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1.
Int J Mol Sci ; 22(15)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34360555

RESUMO

Human cytosolic prolyl-tRNA synthetase (HcProRS) catalyses the formation of the prolyl-tRNAPro, playing an important role in protein synthesis. Inhibition of HcProRS activity has been shown to have potential benefits in the treatment of fibrosis, autoimmune diseases and cancer. Recently, potent pyrazinamide-based inhibitors were identified by a high-throughput screening (HTS) method, but no further elaboration was reported. The pyrazinamide core is a bioactive fragment found in numerous clinically validated drugs and has been subjected to various modifications. Therefore, we applied a virtual screening protocol to our in-house library of pyrazinamide-containing small molecules, searching for potential novel HcProRS inhibitors. We identified a series of 3-benzylaminopyrazine-2-carboxamide derivatives as positive hits. Five of them were confirmed by a thermal shift assay (TSA) with the best compounds 3b and 3c showing EC50 values of 3.77 and 7.34 µM, respectively, in the presence of 1 mM of proline (Pro) and 3.45 µM enzyme concentration. Co-crystal structures of HcProRS in complex with these compounds and Pro confirmed the initial docking studies and show how the Pro facilitates binding of the ligands that compete with ATP substrate. Modelling 3b into other human class II aminoacyl-tRNA synthetases (aaRSs) indicated that the subtle differences in the ATP binding site of these enzymes likely contribute to its potential selective binding of HcProRS. Taken together, this study successfully identified novel HcProRS binders from our anti-tuberculosis in-house compound library, displaying opportunities for repurposing old drug candidates for new applications such as therapeutics in HcProRS-related diseases.


Assuntos
Trifosfato de Adenosina/metabolismo , Aminoacil-tRNA Sintetases/antagonistas & inibidores , Bioensaio/métodos , Simulação por Computador , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Pirazinamida/química , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/isolamento & purificação , Humanos , Ligantes , Modelos Moleculares , Conformação Proteica
2.
Bioorg Chem ; 110: 104806, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33799176

RESUMO

Aminoacyl-tRNA synthetases (aaRSs) are crucial for the correct assembly of amino acids to cognate tRNA to maintain the fidelity of proteosynthesis. AaRSs have become a hot target in antimicrobial research. Three aaRS inhibitors are already in clinical practice; antibacterial mupirocin inhibits the synthetic site of isoleucyl-tRNA synthetase, antifungal tavaborole inhibits the editing site of leucyl-tRNA synthetase, and antiprotozoal halofuginone inhibits proline-tRNA synthetase. According to the World Health Organization, tuberculosis globally remains the leading cause of death from a single infectious agent. The rising incidence of multidrug-resistant tuberculosis is alarming and urges the search for new antimycobacterial compounds, preferably with yet unexploited mechanism of action. In this literature review, we have covered the up-to-date state in the field of inhibitors of mycobacterial aaRSs. The most studied aaRS in mycobacteria is LeuRS with at least four structural types of inhibitors, followed by TyrRS and AspRS. Inhibitors of MetRS, LysRS, and PheRS were addressed in a single significant study each. In many cases, the enzyme inhibition activity translated into micromolar or submicromolar inhibition of growth of mycobacteria. The most promising aaRS inhibitor as an antimycobacterial compound is GSK656 (compound 8), the only aaRS inhibitor in clinical trials (Phase IIa) for systemic use against tuberculosis. GSK656 is orally available and shares the oxaborole tRNA-trapping mechanism of action with antifungal tavaborole.


Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Aminoacil-tRNA Sintetases/metabolismo , Antibacterianos/síntese química , Antibacterianos/química , Bactérias/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade
3.
Int J Mol Sci ; 22(4)2021 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-33578647

RESUMO

Aminoacyl-tRNA synthetases (aaRSs) catalyze the esterification of tRNA with a cognate amino acid and are essential enzymes in all three kingdoms of life. Due to their important role in the translation of the genetic code, aaRSs have been recognized as suitable targets for the development of small molecule anti-infectives. In this review, following a concise discussion of aaRS catalytic and proof-reading activities, the various inhibitory mechanisms of reported natural and synthetic aaRS inhibitors are discussed. Using the expanding repository of ligand-bound X-ray crystal structures, we classified these compounds based on their binding sites, focusing on their ability to compete with the association of one, or more of the canonical aaRS substrates. In parallel, we examined the determinants of species-selectivity and discuss potential resistance mechanisms of some of the inhibitor classes. Combined, this structural perspective highlights the opportunities for further exploration of the aaRS enzyme family as antimicrobial targets.


Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Anti-Infecciosos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Animais , Anti-Infecciosos/química , Sítios de Ligação/efeitos dos fármacos , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Terapia de Alvo Molecular
4.
Molecules ; 25(20)2020 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-33081246

RESUMO

Aminoacyl-tRNA synthetases (aaRSs) have become viable targets for the development of antimicrobial agents due to their crucial role in protein translation. A series of six amino acids were coupled to the purine-like 7-amino-5-hydroxymethylbenzimidazole nucleoside analogue following an optimized synthetic pathway. These compounds were designed as aaRS inhibitors and can be considered as 1,3-dideazaadenine analogues carrying a 2-hydroxymethyl substituent. Despite our intentions to obtain N1-glycosylated 4-aminobenzimidazole congeners, resembling the natural purine nucleosides glycosylated at the N9-position, we obtained the N3-glycosylated benzimidazole derivatives as the major products, resembling the respective purine N7-glycosylated nucleosides. A series of X-ray crystal structures of class I and II aaRSs in complex with newly synthesized compounds revealed interesting interactions of these "base-flipped" analogues with their targets. While the exocyclic amine of the flipped base mimics the reciprocal interaction of the N3-purine atom of aminoacyl-sulfamoyl adenosine (aaSA) congeners, the hydroxymethyl substituent of the flipped base apparently loses part of the standard interactions of the adenine N1 and the N6-amine as seen with aaSA analogues. Upon the evaluation of the inhibitory potency of the newly obtained analogues, nanomolar inhibitory activities were noted for the leucine and isoleucine analogues targeting class I aaRS enzymes, while rather weak inhibitory activity against the corresponding class II aaRSs was observed. This class bias could be further explained by detailed structural analysis.


Assuntos
Aminoacil-tRNA Sintetases/ultraestrutura , Benzimidazóis/química , Inibidores Enzimáticos/síntese química , Ribonucleosídeos/química , Aminoacil-tRNA Sintetases/antagonistas & inibidores , Aminoacil-tRNA Sintetases/química , Benzimidazóis/síntese química , Benzimidazóis/farmacologia , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Neisseria gonorrhoeae/química , Neisseria gonorrhoeae/enzimologia , Neisseria gonorrhoeae/patogenicidade , Conformação Proteica/efeitos dos fármacos , Relação Estrutura-Atividade
5.
Circ Res ; 127(6): 827-846, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32611237

RESUMO

RATIONALE: Increased protein synthesis of profibrotic genes is a common feature in cardiac fibrosis and heart failure. Despite this observation, critical factors and molecular mechanisms for translational control of profibrotic genes during cardiac fibrosis remain unclear. OBJECTIVE: To investigate the role of a bifunctional ARS (aminoacyl-tRNA synthetase), EPRS (glutamyl-prolyl-tRNA synthetase) in translational control of cardiac fibrosis. METHODS AND RESULTS: Results from reanalyses of multiple publicly available data sets of human and mouse heart failure, demonstrated that EPRS acted as an integrated node among the ARSs in various cardiac pathogenic processes. We confirmed that EPRS was induced at mRNA and protein levels (≈1.5-2.5-fold increase) in failing hearts compared with nonfailing hearts using our cohort of human and mouse heart samples. Genetic knockout of one allele of Eprs globally (Eprs+/-) using CRISPR-Cas9 technology or in a Postn-Cre-dependent manner (Eprsflox/+; PostnMCM/+) strongly reduces cardiac fibrosis (≈50% reduction) in isoproterenol-, transverse aortic constriction-, and myocardial infarction (MI)-induced heart failure mouse models. Inhibition of EPRS using a PRS (prolyl-tRNA synthetase)-specific inhibitor, halofuginone, significantly decreases translation efficiency (TE) of proline-rich collagens in cardiac fibroblasts as well as TGF-ß (transforming growth factor-ß)-activated myofibroblasts. Overexpression of EPRS increases collagen protein expression in primary cardiac fibroblasts under TGF-ß stimulation. Using transcriptome-wide RNA-Seq and polysome profiling-Seq in halofuginone-treated fibroblasts, we identified multiple novel Pro-rich genes in addition to collagens, such as Ltbp2 (latent TGF-ß-binding protein 2) and Sulf1 (sulfatase 1), which are translationally regulated by EPRS. SULF1 is highly enriched in human and mouse myofibroblasts. In the primary cardiac fibroblast culture system, siRNA-mediated knockdown of SULF1 attenuates cardiac myofibroblast activation and collagen deposition. Overexpression of SULF1 promotes TGF-ß-induced myofibroblast activation and partially antagonizes anti-fibrotic effects of halofuginone treatment. CONCLUSIONS: Our results indicate that EPRS preferentially controls translational activation of proline codon rich profibrotic genes in cardiac fibroblasts and augments pathological cardiac remodeling. Graphical Abstract: A graphical abstract is available for this article.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Insuficiência Cardíaca/enzimologia , Miócitos Cardíacos/enzimologia , Miofibroblastos/enzimologia , Biossíntese de Proteínas , Aminoacil-tRNA Sintetases/antagonistas & inibidores , Aminoacil-tRNA Sintetases/genética , Animais , Estudos de Casos e Controles , Colágeno/biossíntese , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Feminino , Fibrose , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Humanos , Proteínas de Ligação a TGF-beta Latente/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/patologia , Células NIH 3T3 , Domínios Proteicos Ricos em Prolina , Biossíntese de Proteínas/efeitos dos fármacos , Transdução de Sinais , Sulfotransferases/biossíntese , Sulfotransferases/genética
6.
Int J Mol Sci ; 21(11)2020 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-32471245

