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.

Investigation of Meat from Ostriches Raised and Slaughtered in Bavaria, Germany: Microbiological Quality and Antimicrobial Resistance.

Ostrich meat is characterized by high nutritional value; however, it remains an exotic product in most countries worldwide. In Europe, only few data are available regarding its microbial contamination, prevalence of antimicrobial-resistant bacteria, and safety. Therefore, this study aimed to investigate the microbiological quality and safety of ostrich meat samples (n = 55), each from one animal, produced in Bavaria, Germany. The provided microbiological status of ostrich meat included mesophilic aerobic bacteria, Enterobacteria, and mesophilic yeast and molds. In terms of food safety, all meat samples were negative for Salmonella spp. and Trichinella spp. Additionally, meat samples and a further 30 stool samples from 30 individuals were investigated for Shiga toxin-producing Escherichia coli genes, with two meat samples that were qPCR-positive. Antimicrobial-resistant Enterobacteriaceae, Enterococcus faecalis, and Enterococcus faecium strains were from meat and stool samples also analyzed; 13 potentially resistant Enterobacteriaceae (meat samples) and 4 Enterococcus faecium (stool samples) were isolated, and their susceptibility against 29 and 14 antimicrobials, respectively, was characterized. The results of this study provide an overview of microbial loads and food safety aspects that may be used as baseline data for the ostrich meat industry to improve their hygienic quality. However, the implementation of monitoring programs is recommended, and microbiological standards for ostrich meat production should be established.

AbdomenNet: deep neural network for abdominal organ segmentation in epidemiologic imaging studies.

BACKGROUND: Whole-body imaging has recently been added to large-scale epidemiological studies providing novel opportunities for investigating abdominal organs. However, the segmentation of these organs is required beforehand, which is time consuming, particularly on such a large scale. METHODS: We introduce AbdomentNet, a deep neural network for the automated segmentation of abdominal organs on two-point Dixon MRI scans. A pre-processing pipeline enables to process MRI scans from different imaging studies, namely the German National Cohort, UK Biobank, and Kohorte im Raum Augsburg. We chose a total of 61 MRI scans across the three studies for training an ensemble of segmentation networks, which segment eight abdominal organs. Our network presents a novel combination of octave convolutions and squeeze and excitation layers, as well as training with stochastic weight averaging. RESULTS: Our experiments demonstrate that it is beneficial to combine data from different imaging studies to train deep neural networks in contrast to training separate networks. Combining the water and opposed-phase contrasts of the Dixon sequence as input channels, yields the highest segmentation accuracy, compared to single contrast inputs. The mean Dice similarity coefficient is above 0.9 for larger organs liver, spleen, and kidneys, and 0.71 and 0.74 for gallbladder and pancreas, respectively. CONCLUSIONS: Our fully automated pipeline provides high-quality segmentations of abdominal organs across population studies. In contrast, a network that is only trained on a single dataset does not generalize well to other datasets.

Dual-targeted hit identification using pharmacophore screening.

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.

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.

Identification of Mycobacterium tuberculosis leucyl-tRNA synthetase (LeuRS) inhibitors among the derivatives of 5-phenylamino-2H-[1,2,4]triazin-3-one.

The increase of antibiotic resistance amongst Mycobacterium tuberculosis strains has become one of the most pressing problems of modern medicine. Therefore, the search of antibiotics against M. tuberculosis with novel mechanisms of action is very important. We have identified inhibitors of M. tuberculosis leucyl-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 inhibit M. tuberculosis LeuRS with IC50 of 7.6 µÐœ and 7.2 µÐœ, respectively. It was established that the inhibitory activity of compounds against pathogenic LeuRS is 10-fold better, than for human enzyme.

Discovery of potent anti-tuberculosis agents targeting leucyl-tRNA synthetase.

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 IC50 of 6µM. 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 IC50 of 2.27µM. 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 IC50=10.01µM and IC90=13.53µM.

Effective plaque removal with a new 8 French-compatible atherectomy catheter.

The purpose of this study was to evaluate the safety and efficacy of the new 8 Fr guide catheter-compatible Flexicut directional atherectomy device and to compare it with the conventional Atherocath GTO catheter. The 6 Fr Flexicut catheter has a larger cutting window and a titanium nitride-coated cutter to effect more tissue removal as well as treat mildly calcified lesions. A group of 143 lesions in 117 consecutive patients treated with the Flexicut catheter in four centers were compared with a control group of 277 lesions in 212 consecutive patients treated with the GTO device. Postatherectomy luminal diameters were larger (2.92 +/- 0.79 vs. 2.52 +/- 0.64 mm; P < 0.0001), with more luminal gain (relative gain: 0.58 +/- 0.24 vs. 0.48 +/- 0.25; P = 0.0007) using fewer directional coronary atherectomy (DCA) cuts (12 +/- 7 vs. 16 +/- 9; P = 0.0001) in the Flexicut group. A residual diameter stenosis < 20% immediately after DCA was obtained in 77% of the lesions in the Flexicut group vs. 45% in the GTO group (P < 0.0001). Histology in the former group revealed large calcium speckles in the retrieved specimens. In the Flexicut group, there was a lower incidence of access site complications and damage to the coronary ostium (2.5% vs. 7.5%; P = 0.08). The new Flexicut catheter is more effective than the conventional GTO catheter with a trend for reduced guiding catheter-related complications.