. Thus, indicating that CP1 domain was imperative for both aminoacylation and editing activities of LRS. Binding studies with different amino acids indicated selectivity of isoleucine by CP1 domain over other amino acids. These studies also indicated high affinity of AN2690 with the editing domain. Molecular docking studies indicated that AN2690-CP1 domain complex was stabilized by hydrogen bonding and hydrophobic interactions resulting in high binding affinity between the two. Our data suggests CP1 is crucial for the function of L. donovani LRS. [ABSTRACT FROM AUTHOR]

Adipocytes constitute a major component of the tumour microenvironment. Numerous studies have shown that adipocytes promote aggressiveness and invasion by stimulating cancer cells proliferation and modulating their metabolism. Herein, we reported that Notch3 promotes mouse 3T3‐L1 pre‐adipocytes differentiation by performing the integrative transcriptome and TMT‐based proteomic analyses. The results revealed that aminoacyl‐tRNA_biosynthesis pathway was significantly influenced with Nocth3 change during 3T3‐L1 pre‐adipocytes differentiation, and the expression of LARS in this pathway was positively correlated with Notch3. Published studies have shown that LARS is a sensor of leucine that regulates the mTOR pathway activity, and the latter involves in adipogenesis. We therefore supposed that Notch3 might promote 3T3‐L1 pre‐adipocytes differentiation by up‐regulating LARS expression and activating mTOR pathway. CHIP and luciferase activity assay uncovered that Notch3 could transcriptionally regulate the expression of LARS gene. Oil Red staining identified a positive correlation between Notch3 expression and adipocytic differentiation. The activation of mTOR pathway caused by Notch3 overexpression could be attenuated by knocking down LARS expression. Altogether, our study revealed that Notch3 promotes adipocytic differentiation of 3T3‐L1 pre‐adipocytes cells by up‐regulating LARS expression and activating the mTOR pathway, which might be an emerging target for obesity treatment. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis infection remains a major cause of global morbidity and mortality due to the increase of antibiotics resistance. Dual/multi-target drug discovery is a promising approach to overcome bacterial resistance. In this study, we built ligand-based pharmacophore models and performed pharmacophore screening in order to identify hit compounds targeting simultaneously two enzymes—M. tuberculosis leucyl-tRNA synthetase (LeuRS) and methionyl-tRNA synthetase (MetRS). In vitro aminoacylation assay revealed five compounds from different chemical classes inhibiting both enzymes. Among them the most active compound—3-(3-chloro-4-methoxy-phenyl)-5-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-3H-[1,2,3]triazol-4-ylamine (1) inhibits mycobacterial LeuRS and MetRS with IC50 values of 13 µM and 13.8 µM, respectively. Molecular modeling study indicated that compound 1 has similar binding mode with the active sites of both aminoacyl-tRNA synthetases and can be valuable compound for further chemical optimization in order to find promising antituberculosis agents. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract Leucyl-tRNA synthetase (LRS) plays an important role in amino acid-dependent mTORC1 signaling, which is known to be associated with cellular metabolism and proliferation. Therefore, LRS-targeting small molecules that can suppress mTORC1 activation may provide an alternative strategy to current anticancer therapy. In this work, we developed a library of leucyladenylate sulfate analogues by extensively modifying three different pharmacophoric regions comprising adenine, ribose and leucine. Several effective compounds were identified by cell-based mTORC1 activation assays and further tested for anticancer activity. The selected compounds mostly exhibited selective cytotoxicity toward five different cancer cell lines, supporting the hypothesis that the LRS-mediated mTORC1 pathway is a promising alternative target to current therapeutic approaches. [ABSTRACT FROM AUTHOR]

