Identification of novel antistaphylococcal hit compounds.

Staphylococcus aureus is one of the most common nosocomial biofilm-forming pathogens worldwide that has developed resistance mechanisms against majority of the antibiotics. Therefore, the search of novel antistaphylococcal agents with unexploited mechanisms of action, especially with antibiofilm activity, is of great interest. Seryl-tRNA synthetase is recognized as a promising drug target for the development of antibacterials. We have carried out molecular docking of compounds with antistaphycoccal activity, which were earlier found by us using phenotypic screening, into synthetic site of S. aureus SerRS and found seven hit compounds with low inhibitory activity. Further, we have performed search of S. aureus SerRS inhibitors among compounds which were previously tested by us for inhibitory activity toward S. aureus ThrRS, that belong to the same class of aminoacyl-tRNA synthetases. Among them six hits were identified. We have selected four compounds for antibacterial study and found that the most active compound 1-methyl-3-(1H-imidazol-1-methyl-2-yl)-5-nitro-1H-indazole has MIC values toward S. aureus multidrug-resistant clinical isolates ranging from 78.12 to 156.2 µg/ml. However, this compound precipitated during anti-biofilm study. Therefore, we used 3-[N'-(2-hydroxy-3-methoxybenzylidene)hydrazino]-6-methyl-4H-[1,2,4]triazin-5-one with better solubility (ClogS value = 2.9) among investigated compounds toward SerRS for anti-biofilm study. It was found that this compound has a significant inhibitory effect on the growth of planktonic and biofilm culture of S. aureus 25923 with MIC value of 32 µg ml-1. At the same time, this compound does not reveal antibacterial activity toward Esherichia coli ATCC 47076. Therefore, this compound can be proposed as effective antiseptic toward multidrug-resistant biofilm-forming S. aureus isolates.

Developing a comprehensive solution aimed to disrupt LARS1/RagD protein-protein interaction.

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.

Probing interactions of aminoacyl-adenylate with Mycobacterium tuberculosis methionyl-tRNA synthetase through in silico site-directed mutagenesis and free energy calculation.

Methionyl-tRNA synthetase (MetRS) is an attractive molecular target for antibiotic discovery. Recently, we have developed several classes of small-molecular inhibitors of Mycobacterium tuberculosis MetRS possessing antibacterial activity. In this article, we performed in silico site-directed mutagenesis of aminoacyl-adenylate binding site of M. tuberculosis MetRS in order to identify crucial amino acid residues for substrate interaction. The umbrella sampling algorithm was used to calculate the binding free energy (ΔG) of these mutated forms with methionyl-adenylate analogue. According to the obtained results, the replacement of Glu24 and Leu293 by alanine leads to the most significant decrease in the binding free energy (ΔG) for adenylate analogue with methionyl-tRNA synthetase indicating increasing of the affinity, which in turn causes the loss of compounds inhibitory activity. Therefore, these amino acid residues can be proposed for further experimental site-directed mutagenesis to confirm binding mode of inhibitors and should be taken into account during chemical optimization to overcome resistance due to mutations.Communicated by Ramaswamy H. Sarma.

Identification of dual-targeted Mycobacterium tuberculosis aminoacyl-tRNA synthetase inhibitors using machine learning.

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 IC50 values of 33 µm and 23.9 µm, respectively. Conclusion: 2-(Quinolin-2-ylsulfanyl)-acetamide scaffold can be useful for further research.

Rational Design of Hit Compounds Targeting Staphylococcus aureus Threonyl-tRNA Synthetase.

Staphylococcus aureus is one of the most dangerous nosocomial pathogens which cause a wide variety of hospital-acquired infectious diseases. S. aureus is considered as a superbug due to the development of multidrug resistance to all current therapeutic regimens. Therefore, the discovery of antibiotics with novel mechanisms of action to combat staphylococcal infections is of high priority for modern medicinal chemistry. Nowadays, aminoacyl-tRNA synthetases are considered as promising molecular targets for antibiotic development. In the present study, we used for the first time S. aureus threonyl-tRNA synthetase (ThrRS) as a molecular target. Recombinant S. aureus ThrRS was obtained in the soluble form in a sufficient amount for inhibitor screening assay. Using the molecular docking approach, we selected 180 compounds for investigation of inhibitory activity toward ThrRS. Among the tested compounds, we identified five inhibitors from different chemical classes decreasing the activity of ThrRS by more than 70% at a concentration of 100 µM. The most active compound 2,4-dibromo-6-{[4-(4-nitro-phenyl)-thiazol-2-yl]-hydrazonomethyl}-phenol has an IC50 value of 56.5 ± 3.5 µM. These compounds are not cytotoxic toward eukaryotic cells HEK293 (EC50 > 100 µM) and can be useful for further optimization and biological research.