RESUMO

Recently, there has been increased interest in aminoacyl tRNA synthetases (aaRSs) as potential malarial drug targets. These enzymes play a key role in protein translation by the addition of amino acids to their cognate tRNA. The aaRSs are present in all Plasmodium life cycle stages, and thus present an attractive malarial drug target. Prolyl tRNA synthetase is a class II aaRS that functions in charging tRNA with proline. Various inhibitors against Plasmodium falciparum ProRS (PfProRS) active site have been designed. However, none have gone through clinical trials as they have been found to be highly toxic to human cells. Recently, a possible allosteric site was reported in PfProRS with two possible allosteric modulators: glyburide and TCMDC-124506. In this study, we sought to identify novel selective inhibitors targeting PfProRS active site and possible novel allosteric modulators of this enzyme. To achieve this, virtual screening of South African natural compounds against PfProRS and the human homologue was carried out using AutoDock Vina. The modulation of protein motions by ligand binding was studied by molecular dynamics (MD) using the GROningen MAchine for Chemical Simulations (GROMACS) tool. To further analyse the protein global motions and energetic changes upon ligand binding, principal component analysis (PCA), and free energy landscape (FEL) calculations were performed. Further, to understand the effect of ligand binding on the protein communication, dynamic residue network (DRN) analysis of the MD trajectories was carried out using the MD-TASK tool. A total of ten potential natural hit compounds were identified with strong binding energy scores. Binding of ligands to the protein caused observable global and residue level changes. Dynamic residue network calculations showed increase in betweenness centrality (BC) metric of residues at the allosteric site implying these residues are important in protein communication. A loop region at the catalytic domain between residues 300 and 350 and the anticodon binding domain showed significant contributions to both PC1 and PC2. Large motions were observed at a loop in the Z-domain between residues 697 and 710 which was also in agreement with RMSF calculations that showed increase in flexibility of residues in this region. Residues in this loop region are implicated in ATP binding and thus a change in dynamics may affect ATP binding affinity. Free energy landscape (FEL) calculations showed that the holo protein (protein-ADN complex) and PfProRS-SANC184 complexes were stable, as shown by the low energy with very few intermediates and hardly distinguishable low energy barriers. In addition, FEL results agreed with backbone RMSD distribution plots where stable complexes showed a normal RMSD distribution while unstable complexes had multimodal RMSD distribution. The betweenness centrality metric showed a loss of functional importance of key ATP binding site residues upon allosteric ligand binding. The deep basins in average L observed at the allosteric region imply that there is high accessibility of residues at this region. To further analyse BC and average L metrics data, we calculated the ΔBC and ΔL values by taking each value in the holo protein BC or L matrix less the corresponding value in the ligand-bound complex BC or L matrix. Interestingly, in allosteric complexes, residues located in a loop region implicated in ATP binding had negative ΔL values while in orthosteric complexes these residues had positive ΔL values. An increase in contact frequency between residues Ser263, Thr267, Tyr285, and Leu707 at the allosteric site and residues Thr397, Pro398, Thr402, and Gln395 at the ATP binding TXE loop was observed. In summary, this study identified five potential orthosteric inhibitors and five allosteric modulators against PfProRS. Allosteric modulators changed ATP binding site dynamics, as shown by RMSF, PCA, and DRN calculations. Changes in dynamics of the ATP binding site and increased contact frequency between residues at the proposed allosteric site and the ATP binding site may explain how allosteric modulators distort the ATP binding site and thus might inhibit PfProRS. The scaffolds of the identified hits in the study can be used as a starting point for antimalarial inhibitor development with low human cytotoxicity.