A series of novel 7-oxo-7H-thiazolo[3,2-b]-1,2,4-triazine-2-carboxylic acid derivatives was synthesized in good yields by a multi-step procedure that included the generation of the S-alkylated derivatives from 6-substituted arylmethyl-3-mercapto-1,2,4-triazin-5-ones with ethyl 2-chloroacetoacetate, intramolecular cyclization with microwave irradiation, hydrolysis and amidation. All of the target compounds were fully characterized through 1H-NMR, 13C-NMR and HRMS spectra. The intramolecular cyclization occurred regioselectively at the N2-position of 1,2,4-triazine ring, which was confirmed by compound 3e using single-crystal X-ray diffraction analysis. The antibacterial and antitubercular activities of the target compounds were evaluated. Compared with Ciprofloxacin and Rifampicin, compounds 5d, 5f and 5g containing the terminal amide fragment exhibited broad spectrum antibacterial activity, and carboxylic acid derivatives or its corresponding ethyl esters had less effect on antibacterial properties. The most potent compound 5f also displayed excellent in vitro antitubercular activity against Mycobacterium smegmatis (minimum inhibitory concentration (MIC) = 50 μg/mL) and better growth inhibition activity of leucyl-tRNA synthetase (78.24 ± 4.05% at 15 μg/mL).

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.

The accuracy of protein synthesis is provided by the editing functions of aminoacyl-tRNA synthetases (aaRSs), a mechanism that eliminates misactivated amino acids or mischarged tRNAs. Despite research efforts, some molecular bases of these mechanisms are still unclear. The post-transfer editing pathway of leucyl-tRNA synthetase (LeuRS) carried out in a special insertion domain (the Connective Polypeptide 1 or CP1), as editing domain. Recently, it was shown by in vivo studies and was supported by mutagenesis, and the kinetics approaches that the CP1 domain of LeuRS has discriminatory power for different substrates. The goal of this work is to investigate the structural basis for amino acid recognition of LeuRS post-transfer editing processes with molecular dynamics (MD) simulation method. To pursue this aim, the molecular modeling studies on Thermus thermophiles LeuRS (LeuRSTT) with two post-transfer substrates (norvalyl-tRNA Leu and isoleucyl-tRNA Leu ) was performed. Our results revealed that post-transfer substrate norvalyl-tRNA Leu is more favorable. Moreover, the MD simulations show that branched side chain of Ile-A76 cannot allow water molecules to get close, which leads to a significant decrease in the rate of hydrolysis. Finally, the study showed that site mutation Asp347Ala has elucidated a number of fine structural differences in the binding mode of two post-transfer substrates to the active centre of LeuRS editing domain and two conserved threonines, namely Thr247 and Thr248, are responsible for the amino acid selection through the interaction with substrates. [ABSTRACT FROM AUTHOR]

A protein synthesis enzyme, leucyl-tRNA synthetase (LRS), serves as a leucine sensor for the mechanistic target of rapamycin complex 1 (mTORC1), which is a central effector for protein synthesis, metabolism, autophagy, and cell growth. However, its significance in mTORC1 signaling and cancer growth and its functional relationship with other suggested leucine signal mediators are not wellunderstood. Here we show the kinetics of the Rag GTPase cycle during leucine signaling and that LRS serves as an initiating "ON" switch via GTP hydrolysis of RagD that drives the entire Rag GTPase cycle, whereas Sestrin2 functions as an "OFF" switch by controlling GTP hydrolysis of RagB in the Rag GTPase--mTORC1 axis. The LRS--RagD axis showed a positive correlation with mTORC1 activity in cancer tissues and cells. The GTP--GDP cycle of the RagD--RagB pair, rather than the RagC--RagA pair, is critical for leucine-induced mTORC1 activation. The active RagD--RagB pair can overcome the absence of the RagC--RagA pair, but the opposite is not the case. This work suggests that the GTPase cycle of RagD--RagB coordinated by LRS and Sestrin2 is critical for controlling mTORC1 activation, and thus will extend the current understanding of the amino acidsensing mechanism. [ABSTRACT FROM AUTHOR]