Discovery of novel antituberculosis agents among 3-phenyl-5-(1-phenyl-1H-[1,2,3]triazol-4-yl)-[1,2,4]oxadiazole derivatives targeting aminoacyl-tRNA synthetases.

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.

Effect of Charge Distribution in a Modified tRNA Substrate on Pre-Reaction Protein-tRNA Complex Geometry.

An important aspect of molecular mechanics simulations of a protein structure and ligand binding often involves the generation of reliable force fields for nonstandard residues and ligands. We consider the aminoacyl-tRNA synthetase (AaRS) system that involves nucleic acid and amino acid derivatives, obtaining force field atomic charges using the restrained electrostatic potential (RESP) approach. These charges are shown to predict observed properties of the post-transfer editing reaction in this system, in contrast to simulations performed using approximate charges conceived based upon standard charges for related systems present in force field databases. In particular, the simulations predicted key properties induced by mutation. The approach taken for generating the RESP charges retains established charges for known fragments, defining new charges only for the novel chemical features present in the modified residues. This approach is of general relevance for the design of force fields for pharmacological applications, and indeed the AaRS target system is itself relevant to antibiotics development.

Stereospecificity control in aminoacyl-tRNA-synthetases: new evidence of d-amino acids activation and editing.

The homochirality of amino acids is vital for the functioning of the translation apparatus. l-Amino acids predominate in proteins and d-amino acids usually represent diverse regulatory functional physiological roles in both pro- and eukaryotes. Aminoacyl-tRNA-synthetases (aaRSs) ensure activation of proteinogenic or nonproteinogenic amino acids and attach them to cognate or noncognate tRNAs. Although many editing mechanisms by aaRSs have been described, data about the protective role of aaRSs in d-amino acids incorporation remained unknown. Tyrosyl- and alanyl-tRNA-synthetases were represented as distinct members of this enzyme family. To study the potential to bind and edit noncognate substrates, Thermus thermophilus alanyl-tRNA-synthetase (AlaRS) and tyrosyl-tRNA-synthetase were investigated in the context of d-amino acids recognition. Here, we showed that d-alanine was effectively activated by AlaRS and d-Ala-tRNAAla, formed during the erroneous aminoacylation, was edited by AlaRS. On the other hand, it turned out that d-aminoacyl-tRNA-deacylase (DTD), which usually hydrolyzes d-aminoacyl-tRNAs, was inactive against d-Ala-tRNAAla. To support the finding about DTD, computational docking and molecular dynamics simulations were run. Overall, our work illustrates the novel function of the AlaRS editing domain in stereospecificity control during translation together with trans-editing factor DTD. Thus, we propose different evolutionary strategies for the maintenance of chiral selectivity during translation.

Substrate-assisted mechanism of catalytic hydrolysis of misaminoacylated tRNA required for protein synthesis fidelity.

d-aminoacyl-tRNA-deacylase (DTD) prevents the incorporation of d-amino acids into proteins during translation by hydrolyzing the ester bond between mistakenly attached amino acids and tRNAs. Despite extensive study of this proofreading enzyme, the precise catalytic mechanism remains unknown. Here, a combination of biochemical and computational investigations has enabled the discovery of a new substrate-assisted mechanism of d-Tyr-tRNATyr hydrolysis by Thermus thermophilus DTD. Several functional elements of the substrate, misacylated tRNA, participate in the catalysis. During the hydrolytic reaction, the 2'-OH group of the А76 residue of d-Tyr-tRNATyr forms a hydrogen bond with a carbonyl group of the tyrosine residue, stabilizing the transition-state intermediate. Two water molecules participate in this reaction, attacking and assisting ones, resulting in a significant decrease in the activation energy of the rate-limiting step. The amino group of the d-Tyr aminoacyl moiety is unprotonated and serves as a general base, abstracting the proton from the assisting water molecule and forming a more nucleophilic ester-attacking species. Quantum chemical methodology was used to investigate the mechanism of hydrolysis. The DFT-calculated deacylation reaction is in full agreement with the experimental data. The Gibbs activation energies for the first and second steps were 10.52 and 1.05 kcal/mol, respectively, highlighting that the first step of the hydrolysis process is the rate-limiting step. Several amino acid residues of the enzyme participate in the coordination of the substrate and water molecules. Thus, the present work provides new insights into the proofreading details of misacylated tRNAs and can be extended to other systems important for translation fidelity.