Assuntos
Sítio Alostérico , Aminoacil-tRNA Sintetases/antagonistas & inibidores , Produtos Biológicos/farmacologia , Inibidores Enzimáticos/farmacologia , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/antagonistas & inibidores , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Produtos Biológicos/química , Domínio Catalítico , Inibidores Enzimáticos/química , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo
7.
Proc Natl Acad Sci U S A ; 117(16): 8900-8911, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32253314

RESUMO

Signaling pathways that sense amino acid abundance are integral to tissue homeostasis and cellular defense. Our laboratory has previously shown that halofuginone (HF) inhibits the prolyl-tRNA synthetase catalytic activity of glutamyl-prolyl-tRNA synthetase (EPRS), thereby activating the amino acid response (AAR). We now show that HF treatment selectively inhibits inflammatory responses in diverse cell types and that these therapeutic benefits occur in cells that lack GCN2, the signature effector of the AAR. Depletion of arginine, histidine, or lysine from cultured fibroblast-like synoviocytes recapitulates key aspects of HF treatment, without utilizing GCN2 or mammalian target of rapamycin complex 1 pathway signaling. Like HF, the threonyl-tRNA synthetase inhibitor borrelidin suppresses the induction of tissue remodeling and inflammatory mediators in cytokine-stimulated fibroblast-like synoviocytes without GCN2, but both aminoacyl-tRNA synthetase (aaRS) inhibitors are sensitive to the removal of GCN1. GCN1, an upstream component of the AAR pathway, binds to ribosomes and is required for GCN2 activation. These observations indicate that aaRS inhibitors, like HF, can modulate inflammatory response without the AAR/GCN2 signaling cassette, and that GCN1 has a role that is distinct from its activation of GCN2. We propose that GCN1 participates in a previously unrecognized amino acid sensor pathway that branches from the canonical AAR.


Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Anti-Inflamatórios/farmacologia , Artrite Reumatoide/tratamento farmacológico , Piperidinas/farmacologia , Quinazolinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Animais , Anti-Inflamatórios/uso terapêutico , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Artrite Reumatoide/cirurgia , Linhagem Celular , Fibroblastos , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , Pulmão/citologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Knockout , Piperidinas/uso terapêutico , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Quinazolinonas/uso terapêutico , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA-Seq , Transdução de Sinais/imunologia , Membrana Sinovial/citologia , Membrana Sinovial/patologia , Sinoviócitos , Transativadores/genética , Transativadores/metabolismo
8.
J Med Chem ; 63(8): 3908-3914, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32208684

RESUMO

Aminoacyl-tRNA synthetase interacting multifunctional proteins (AIMPs) have recently been considered novel therapeutic targets in several cancers. In this publication we report the development of novel 2-aminophenylpyrimidines as new AIMP2-DX2 inhibitors. In particular, aminophenylpyrimidine 3 not only exhibited promising in vitro and in vivo potency but also exerted selective inhibition of H460 and A549 cells and AIMP2-DX2 rather than WI-26 cells and AIMP2. Aminophenylpyrimidine 3 offers possible therapeutic potential in the treatment of lung cancer.


Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Células A549 , Aminoacil-tRNA Sintetases/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Nucleares/metabolismo , Pirimidinas/química , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
9.
Infect Immun ; 88(4)2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-31964747

RESUMO

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections, and Chlamydia pneumoniae causes community-acquired respiratory infections. In vivo, the host immune system will release gamma interferon (IFN-γ) to combat infection. IFN-γ activates human cells to produce the tryptophan (Trp)-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO). Consequently, there is a reduction in cytosolic Trp in IFN-γ-activated host cells. In evolving to obligate intracellular dependence, Chlamydia has significantly reduced its genome size and content, as it relies on the host cell for various nutrients. Importantly, C. trachomatis and C. pneumoniae are Trp auxotrophs and are starved for this essential nutrient when the human host cell is exposed to IFN-γ. To survive this, chlamydiae enter an alternative developmental state referred to as persistence. Chlamydial persistence is characterized by a halt in the division cycle, aberrant morphology, and, in the case of IFN-γ-induced persistence, Trp codon-dependent changes in transcription. We hypothesize that these changes in transcription are dependent on the particular amino acid starvation state. To investigate the chlamydial response mechanisms acting when other amino acids become limiting, we tested the efficacy of prokaryote-specific tRNA synthetase inhibitors, indolmycin and AN3365, to mimic starvation of Trp and leucine, respectively. We show that these drugs block chlamydial growth and induce changes in morphology and transcription consistent with persistence. Importantly, growth inhibition was reversed when the compounds were removed from the medium. With these data, we find that indolmycin and AN3365 are valid tools that can be used to mimic the persistent state independently of IFN-γ.


Assuntos
Adaptação Fisiológica , Aminoacil-tRNA Sintetases/antagonistas & inibidores , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/crescimento & desenvolvimento , Chlamydophila pneumoniae/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Triptofano/metabolismo , Linhagem Celular , Chlamydia trachomatis/citologia , Chlamydia trachomatis/efeitos dos fármacos , Chlamydia trachomatis/enzimologia , Chlamydophila pneumoniae/citologia , Chlamydophila pneumoniae/efeitos dos fármacos , Chlamydophila pneumoniae/enzimologia , Inibidores Enzimáticos/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Indóis/metabolismo , Leucina/metabolismo , Modelos Biológicos , Transcrição Genética
10.
Protein Sci ; 29(4): 905-918, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31833153