Mammalian target of rapamycin (mTOR) signaling controls skeletal muscle cell differentiation, growth, and metabolism by sensing the intracellular energy status and nutrients. Recently, leucyl-tRNA synthetase (Lars) was identified as an intracellular sensor of leucine involved in the activation of mTOR signaling. However, there is still no evidence for the activation of mTOR signaling by Lars and its physiological roles in skeletal muscle cells. In this study, we determined the potential roles of Lars for the activation of mTOR signaling, skeletal muscle cell differentiation, hypertrophy, and metabolism using small interfering (si)-RNA knockdown. siRNA-mediated knockdown of Lars decreased phosphorylated p70 S6 kinase and inhibited the differentiation of C2C12 mouse myoblasts into myotubes, as evidenced by a decreased fusion index and decreased mRNA and protein expression levels of myogenic markers. Importantly, si-Lars decreased the level of Insulin-like growth factor 2 ( Igf2 ) mRNA expression from the early stages of differentiation, indicating the possibility of an association between the mTOR–IGF2 axis and Lars. However, Lars knockdown did not decrease phosphorylated mTOR in differentiated myotubes, nor did it affect the hypertrophy of myotubes as evidenced by measuring their diameters and detecting the mRNA and protein expression of hypertrophy markers. Similarly, an extracellular flux analyzer showed that Lars knockdown did not affect the metabolism (glycolysis and mitochondrial respiration) of myotubes. These results demonstrate that Lars is required for skeletal muscle differentiation through the activation of mTOR signaling, but not for hypertrophy or metabolic alteration of myotubes. [ABSTRACT FROM AUTHOR]

The intrinsic editing capacities of aminoacyl-tRNA synthetases ensure a high-fidelity translation of the amino acids that possess effective non-cognate aminoacylation surrogates. The dominant error-correction pathway comprises deacylation of misaminoacylated tRNA within the aminoacyl-tRNA synthetase editing site. To assess the origin of specificity of Escherichia coli leucyl-tRNA synthetase (LeuRS) against the cognate aminoacylation product in editing, we followed binding and catalysis independently using cognate leucyl- and non-cognate norvalyl-tRNA Leu and their non-hydrolyzable analogues. We found that the amino acid part (leucine versus norvaline) of (mis)aminoacyl-tRNAs can contribute approximately 10-fold to ground-state discrimination at the editing site. In sharp contrast, the rate of deacylation of leucyl- and norvalyl-tRNA Leu differed by about 10 4 -fold. We further established the critical role for the A76 3′-OH group of the tRNA Leu in post-transfer editing, which supports the substrate-assisted deacylation mechanism. Interestingly, the abrogation of the LeuRS specificity determinant threonine 252 did not improve the affinity of the editing site for the cognate leucine as expected, but instead substantially enhanced the rate of leucyl-tRNA Leu hydrolysis. In line with that, molecular dynamics simulations revealed that the wild-type enzyme, but not the T252A mutant, enforced leucine to adopt the side-chain conformation that promotes the steric exclusion of a putative catalytic water. Our data demonstrated that the LeuRS editing site exhibits amino acid specificity of kinetic origin, arguing against the anticipated prominent role of steric exclusion in the rejection of leucine. This feature distinguishes editing from the synthetic site, which relies on ground-state discrimination in amino acid selection. [ABSTRACT FROM AUTHOR]

Leucyl-tRNA synthetase (LRS) plays major roles in providing leucine-tRNA and activating mechanistic target of rapamycin complex 1 (mTORC1) through intracellular leucine sensing. mTORC1 activated by amino acids affects the influence on physiology functions including cell proliferation, protein synthesis and autophagy in various organisms. Biochemical results demonstrating leucine sensing have been published, but visual results are lacking. Therefore, we observed the location of LRS with and without leucine using stimulated emission depletion (STED) microscopy one of the super-resolution microscopy and transmission electron microscopy (TEM). This revealed that LRS was translocated to the lysosome on addition of leucine. The translocation was inhibited by treatment with compound BC-LI-0186, disrupting the interaction between RagD and LRS. Immuno-TEM revealed a clear decrease in LRS translocation to the lysosome on addition of the inhibitor. This direct visualization of leucine sensing and LRS translocation to the lysosome was related to mTORC1 activation. To study the relationship between mTORC1 activation and LRS translocation, we monitored the change in autophagy for each condition using TEM and CLSM. The results showed a decrease in autophagy on addition of leucine, demonstrating crosstalk between leucine sensing, LRS translocation, RagD interaction, and mTORC1 activation. [ABSTRACT FROM AUTHOR]