Dual-target inhibitors of mycobacterial aminoacyl-tRNA synthetases among N-benzylidene-N'-thiazol-2-yl-hydrazines.

Effective treatment of tuberculosis is challenged by the rapid development of Mycobacterium tuberculosis (Mtb) multidrug resistance that presumably could be overcome with novel multi-target drugs. Aminoacyl-tRNA synthetases (AARSs) are an essential part of protein biosynthesis machinery and attractive targets for drug discovery. Here, we experimentally verify a hypothesis of simultaneous targeting of structurally related AARSs by a single inhibitor. We previously identified a new class of mycobacterial leucyl-tRNA synthetase inhibitors, N-benzylidene-N'-thiazol-2-yl-hydrazines. Molecular docking of a library of novel N-benzylidene-N'-thiazol-2-yl-hydrazine derivatives into active sites of M. tuberculosis LeuRS (MtbLeuRS) and MetRS (MtbMetRS) resulted in a panel of the best ranking compounds, which were then evaluated for enzymatic potency. Screening data revealed 11 compounds active against MtbLeuRS and 28 compounds active against MtbMetRS. The hit compounds display dual inhibitory potency as demonstrated by IC50 values for both enzymes. Compound 3 is active against Mtb H37Rv cells in in vitro bioassays.

The Dual Role of the 2'-OH Group of A76 tRNATyr in the Prevention of d-tyrosine Mistranslation.

Aminoacyl-tRNA-synthetases are crucial enzymes for initiation step of translation. Possessing editing activity, they protect living cells from misincorporation of non-cognate and non-proteinogenic amino acids into proteins. Tyrosyl-tRNA synthetase (TyrRS) does not have such editing properties, but it shares weak stereospecificity in recognition of d-/l-tyrosine (Tyr). Nevertheless, an additional enzyme, d-aminoacyl-tRNA-deacylase (DTD), exists to overcome these deficiencies. The precise catalytic role of hydroxyl groups of the tRNATyr A76 in the catalysis by TyrRS and DTD remained unknown. To address this issue, [32P]-labeled tRNATyr substrates have been tested in aminoacylation and deacylation assays. TyrRS demonstrates similar activity in charging the 2' and 3'-OH groups of A76 with l-Tyr. This synthetase can effectively use both OH groups as primary sites for aminoacylation with l-Tyr, but demonstrates severe preference toward 2'-OH, in charging with d-Tyr. In both cases, the catalysis is not substrate-assisted: neither the 2'-OH nor the 3'-OH group assists catalysis. In contrast, DTD catalyzes deacylation of d-Tyr-tRNATyr specifically from the 3'-OH group, while the 2'-OH assists in this hydrolysis.

A molecular dynamics simulation study of amino acid selectivity of LeuRS editing domain from Thermus thermophilus.

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-tRNALeu and isoleucyl-tRNALeu) was performed. Our results revealed that post-transfer substrate norvalyl-tRNALeu 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.

A new mechanism of post-transfer editing by aminoacyl-tRNA synthetases: catalysis of hydrolytic reaction by bacterial-type prolyl-tRNA synthetase.

Aminoacyl tRNA synthetases are enzymes that specifically attach amino acids to cognate tRNAs for use in the ribosomal stage of translation. For many aminoacyl tRNA synthetases, the required level of amino acid specificity is achieved either by specific hydrolysis of misactivated aminoacyl-adenylate intermediate (pre-transfer editing) or by hydrolysis of the mischarged aminoacyl-tRNA (post-transfer editing). To investigate the mechanism of post-transfer editing of alanine by prolyl-tRNA synthetase from the pathogenic bacteria Enterococcus faecalis, we used molecular modeling, molecular dynamic simulations, quantum mechanical (QM) calculations, site-directed mutagenesis of the enzyme, and tRNA modification. The results support a new tRNA-assisted mechanism of hydrolysis of misacylated Ala-tRNAPro. The most important functional element of this catalytic mechanism is the 2'-OH group of the terminal adenosine 76 of Ala-tRNAPro, which forms an intramolecular hydrogen bond with the carbonyl group of the alanine residue, strongly facilitating hydrolysis. Hydrolysis was shown by QM methods to proceed via a general acid-base catalysis mechanism involving two functionally distinct water molecules. The transition state of the reaction was identified. Amino acid residues of the editing active site participate in the coordination of substrate and both attacking and assisting water molecules, performing the proton transfer to the 3'-O atom of A76.