RESUMO

Pseudomonas aeruginosa has a high potential for developing resistance to multiple antibiotics. The gene (glnS) encoding glutaminyl-tRNA synthetase (GlnRS) from P. aeruginosa was cloned and the resulting protein characterized. GlnRS was kinetically evaluated and the KM and kcat obs , governing interactions with tRNA, were 1.0 µM and 0.15 s-1 , respectively. The crystal structure of the α2 form of P. aeruginosa GlnRS was solved to 1.9 Å resolution. The amino acid sequence and structure of P. aeruginosa GlnRS were analyzed and compared to that of GlnRS from Escherichia coli. Amino acids that interact with ATP, glutamine, and tRNA are well conserved and structure overlays indicate that both GlnRS proteins conform to a similar three-dimensional structure. GlnRS was developed into a screening platform using scintillation proximity assay technology and used to screen ~2,000 chemical compounds. Three inhibitory compounds were identified and analyzed for enzymatic inhibition as well as minimum inhibitory concentrations against clinically relevant bacterial strains. Two of the compounds, BM02E04 and BM04H03, were selected for further studies. These compounds displayed broad-spectrum antibacterial activity and exhibited moderate inhibitory activity against mutant efflux deficient strains of P. aeruginosa and E. coli. Growth of wild-type strains was unaffected, indicating that efflux was likely responsible for the lack of sensitivity. The global mode of action was determined using time-kill kinetics. BM04H03 did not inhibit the growth of human cell cultures at any concentration and BM02E04 only inhibit cultures at the highest concentration tested (400 µg/ml). In conclusion, GlnRS from P. aeruginosa is shown to have a structure similar to that of E. coli GlnRS and two natural product compounds were identified as inhibitors of P. aeruginosa GlnRS with the potential for utility as lead candidates in antibacterial drug development in a time of increased antibiotic resistance.


Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Antibacterianos/química , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Cinética , Testes de Sensibilidade Microbiana , Estrutura Molecular , Pseudomonas aeruginosa/enzimologia
11.
Proc Natl Acad Sci U S A ; 116(29): 14614-14619, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31262815

RESUMO

Aberrant MYC oncogene activation is one of the most prevalent characteristics of cancer. By overlapping datasets of Drosophila genes that are insulin-responsive and also regulate nucleolus size, we enriched for Myc target genes required for cellular biosynthesis. Among these, we identified the aminoacyl tRNA synthetases (aaRSs) as essential mediators of Myc growth control in Drosophila and found that their pharmacologic inhibition is sufficient to kill MYC-overexpressing human cells, indicating that aaRS inhibitors might be used to selectively target MYC-driven cancers. We suggest a general principle in which oncogenic increases in cellular biosynthesis sensitize cells to disruption of protein homeostasis.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Fatores de Transcrição/metabolismo , Aminoacil-tRNA Sintetases/antagonistas & inibidores , Animais , Animais Geneticamente Modificados , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Células Epiteliais , Feminino , Humanos , Insulina/metabolismo , Masculino , Neoplasias/genética , Neoplasias/patologia , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Transcrição/genética
12.
Eur J Med Chem ; 174: 252-264, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31048140

RESUMO

The superfamily of adenylate-forming enzymes all share a common chemistry. They activate a carboxylate group, on a specific substrate, by catalyzing the formation of a high energy mixed phosphoanhydride-linked nucleoside intermediate. Members of this diverse enzymatic family play key roles in a variety of metabolic pathways and therefore many have been regarded as drug targets. A generic approach to inhibit such enzymes is the use of non-hydrolysable sulfur-based bioisosteres of the adenylate intermediate. Here we compare the activity of compounds containing a sulfamoyl and sulfonamide linker respectively. An improved synthetic strategy was developed to generate inhibitors containing the latter that target isoleucyl- (IleRS) and seryl-tRNA synthetase (SerRS), two structurally distinct representatives of Class I and II aminoacyl-tRNA synthetases (aaRSs). These enzymes attach their respective amino acid to its cognate tRNA and are indispensable for protein translation. Evaluation of the ability of the two similar isosteres to inhibit serRS revealed a remarkable difference, with an almost complete loss of activity for seryl-sulfonamide 15 (SerSoHA) compared to its sulfamoyl analogue (SerSA), while inhibition of IleRS was unaffected. To explain these observations, we have determined a 2.1 Šcrystal structure of Klebsiella pneumoniae SerRS in complex with SerSA. Using this structure as a template, modelling of 15 in the active site predicts an unfavourable eclipsed conformation. We extended the same modelling strategy to representative members of the whole adenylate-forming enzyme superfamily, and were able to disclose a new classification system for adenylating enzymes, based on their protein fold. The results suggest that, other than for the structural and functional orthologues of the Class II aaRSs, the O to C substitution within the sulfur-sugar link should generally preserve the inhibitory potency.