Aminoacyl-tRNA synthetases (aaRSs) are part of the cellular translation machinery and as such, they are essential enzymes for every known cell. Due to their ubiquitous nature, their evolutionary history has been intensely researched to better understand the origins of life on a molecular level. Herein, we examine the evolutionary relatedness of leucyl-tRNA synthetases (LeuRS) from each major eukaryotic branch through the speciation process. This research effort was centered on amino acid sequence data as well as generating homology protein models for each LeuRS enzyme. Comparative analysis of this sequence and structural data for LeuRS amongst eukaryotes has indicated a high level of conservation within the active sites of these enzymes. Phylogenetic analysis confirmed this high degree of conservation as well as established evolutionary relatedness between these LeuRS enzymes. Based on this data, vertical gene transfer propagated LeuRS throughout the eukaryotic domain. Horizontal gene transfer and domain acquisition events were not observed within the eukaryotic organisms studied. Our data also highlighted LeuRS adaptation through the speciation process due to slight variability of scaffolding residues outside of the active site regions. We hypothesize that this variability may be due to mechanistic differences amongst LeuRS enzymes that have assumed nontranslational functionality through the evolutionary process. [ABSTRACT FROM AUTHOR]

There is an urgent need to develop new and safer antitubercular agents that possess a novel mode of action. We synthesized and evaluated a novel series of 3-aminomethyl 4-halogen benzoxaboroles as Mycobacterium tuberculosis (Mtb) leucyl-tRNA synthetase (LeuRS) inhibitors. A number of Mtb LeuRS inhibitors were identified that demonstrated good antitubercular activity with high selectivity over human mitochondrial and cytoplasmic LeuRS. Further evaluation of these Mtb LeuRS inhibitors by in vivo pharmacokinetics (PK) and murine tuberculosis (TB) efficacy models led to the discovery of GSK3036656 (abbreviated as GSK656). This molecule shows potent inhibition of Mtb LeuRS (IC50 = 0.20 µM) and in vitro antitubercular activity (Mtb H37Rv MIC = 0.08 µM). Additionally, it is highly selective for the Mtb LeuRS enzyme with IC50 of >300 µM and 132 µM for human mitochondrial LeuRS and human cytoplasmic LeuRS, respectively. In addition, it exhibits remarkable PK profiles and efficacy against Mtb in mouse TB infection models with superior tolerability over initial leads. This compound has been progressed to clinical development for the treatment of tuberculosis. [ABSTRACT FROM AUTHOR]

Aminoacyl-tRNA synthetases (aaRSs) play important roles in maintaining the accuracy of protein synthesis. Some aaRSs accomplish this via editing mechanisms, among which leucyl-tRNA synthetase (LeuRS) edits non-cognate amino acid norvaline mainly by post-transfer editing. However, the molecular basis for this pathway for eukaryotic and archaeal LeuRS remain unclear. In this study, a complex of archaeal P. horikoshii LeuRS ( Ph LeuRS) with misacylated tRNA Leu was modeled wherever tRNA’s acceptor stem was oriented directly into the editing site. To understand the distinctive features of organization we reconstructed a complex of Ph LeuRS with tRNA and visualize post-transfer editing interactions mode by performing molecular dynamics (MD) simulation studies. To study molecular basis for substrate selectivity by Ph LeuRS’s editing site we utilized MD simulation of the entire LeuRS complexes using a diverse charged form of tRNAs, namely norvalyl-tRNA Leu and isoleucyl-tRNA Leu . In general, the editing site organization of LeuRS from P.horikoshii has much in common with bacterial LeuRS. The MD simulation results revealed that the post-transfer editing substrate norvalyl-A76, binds more strongly than isoleucyl-A76. Moreover, the branched side chain of isoleucine prevents water molecules from being closer and hence the hydrolysis reaction slows significantly. To investigate a possible mechanism of the post-transfer editing reaction, by Ph LeuRS we have determined that two water molecules (the attacking and assisting water molecules) are localized near the carbonyl group of the amino acid to be cleaved off. These water molecules approach the substrate from the opposite side to that observed for Thermus thermophilus LeuRS ( Tt LeuRS). Based on the results obtained, it was suggested that the post-transfer editing mechanism of Ph LeuRS differs from that of prokaryotic Tt LeuRS. [ABSTRACT FROM AUTHOR]