The crystal structure of leucyl-tRNA synthetase complexed with tRNALeu in the post-transfer-editing conformation.

Leucyl-tRNA synthetase (LeuRS) has a specific post-transfer editing activity directed against mischarged isoleucine and similar noncognate amino acids. We describe the post-transfer-editing and product complexes of Thermus thermophilus LeuRS (LeuRSTT) with tRNA(Leu) at 2.9- to 3.3-A resolution. In the post-transfer-editing configuration, A76 binds in the editing active site exactly as previously found for the adenosine moiety of a small-molecule editing-substrate analog. The 60 C-terminal residues of LeuRSTT, unseen in previous structures, fold into a compact domain flexibly linked to the rest of the molecule and interacting with the G19-C56 tertiary base pair of tRNA(Leu). LeuRS recognition of tRNA(Leu) depends essentially on tRNA shape rather than base-specific interactions. The structures show that considerable domain rotations, notably of the editing domain, accompany the tRNA-3' end dynamics associated successively with aminoacylation, post-transfer editing and product release.

Structural basis for orthogonal tRNA specificities of tyrosyl-tRNA synthetases for genetic code expansion.

The archaeal/eukaryotic tyrosyl-tRNA synthetase (TyrRS)-tRNA(Tyr) pairs do not cross-react with their bacterial counterparts. This 'orthogonal' condition is essential for using the archaeal pair to expand the bacterial genetic code. In this study, the structure of the Methanococcus jannaschii TyrRS-tRNA(Tyr)-L-tyrosine complex, solved at a resolution of 1.95 A, reveals that this archaeal TyrRS strictly recognizes the C1-G72 base pair, whereas the bacterial TyrRS recognizes the G1-C72 in a different manner using different residues. These diverse tRNA recognition modes form the basis for the orthogonality. The common tRNA(Tyr) identity determinants (the discriminator, A73 and the anticodon residues) are also recognized in manners different from those of the bacterial TyrRS. Based on this finding, we created a mutant TyrRS that aminoacylates the amber suppressor tRNA with C34 65 times more efficiently than does the wild-type enzyme.

Structural and mechanistic basis of pre- and posttransfer editing by leucyl-tRNA synthetase.

The aminoacyl-tRNA synthetases link tRNAs with their cognate amino acid. In some cases, their fidelity relies on hydrolytic editing that destroys incorrectly activated amino acids or mischarged tRNAs. We present structures of leucyl-tRNA synthetase complexed with analogs of the distinct pre- and posttransfer editing substrates. The editing active site binds the two different substrates using a single amino acid discriminatory pocket while preserving the same mode of adenine recognition. This suggests a similar mechanism of hydrolysis for both editing substrates that depends on a key, completely conserved aspartic acid, which interacts with the alpha-amino group of the noncognate amino acid and positions both substrates for hydrolysis. Our results demonstrate the economy by which a single active site accommodates two distinct substrates in a proofreading process critical to the fidelity of protein synthesis.

Class I tyrosyl-tRNA synthetase has a class II mode of cognate tRNA recognition.

Bacterial tyrosyl-tRNA synthetases (TyrRS) possess a flexibly linked C-terminal domain of approximately 80 residues, which has hitherto been disordered in crystal structures of the enzyme. We have determined the structure of Thermus thermophilus TyrRS at 2.0 A resolution in a crystal form in which the C-terminal domain is ordered, and confirm that the fold is similar to part of the C-terminal domain of ribosomal protein S4. We have also determined the structure at 2.9 A resolution of the complex of T.thermophilus TyrRS with cognate tRNA(tyr)(G Psi A). In this structure, the C-terminal domain binds between the characteristic long variable arm of the tRNA and the anti-codon stem, thus recognizing the unique shape of the tRNA. The anticodon bases have a novel conformation with A-36 stacked on G-34, and both G-34 and Psi-35 are base-specifically recognized. The tRNA binds across the two subunits of the dimeric enzyme and, remarkably, the mode of recognition of the class I TyrRS for its cognate tRNA resembles that of a class II synthetase in being from the major groove side of the acceptor stem.