Identification of Novel Dual-targeting Inhibitors of Aminoacyl-tRNA Synthetases in Mycobacterium tuberculosis

Antibiotic resistance is a major problem of tuberculosis treatment. This provides the stimulus for the search of novel molecular targets and approaches to reduce or forestall resistance emergence in Mycobacterium tuberculosis. Earlier, we discovered a novel small-molecular inhibitor among 3-phenyl-5-(1-phenyl-1H-[1,2,3]triazol-4-yl)-[1,2,4]oxadiazoles targeting simultaneously two enzymes—mycobacterial leucyl-tRNA synthetase (LeuRS) and methionyl-tRNA synthetase (MetRS), which are promising molecular targets for antibiotic development. Unfortunately, the identified inhibitor does not reveal antibacterial activity toward M. tuberculosis. This study aims to develop novel aminoacyl-tRNA synthetase inhibitors among this chemical class with antibacterial activity toward resistant strains of M. tuberculosis. We performed molecular docking of the library of 3-phenyl-5-(1-phenyl-1H-[1,2,3]triazol-4-yl)-[1,2,4]oxadiazole derivatives and selected 41 compounds for investigation of their inhibitory activity toward MetRS and LeuRS in aminoacylation assay and antibacterial activity toward M. tuberculosis strains using microdilution assay. In vitro screening resulted in 10 compounds active against MetRS and 3 compounds active against LeuRS. Structure-related relationships (SAR) were established. The antibacterial screening revealed 4 compounds active toward M. tuberculosis mono-resistant strains in the range of concentrations 2–20 mg/L. Among these compounds, only one compound 27 has significant enzyme inhibitory activity toward mycobacterial MetRS (IC50 = 148.5 µM). The MIC for this compound toward M. tuberculosis H37Rv strain is 12.5 µM. This compound is not cytotoxic to human HEK293 and HepG2 cell lines. Therefore, 3-phenyl-5-(1-phenyl-1H-[1,2,3]triazol-4-yl)-[1,2,4]oxadiazole derivatives can be used for further chemical optimization and biological research to find non-toxic antituberculosis agents with a novel mechanism of action.

Leucyl-tRNA synthetase (LeuRS) performs dual essential roles in group I intron RNA splicing as well as protein synthesis within the yeast mitochondria. Deletions of the C terminus differentially impact the two functions of the enzyme in splicing and aminoacylation in vivo. Herein, we determined that a fiveamino acid C-terminal deletion of LeuRS, which does not complement a null strain, can form a ternary complex with the bI4 intron and its maturase splicing partner. However, the complex fails to stimulate splicing activity. The x-ray co-crystal structure of LeuRS showed that a C-terminal extension of about 60 amino acids forms a discrete domain, which is unique among the LeuRSs and interacts with the corner of the L-shaped tRNALeu. Interestingly, deletion of the entire yeast mitochondrial LeuRS C-terminal domain enhanced its aminoacylation and amino acid editing activities. In striking contrast, deletion of the corresponding C-terminal domain of Escherichia coli LeuRS abolished aminoacylation of tRNALeu and also amino acid editing of mischarged tRNA molecules. These results suggest that the role of the leucine-specific C-terminal domain in tRNA recognition for aminoacylation and amino acid editing has adapted differentially and with surprisingly opposite effects. We propose that the secondary role of yeast mitochondrial LeuRS in RNA splicing has impacted the functional evolution of this critical C-terminal domain.

The macromolecular tRNA synthetase complex consists of nine different enzymes and three non-enzymatic factors. This complex was recently shown to be a novel signalosome, since many of its components are involved in signaling pathways in addition to their catalytic roles in protein synthesis. The structural organization and dynamic relationships of the components of the complex are not well understood. Here we performed a systematic depletion analysis to determine the effects of structural intimacy and the turnover of the components. The results showed that the stability of some components depended on their neighbors. Lysyl-tRNA synthetase was most independent of other components for its stability whereas it was most required for the stability of other components. Arginyl- and methionyl-tRNA synthetases had the opposite characteristics. Thus, the systematic depletion of the components revealed the functional reason for the complex formation and the assembly pattern of these multi-functional enzymes and their associated factors.