Leucyl-tRNA synthetase (LRS) has been reported to be a possible mediator of intracellular amino acids signaling to mTORC1. Given that mTORC1 is associated with cell proliferation and tumorigenesis, the LRS-mediated mTORC1 pathway may offer an alternative strategy in anticancer therapy. In this study, we developed a series of simplified analogues of leucyladenylate sulfamate ( 1 ) as LRS-targeted mTORC1 inhibitors. We replaced the adenylate group with a N -(3,4-dimethoxybenzyl)benzenesulfonamide ( 2a ) or a N -(2-phenoxyethyl)benzenesulfonamide groups ( 2b ) that can maintain specific binding, but has more favorable physicochemical properties such as reduced polarity and asymmetric centers. Among these simplified analogues, compound 16 and its constrained analogue 22 effectively inhibited S6K phosphorylation in a dose-dependent manner and exhibited cancer cell specific cytotoxicity against six different types of cancer cells. This result supports that LRS is a viable target for novel anticancer therapy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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]

Perrault syndrome is a rare autosomal recessive disorder characterized by sensorineural hearing loss ( SNHL) in both sexes and primary ovarian insufficiency in 46, XX karyotype females. Biallelic variants in five genes are reported to be causative: HSD17B4, HARS2, LARS2, CLPP and C10orf2. Here we present eight families affected by Perrault syndrome. In five families we identified novel or previously reported variants in HSD17B4, LARS2, CLPP and C10orf2. The proband from each family was whole exome sequenced and variants confirmed by Sanger sequencing. A female was compound heterozygous for a known, p.( Gly16Ser) and novel, p.( Val82Phe) variant in D-bifunctional protein ( HSD17B4). A family was homozygous for mitochondrial leucyl aminocyl tRNA synthetase ( mtLeuRS) ( LARS2) p.( Thr522Asn), previously associated with Perrault syndrome. A further family was compound heterozygous for mtLeuRS, p.( Thr522Asn) and a novel variant, p.( Met117Ile). Affected individuals with LARS2 variants had low frequency SNHL, a feature previously described in Perrault syndrome. A female with significant neurological disability was compound heterozygous for p.( Arg323Gln) and p.( Asn399Ser) variants in Twinkle ( C10orf2). A male was homozygous for a novel variant in CLPP, p.( Cys144Arg). In three families there were no putative pathogenic variants in these genes confirming additional disease-causing genes remain unidentified. We have expanded the spectrum of disease-causing variants associated with Perrault syndrome. [ABSTRACT FROM AUTHOR]

Recent studies indicate that LRS may act as a leucine sensor for the mTORC1 pathway, potentially providing an alternative strategy to overcome rapamycin resistance in cancer treatments. In this study, we developed leucyladenylate sulfamate derivatives as LRS-targeted mTORC1 inhibitors. Compound 18 selectively inhibited LRS-mediated mTORC1 activation and exerted specific cytotoxicity against colon cancer cells with a hyperactive mTORC1, suggesting that 18 may offer a novel treatment option for human colorectal cancer. [ABSTRACT FROM AUTHOR]