Assuntos
Adenosina/análogos & derivados , Aminoacil-tRNA Sintetases/antagonistas & inibidores , Inibidores Enzimáticos/química , Sulfonamidas/química , Adenosina/síntese química , Aminoacil-tRNA Sintetases/química , Aminoacilação , Bacillus subtilis/enzimologia , Domínio Catalítico , Dickeya chrysanthemi/enzimologia , Inibidores Enzimáticos/síntese química , Klebsiella pneumoniae/enzimologia , Modelos Moleculares , Mycobacterium tuberculosis/enzimologia , Sulfolobus/enzimologia , Sulfonamidas/síntese química , Thermus thermophilus/enzimologia
13.
Respir Med ; 152: 44-50, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31128609

RESUMO

BACKGROUND AND PURPOSE: Patients with anti-aminoacyl-tRNA synthetase (ARS) antibodies frequently experience complications of interstitial pneumonia (ARS-IP), and the computed tomography (CT) of ARS-IP frequently shows nonspecific interstitial pneumonia (NSIP) pattern. The CT pattern of ARS-IP might be different from that of idiopathic IP. However, the clinical differences in patients with ARS-IP and idiopathic IP showing the similar CT patterns have not yet been well studied. The objective of this study was to evaluate the clinical differences between patients with ARS-NSIP and idiopathic NSIP (I-NSIP). METHODS: Two groups of 34 patients each, with ARS-NSIP and I-NSIP, who visited Hiroshima University Hospital between January 2005 and December 2017, were enrolled. Clinical features and outcomes were retrospectively compared between the two groups. RESULTS: The ARS-NSIP group included more female patients and significantly younger patients than the I-NSIP group. The percentage of lymphocytes in bronchoalveolar lavage fluid (BALF) was significantly higher, and the CD4/CD8 ratio in BALF was significantly lower in the ARS-NSIP group compared with the I-NSIP group. The proportion of patients with traction bronchiectasis detected by CT was significantly higher in I-NSIP compared with ARS-NSIP. The number of patients who received corticosteroid and/or immunosuppressant therapy was significantly larger in the ARS-NSIP group than in the I-NSIP group. In addition, the patients in the I-NSIP group who underwent the immunosuppressive therapy demonstrated shorter survival than those who underwent no treatment; this tendency was not observed in the ARS-NSIP group. The 10-year survival rate of patients in the ARS-NSIP group was significantly higher than that of patients in the I-NSIP group (91.8% vs. 43.0%; log-rank, p = 0.012). The multivariate survival analysis revealed that positive anti-ARS antibody was an independent favorable prognostic factor in the patients with NSIP (OR, [95% CI]:0.12 [0.02-0.55], p = 0.013). CONCLUSIONS: Patients with ARS-NSIP had a significantly better prognosis than those with I-NSIP; this may be associated with the sensitivity to immunosuppressive therapies, and the different findings of BALF and HRCT between the two groups.


Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Líquido da Lavagem Broncoalveolar/imunologia , Pneumonias Intersticiais Idiopáticas/imunologia , Doenças Pulmonares Intersticiais/imunologia , Corticosteroides/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Aminoacil-tRNA Sintetases/imunologia , Autoanticorpos/imunologia , Bronquiectasia/diagnóstico por imagem , Bronquiectasia/epidemiologia , Relação CD4-CD8/métodos , Feminino , Humanos , Pneumonias Intersticiais Idiopáticas/diagnóstico por imagem , Pneumonias Intersticiais Idiopáticas/tratamento farmacológico , Pneumonias Intersticiais Idiopáticas/fisiopatologia , Imunossupressores/uso terapêutico , Japão/epidemiologia , Doenças Pulmonares Intersticiais/diagnóstico por imagem , Doenças Pulmonares Intersticiais/tratamento farmacológico , Doenças Pulmonares Intersticiais/fisiopatologia , Linfócitos/citologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Análise de Sobrevida , Taxa de Sobrevida , Tomografia Computadorizada por Raios X/métodos
14.
Eur J Med Chem ; 173: 154-166, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30995568

RESUMO

Aminoacyl-tRNA synthetases (aaRSs) catalyse the ATP-dependent coupling of an amino acid to its cognate tRNA. Being vital for protein translation aaRSs are considered a promising target for the development of novel antimicrobial agents. 5'-O-(N-aminoacyl)-sulfamoyl adenosine (aaSA) is a non-hydrolysable analogue of the aaRS reaction intermediate that has been shown to be a potent inhibitor of this enzyme family but is prone to chemical instability and enzymatic modification. In an attempt to improve the molecular properties of this scaffold we synthesized a series of base substituted aaSA analogues comprising cytosine, uracil and N3-methyluracil targeting leucyl-, tyrosyl- and isoleucyl-tRNA synthetases. In in vitro assays seven out of the nine inhibitors demonstrated Kiapp values in the low nanomolar range. To complement the biochemical studies, X-ray crystallographic structures of Neisseria gonorrhoeae leucyl-tRNA synthetase and Escherichia coli tyrosyl-tRNA synthetase in complex with the newly synthesized compounds were determined. These highlighted a subtle interplay between the base moiety and the target enzyme in defining relative inhibitory activity. Encouraged by this data we investigated if the pyrimidine congeners could escape a natural resistance mechanism, involving acetylation of the amine of the aminoacyl group by the bacterial N-acetyltransferases RimL and YhhY. With RimL the pyrimidine congeners were less susceptible to inactivation compared to the equivalent aaSA, whereas with YhhY the converse was true. Combined the various insights resulting from this study will pave the way for the further rational design of aaRS inhibitors.


Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Nucleosídeos/farmacologia , Pirimidinas/farmacologia , Aminoacil-tRNA Sintetases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/síntese química , Escherichia coli/citologia , Escherichia coli/enzimologia , Estrutura Molecular , Nucleosídeos/análise , Nucleosídeos/síntese química , Pirimidinas/análise , Pirimidinas/síntese química , Relação Estrutura-Atividade
15.
Malar J ; 18(1): 34, 2019 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-30728021

RESUMO

BACKGROUND: Treatment of parasitic diseases has been challenging due to evolution of drug resistant parasites, and thus there is need to identify new class of drugs and drug targets. Protein translation is important for survival of malarial parasite, Plasmodium, and the pathway is present in all of its life cycle stages. Aminoacyl tRNA synthetases are primary enzymes in protein translation as they catalyse amino acid addition to the cognate tRNA. This study sought to understand differences between Plasmodium and human aminoacyl tRNA synthetases through bioinformatics analysis. METHODS: Plasmodium berghei, Plasmodium falciparum, Plasmodium fragile, Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Plasmodium yoelii and human aminoacyl tRNA synthetase sequences were retrieved from UniProt database and grouped into 20 families based on amino acid specificity. These families were further divided into two classes. Both families and classes were analysed. Motif discovery was carried out using the MEME software, sequence identity calculation was done using an in-house Python script, multiple sequence alignments were performed using PROMALS3D and TCOFFEE tools, and phylogenetic tree calculations were performed using MEGA vs 7.0 tool. Possible alternative binding sites were predicted using FTMap webserver and SiteMap tool. RESULTS: Motif discovery revealed Plasmodium-specific motifs while phylogenetic tree calculations showed that Plasmodium proteins have different evolutionary history to the human homologues. Human aaRSs sequences showed low sequence identity (below 40%) compared to Plasmodium sequences. Prediction of alternative binding sites revealed potential druggable sites in PfArgRS, PfMetRS and PfProRS at regions that are weakly conserved when compared to the human homologues. Multiple sequence analysis, motif discovery, pairwise sequence identity calculations and phylogenetic tree analysis showed significant differences between parasite and human aaRSs proteins despite functional and structural conservation. These differences may provide a basis for further exploration of Plasmodium aminoacyl tRNA synthetases as potential drug targets. CONCLUSION: This study showed that, despite, functional and structural conservation, Plasmodium aaRSs have key differences from the human homologues. These differences in Plasmodium aaRSs can be targeted to develop anti-malarial drugs with less toxicity to the host.


Assuntos
Aminoacil-tRNA Sintetases/genética , Antimaláricos/farmacologia , Plasmodium/genética , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/antagonistas & inibidores , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Biologia Computacional , Humanos , Filogenia , Plasmodium/efeitos dos fármacos , Plasmodium/enzimologia , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência
16.
Protein Sci ; 28(4): 727-737, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30666738

RESUMO

Pseudomonas aeruginosa is an opportunistic multi-drug resistant pathogen implicated as a causative agent in nosocomial and community acquired bacterial infections. The gene encoding prolyl-tRNA synthetase (ProRS) from P. aeruginosa was overexpressed in Escherichia coli and the resulting protein was characterized. ProRS was kinetically evaluated and the KM values for interactions with ATP, proline, and tRNA were 154, 122, and 5.5 µM, respectively. The turn-over numbers, kcat obs , for interactions with these substrates were calculated to be 5.5, 6.3, and 0.2 s-1 , respectively. The crystal structure of the α2 form of P. aeruginosa ProRS was solved to 2.60 Å resolution. The amino acid sequence and X-ray crystal structure of P. aeruginosa ProRS was analyzed and compared with homologs in which the crystal structures have been solved. The amino acids that interact with ATP and proline are well conserved in the active site region and overlay of the crystal structure with ProRS homologs conforms to a similar overall three-dimensional structure. ProRS was developed into a screening platform using scintillation proximity assay (SPA) technology and used to screen 890 chemical compounds, resulting in the identification of two inhibitory compounds, BT06A02 and BT07H05. This work confirms the utility of a screening system based on the functionality of ProRS from P. aeruginosa.