Pharmacophore identification and virtual screening for methionyl-tRNA synthetase inhibitors

Incidences of drug-resistant tuberculosis have become common and are rising at an alarming rate. Aminoacyl t-RNA synthetase has been validated as a newer target against Mycobacterium tuberculosis. Leucyl t-RNA synthetase (LeuRS) is ubiquitously found in all organisms and regulates transcription, protein synthesis, mitochondrial RNA cleavage, and proofreading of matured t-RNA. Leucyl t-RNA synthetase promotes growth and development and is the key enzyme needed for biofilm formation in Mycobacterium. Inhibition of this enzyme could restrict the growth and development of the mycobacterial population. A database consisting of 2734 drug-like molecules was screened against leucyl t-RNA synthetase enzymes through virtual screening. Based on the docking scores and MMGBSA energy values, the top three compounds were selected for molecular dynamics simulation. The druggable nature of the top three hits was confirmed by predicting their pharmacokinetic parameters. The top three hits-compounds 1035 (ZINC000001543916), 1054 (ZINC000001554197), and 2077 (ZINC000008214483)-were evaluated for their binding affinity toward leucyl t-RNA synthetase by an isothermal titration calorimetry study. The inhibitory activity of these compounds was tested against antimycobacterial activity, biofilm formation, and LeuRS gene expression potential. Compound 1054 (Macimorelin) was found to be the most potent molecule, with better antimycobacterial activity, enzyme binding affinity, and significant inhibition of biofilm formation, as well as inhibition of the LeuRS gene expression. Compound 1054, the top hit compound, has the potential to be used as a lead to develop successful leucyl t-RNA synthetase inhibitors.

Pharmacophore-based virtual screening: The discovery of novel methionyl-tRNA synthetase inhibitors

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 13µM and 13.8µM, 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.

Tuberculosis has become the world's most lethal infectious disease. A major challenge in treating tuberculosis is the multidrug resistance of Mycobacterium tuberculosis to existing antibiotics. Therefore, there is an urgent need to discover new antituberculosis agents with unexploited mechanisms of action. The aim of the work was to develop inhibitors of mycobacterial leucyl-tRNA synthetase (LeuRS) with antibacterial activity. The virtual screening of compound collection containing about 250,000 ligands into aminoacyl-adenylate binding site of M. tuberculosis LeuRS was performed with AutoDock 4.2 software. The inhibitory activity of compounds toward recombinant LeuRS was studied in aminoacylation assay using 14C-labeled L-leucine. Antibacterial activity was investigated toward M. tuberculosis H37Rv strain under four different conditions. According to the data of biochemical screening, we have found M. tuberculosis LeuRS inhibitors among N-(5-Benzyl-thiazol-2-yl)-2-(1-phenyl-1H-tetrazol-5-ylsulfanyl)-acetamide deivatives, which decrease enzyme activity with IC50 values in micromolar range. The most promising compound, N-(5-Benzyl-thiazol-2-yl)-2-[4-(4-methoxy-phenyl)-1H-tetrazol-5-ylsulfanyl]-acetamide, reveals potent antibacterial activity with the best minimum inhibitory concentration (MIC) value of 4.7 µM. N-(5-Benzyl-thiazol-2-yl)-2-(1-phenyl-1H-tetrazol-5-ylsulfanyl)-acetamide scaffold can be valuable for further biological research and chemical optimization.

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.

Incubation of liver extracts with MgATP led to complete inactivation of the aminoacyl‐tRNA synthetase complex provided that sodium fluoride was included. If sodium fluoride was omitted no inactivation was observed. The arginyl‐tRNA and isoleucyl‐tRNA synthetases were inactivated more slowly than the leucyl‐tRNA, lysyl‐tRNA and methionyl‐tRNA synthetases. Gel filtration on Sepharose 4B separated the aminoacyl‐tRNA synthetase complex (Ve/Vo= 1.2) from an inactivating factor that required MgATP (Ve/Vo= 2.8).The inactivated aminoacyl‐tRNA synthetase complex was eluted from Sepharose‐4B at the same position as the activated complex, and was resolved from a reactivating factor that required Mg2+ and was inhibited by sodium fluoride (Ve/Vo= 2.5). The arginyi‐tRNA and isoleucyl‐tRNA synthetases were reactivated more rapidly than the leucyl‐tRNA, lysyl‐tRNA and methionyl‐tRNA synthetases.The aminoacyl‐tRNA synthetase complex could also be reactivated using highly purified preparations of either protein phosphatase 1 or protein phosphatase 2A. Inhibitor 2, a specific inhibitor of protein phosphatase 1, prevented reactivation of the complex by protein phosphatase 1, but not by protein phosphatase 2A. The relative rates of reactivation of the different activities of the complex by either protein phosphatase 1 or protein phosphatase 2A were very similar to those observed with the reactivating factor.The aminoacyl‐tRNA synthetase complex was largely in the activated state in livers of normally fed rats, but overnight starvation produced substantial conversion to the inactivated form of the complex. Incubation of hepatocytes from normally fed rats with glucagon and isobutylmethylxanthine for 20 min also caused a substantial decrease in the activation state from approximately 80% to approximately 30%. Conversely, when hepatocytes prepared from starved rats were incubated with insulin for 30 min, almost complete reactivation of the complex was achieved.The results indicate that the aminoacyl‐tRNA synthetase complex is regulated by a phosphorylation dephosphorylation mechanism in vitro and in vivo. The effects of hormones on the activation state of the complex are discussed in the light of the observation that the inactivating factor is not cyclic‐AMP‐dependent protein kinase.