Aminoacyl tRNA synthetases are enzymes involved in the key process of coupling an amino acid to its cognate tRNA. AN3365 is a novel antibiotic that specifically targets leucyl-tRNA synthetase, whose development was halted after evaluation in phase II clinical trials owing to the rapid selection of resistance. In an attempt to bring AN3365 back into the developmental pipeline we have evaluated the efficacy of AN3365 in combination with different classes of antibiotic and characterized its mechanism of action. Although we detect no synergy or antagonism in combination with a range of antibiotic classes, a combination of AN3365 with colistin reduces the accumulation of AN3365-resistant and colistin resistance mutations. We also demonstrate that treatment with AN3365 results in the dramatic accumulation of the alarmone (p)ppGpp, the effector of the stringent response-a key player in antibiotic tolerance. [ABSTRACT FROM AUTHOR]

The increase of antibiotic resistance amongstMycobacterium tuberculosisstrains has become one of the most pressing problems of modern medicine. Therefore, the search of antibiotics againstM. tuberculosiswith novel mechanisms of action is very important. We have identified inhibitors ofM. tuberculosisleucyl-tRNA synthetase (LeuRS) among the derivatives of 5-phenylamino-2H-[1,2,4]triazin-3-one. The most active compounds 5-(5-chloro-2-hydroxy-phenylamino)-6-methyl-2H-[1,2,4]triazin-3-one and 5-(5-chloro-2-hydroxy-phenylamino)-2H-[1,2,4]triazin-3-one inhibitM. tuberculosisLeuRS with IC50of 7.6 μМ and 7.2 μМ, respectively. It was established that the inhibitory activity of compounds against pathogenic LeuRS is 10-fold better, than for human enzyme. [ABSTRACT FROM AUTHOR]

A series of leucinol analogs were investigated as leucyl-tRNA synthetase-targeted mTORC1 inhibitors. Among them, compound 5 , ( S )-4-isobutyloxazolidin-2-one, showed the most potent inhibition on the mTORC1 pathway in a concentration-dependent manner. Compound 5 inhibited downstream phosphorylation of mTORC1 by blocking leucine-sensing ability of LRS, without affecting the catalytic activity of LRS. In addition, compound 5 exhibited cytotoxicity against rapamycin-resistant colon cancer cells, suggesting that LRS has the potential to serve as a novel therapeutic target. [ABSTRACT FROM AUTHOR]

Leucyl-tRNA synthetase catalyzes attachment of leucine amino acid to its cognate tRNA. During the second, aminoacetylation, step of the reaction, the leucyl moiety is transferred from leucyl-adenylate to the terminal A76 adenosine of tRNA. In this work, we have investigated the aminoacetylation step catalyzed by leucyl-tRNA synthase, using ab initio quantum chemical/molecular mechanical hybrid potentials in conjunction with reaction-path-location algorithms and molecular dynamics free energy simulations. We have modeled reaction mechanisms arising from both crystallographic studies and computational work. We invoke various groups as potential proton acceptorsnamely, the phosphate and leucyl amino groups of leucyl-adenylate, the A76 base of tRNA, and the Asp80 and Glu532 residues of the proteinand consider both metal-assisted and metal-free reactions. Free energy calculations indicate that both the phosphate group of leucyl adenylate and Glu532 are not strong bases. This agrees with the results of the quantum chemical/molecular mechanical reaction path calculations which give high free energy barriers for the studied pathways involving these groups. A self-assisted mechanism with the leucyl amino group and Asp80 as proton acceptors is the most likely. Furthermore, in this mechanism the presence of a metal ion coordinated by the phosphate group and Glu532 strongly activates the reaction. [ABSTRACT FROM AUTHOR]

Tuberculosis is a serious infectious disease caused by human pathogen bacteria Mycobacterium tuberculosis . Bacterial drug resistance is a very significant medical problem nowadays and development of novel antibiotics with different mechanisms of action is an important goal of modern medical science. Leucyl-tRNA synthetase (LeuRS) has been recently clinically validated as antimicrobial target. Here we report the discovery of small-molecule inhibitors of M. tuberculosis LeuRS. Using receptor-based virtual screening we have identified six inhibitors of M. tuberculosis LeuRS from two different chemical classes. The most active compound 4-{[4-(4-Bromo-phenyl)-thiazol-2-yl]hydrazonomethyl}-2-methoxy-6-nitro-phenol ( 1 ) inhibits LeuRS with IC 50 of 6 μM. A series of derivatives has been synthesized and evaluated in vitro toward M. tuberculosis LeuRS. It was revealed that the most active compound 2,6-Dibromo-4-{[4-(4-nitro-phenyl)-thiazol-2-yl]-hydrazonomethyl}-phenol inhibits LeuRS with IC 50 of 2.27 μM. All active compounds were tested for antimicrobial effect against M. tuberculosis H37Rv. The compound 1 seems to have the best cell permeability and inhibits growth of pathogenic bacteria with IC 50 = 10.01 μM and IC 90 = 13.53 μM. [ABSTRACT FROM AUTHOR]

Leucyl-tRNA synthetase (LeuRS) is a multidomain enzyme that catalyzes Leu-tRNALeu formation and is classified into bacterial and archaeal/eukaryotic types with significant diversity in the C-terminal domain (CTD). CTDs of both bacterial and archaeal LeuRSs have been reported to recognize tRNALeu through different modes of interaction. In the human pathogen Candida albicans, the cytoplasmic LeuRS (CaLeuRS) is distinguished by its capacity to recognize a uniquely evolved chimeric tRNASer (CatRNASer (CAG)) in addition to its cognate CatRNALeu, leading to CUG codon reassignment. Our previous study showed that eukaryotic but not archaeal LeuRSs recognize this peculiar tRNASer, suggesting the significance of their highly divergent CTDs in tRNASer recognition. The results of this study provided the first evidence of the indispensable function of the CTD of eukaryotic LeuRS in recognizing non-cognate CatRNASer and cognate CatRNALeu. Three lysine residues were identified as involved in mediating enzyme-tRNA interaction in the leucylation process:mutation of all three sites totally ablated the leucylation activity. The importance of the three lysine residues was further verified by gel mobility shift assays and complementation of a yeast leuS gene knock-out strain. [ABSTRACT FROM AUTHOR]

Several aminoacyl-tRNA synthetases have been reported to be overexpressed for charging essential aminoacyl-tRNAs in many cancer types. In this study, we aimed to explore the potential role of leucyl-tRNA synthetase (LARS) as an anticancer target. MTT assay was performed to screen inhibitors to human LARS (hsLARS) from compounds AN2690 and its derivatives, compounds 1-6, in U2OS and SKOV3 cells. The compound with the strongest inhibitory ability was further investigated for its inhibitory effect in cancer cell lines and in an animal tumor model. Additionally, a LARS-rescue experiment was performed to explore the potential target in U2OS using Western blot and flow cytometry. Luciferase reporter assay was designed to analyze the effect of of hsLARS inhibitor on p21 activation. We identified an hsLARS inhibitor (compound 2) that suppressed the proliferation of U2OS and SKOV3 cells in vitro. A LARS-rescue experiment demonstrated that the proliferation inhibition was induced by targeting intracellular LARS. In addition, the hsLARS inhibition was shown to activate the p21 early transcription and promote cell apoptosis, as well as reduce implanted EMT6 tumor progression in mice. Our results suggest that LARS might serve as a potential anticancer target through the p21 signaling pathway and that the nutritional signaling pathway may provide a valuable anticancer strategy for further investigation. [ABSTRACT FROM AUTHOR]