Aminoacyl-tRNA synthetases are a family of enzymes that are responsible for translating the genetic code in the first step of protein synthesis. Some aminoacyl-tRNA synthetases have editing activities to clear their mistakes and enhance fidelity. Leucyl-tRNA synthetases have a hydrolytic active site that resides in a discrete amino acid editing domain called CP1. Mutational analysis within yeast mitochondrial leucyl-tRNA synthetase showed that the enzyme has maintained an editing active site that is competent for post-transfer editing of mischarged tRNA similar to other leucyl-tRNA synthetases. These mutations that altered or abolished leucyl-tRNA synthetase editing were introduced into complementation assays. Cell viability and mitochondrial function were largely unaffected in the presence of high levels of non-leucine amino acids. In contrast, these editing-defective mutations limited cell viability in Escherichia coli. It is possible that the yeast mitochondria have evolved to tolerate lower levels of fidelity in protein synthesis or have developed alternate mechanisms to enhance discrimination of leucine from non-cognate amino acids that can be misactivated by leucyl-tRNA synthetase.

Identification of novel inhibitors of methionyl-tRNA synthetase (MetRS) by virtual screening

Aminoacyl-tRNA synthetases are multidomain enzymes that often possess two activities to ensure translational accuracy. A synthetic active site catalyzes tRNA aminoacylation, while an editing active site hydrolyzes mischarged tRNAs. Prolyl-tRNA synthetases (ProRS) have been shown to misacylate Cys onto tRNA(Pro), but lack a Cys-specific editing function. The synthetase-like Haemophilus influenzae YbaK protein was recently shown to hydrolyze misacylated Cys-tRNA(Pro) in trans. However, the mechanism of specific substrate selection by this single domain hydrolase is unknown. Here, we demonstrate that YbaK alone appears to lack specific tRNA recognition capabilities. Moreover, YbaK cannot compete for aminoacyl-tRNAs in the presence of elongation factor Tu, suggesting that YbaK acts before release of the aminoacyl-tRNA from the synthetase. In support of this idea, cross-linking studies reveal the formation of binary (ProRS.YbaK) and ternary (ProRS.YbaK.tRNA) complexes. The binding constants for the interaction between ProRS and YbaK are 550 nM and 45 nM in the absence and presence of tRNA(Pro), respectively. These results suggest that the specificity of trans-editing by YbaK is ensured through formation of a novel ProRS.YbaK.tRNA complex.

The editing reactions catalyzed by aminoacyl-tRNA synthetases are critical for the faithful protein synthesis by correcting misactivated amino acids and misaminoacylated tRNAs. We report that the isolated editing domain of leucyl-tRNA synthetase from the deep-rooted bacterium Aquifex aeolicus (alphabeta-LeuRS) catalyzes the hydrolytic editing of both mischarged tRNA(Leu) and minihelix(Leu). Within the domain, we have identified a crucial 20-amino-acid peptide that confers editing capacity when transplanted into the inactive Escherichia coli LeuRS editing domain. Likewise, fusion of the beta-subunit of alphabeta-LeuRS to the E. coli editing domain activates its editing function. These results suggest that alphabeta-LeuRS still carries the basic features from a primitive synthetase molecule. It has a remarkable capacity to transfer autonomous active modules, which is consistent with the idea that modern synthetases arose after exchange of small idiosyncratic domains. It also has a unique alphabeta-heterodimeric structure with separated catalytic and tRNA-binding sites. Such an organization supports the tRNA/synthetase coevolution theory that predicts sequential addition of tRNA and synthetase domains.

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