A conserved structural module following the KMSKS catalytic loop exhibits α- α- β- α topology in class Ia and Ib aminoacyl- tRNA synthetases. However, the function of this domain has received little attention. Here, we describe the effect this module has on the aminoacylation and editing capacities of leucyl- tRNA synthetases (LeuRSs) by characterizing the key residues from various species. Mutation of highly conserved basic residues on the third α-helix of this domain impairs the affinity of LeuRS for the anticodon stem of tRNALeu, which decreases both aminoacylation and editing activities.Twoglycine residues on this α-helix contribute to flexibility, leucine activation, and editing of LeuRS from Escherichia coli (EcLeuRS). Acidic residues on the β-strand enhance the editing activity of EcLeuRS and sense the size of the tRNALeu D-loop. Incorporation of these residues stimulates the tRNA-dependent editing activity of the chimeric minimalist enzyme Mycoplasma mobile LeuRS fused to the connective polypeptide 1 editing domain and leucine-specific domain from EcLeuRS. Together, these results reveal the stem contact-fold to be a functional as well as a structural linker between the catalytic site and the tRNA binding domain. Sequence comparison of the EcLeuRS stem contact-fold domain with editing-deficient enzymes suggests that key residues of this module have evolved an adaptive strategy to follow the editing functions of LeuRS. [ABSTRACT FROM AUTHOR]

The Escherichia coli ( E. coli ) leucyl-tRNA synthetase (LeuRS) enzyme is part of the aminoacyl-tRNA synthetase (aaRS) family. LeuRS is an essential enzyme that relies on specialized domains to facilitate the aminoacylation reaction. Herein, we have biochemically characterized a specialized zinc-binding domain 1 (ZN-1). We demonstrate that the ZN-1 domain plays a central role in the catalytic cycle of E. coli LeuRS. The ZN-1 domain, when associated with Zn 2 + , assumes a rigid architecture that is stabilized by thiol groups from the residues C159, C176 and C179. When LeuRS is in the aminoacylation complex, these cysteine residues form an equilateral planar triangular configuration with Zn 2 + , but when LeuRS transitions to the editing conformation, this geometric configuration breaks down. By generating a homology model of LeuRS while in the editing conformation, we conclude that structural changes within the ZN-1 domain play a central role in LeuRS's catalytic cycle. Additionally, we have biochemically shown that C159, C176 and C179 coordinate Zn 2 + and that this interaction is essential for leucylation to occur, but is not essential for deacylation. Furthermore, calculated K d values indicate that the wild-type enzyme binds Zn 2 + to a greater extent than any of the mutant LeuRSs. Lastly, we have shown through secondary structural analysis of our LeuRS enzymes that Zn 2 + is an architectural cornerstone of the ZN-1 domain and that without its geometric coordination the domain collapses. We believe that future research on the ZN-1 domain may reveal a possible Zn 2 + dependent translocation mechanism for charged tRNA Leu . [ABSTRACT FROM AUTHOR]

The fidelity of protein synthesis depends on the capacity of aminoacyl- tRNA synthetases (AARSs) to couple only cognate amino acid- tRNA pairs. If amino acid selectivity is compromised, fidelity can be ensured by an inherent AARS editing activity that hydrolyses mischarged tRNAs. Here, we show that the editing activity of Escherichia coli leucyl- tRNA synthetase (EcLeuRS) is not required to prevent incorrect isoleucine incorporation. Rather, as shown by kinetic, structural and in vivo approaches, the prime biological function of LeuRS editing is to prevent mis-incorporation of the non-standard amino acid norvaline. This conclusion follows from a reassessment of the discriminatory power of LeuRS against isoleucine and the demonstration that a LeuRS editing-deficient E. coli strain grows normally in high concentrations of isoleucine but not under oxygen deprivation conditions when norvaline accumulates to substantial levels. Thus, AARS-based translational quality control is a key feature for bacterial adaptive response to oxygen deprivation. The non-essential role for editing under normal bacterial growth has important implications for the development of resistance to antimicrobial agents targeting the LeuRS editing site. [ABSTRACT FROM AUTHOR]