Assuntos
Aminoacil-tRNA Sintetases/química , Proteínas de Bactérias/química , Pseudomonas aeruginosa/química , Aminoacil-tRNA Sintetases/antagonistas & inibidores , Aminoacil-tRNA Sintetases/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Proteica , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/metabolismo
17.
J Biol Chem ; 294(14): 5365-5385, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30670594

RESUMO

Aminoacyl-tRNA synthetases (ARSs) are universal enzymes that catalyze the attachment of amino acids to the 3' ends of their cognate tRNAs. The resulting aminoacylated tRNAs are escorted to the ribosome where they enter protein synthesis. By specifically matching amino acids to defined anticodon sequences in tRNAs, ARSs are essential to the physical interpretation of the genetic code. In addition to their canonical role in protein synthesis, ARSs are also involved in RNA splicing, transcriptional regulation, translation, and other aspects of cellular homeostasis. Likewise, aminoacylated tRNAs serve as amino acid donors for biosynthetic processes distinct from protein synthesis, including lipid modification and antibiotic biosynthesis. Thanks to the wealth of details on ARS structures and functions and the growing appreciation of their additional roles regulating cellular homeostasis, opportunities for the development of clinically useful ARS inhibitors are emerging to manage microbial and parasite infections. Exploitation of these opportunities has been stimulated by the discovery of new inhibitor frameworks, the use of semi-synthetic approaches combining chemistry and genome engineering, and more powerful techniques for identifying leads from the screening of large chemical libraries. Here, we review the inhibition of ARSs by small molecules, including the various families of natural products, as well as inhibitors developed by either rational design or high-throughput screening as antibiotics and anti-parasitic therapeutics.


Assuntos
Aminoacil-tRNA Sintetases , Antibacterianos , Antiparasitários , Inibidores Enzimáticos , Infecções , Doenças Parasitárias , Aminoacil-tRNA Sintetases/antagonistas & inibidores , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Animais , Antibacterianos/química , Antibacterianos/uso terapêutico , Antiparasitários/química , Antiparasitários/uso terapêutico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Humanos , Infecções/tratamento farmacológico , Infecções/enzimologia , Infecções/genética , Infecções/patologia , Doenças Parasitárias/tratamento farmacológico , Doenças Parasitárias/enzimologia , Doenças Parasitárias/genética , Splicing de RNA/efeitos dos fármacos , RNA de Transferência/genética , RNA de Transferência/metabolismo
18.
Antiviral Res ; 161: 163-168, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30521835

RESUMO

Arboviruses represent a group of pathogens that can spread efficiently throughout human populations by hematophagous arthropod vectors. The mosquito-borne (re)emerging Chikungunya and Dengue viruses belong to the alphavirus and flavivirus genus, respectively, with no approved therapeutics or safe vaccines for humans. Transmitted by the same vector Aedes spp., these viruses cause significant morbidity and mortality in endemic areas. Due to the increasing likelihood of co-circulation and co-infection with viruses, we aimed to identify a pharmacologically targetable host factor that can inhibit multiple viruses and show that a potent antagonist of prolyl tRNA synthetase (halofuginone) suppresses both Chikungunya and Dengue viruses. Host tRNA synthetase inhibition may signify an additional approach to combat present and future epidemic pathogens.


Assuntos
Aedes/enzimologia , Aminoacil-tRNA Sintetases/antagonistas & inibidores , Antivirais/farmacologia , Vírus Chikungunya/efeitos dos fármacos , Vírus da Dengue/efeitos dos fármacos , Piperidinas/farmacologia , Quinazolinonas/farmacologia , Aedes/virologia , Animais , Células Cultivadas , Febre de Chikungunya/virologia , Dengue/virologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/virologia , Prepúcio do Pênis/citologia , Interações entre Hospedeiro e Microrganismos , Humanos , Proteínas de Insetos/antagonistas & inibidores , Masculino , Mosquitos Vetores , Receptor de Interferon alfa e beta/agonistas
19.
Biochem Pharmacol ; 154: 424-434, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29890143

RESUMO

Despite remarkable advances in medical science, infection-associated diseases remain among the leading causes of death worldwide. There is a great deal of interest and concern at the rate at which new pathogens are emerging and causing significant human health problems. Expanding our understanding of how cells regulate signaling networks to defend against invaders and retain cell homeostasis will reveal promising strategies against infection. It has taken scientists decades to appreciate that eukaryotic aminoacyl-tRNA synthetases (ARSs) play a role as global cell signaling mediators to regulate cell homeostasis, beyond their intrinsic function as protein synthesis enzymes. Recent discoveries revealed that ubiquitously expressed standby cytoplasmic ARSs sense and respond to danger signals and regulate immunity against infections, indicating their potential as therapeutic targets for infectious diseases. In this review, we discuss ARS-mediated anti-infectious signaling and the emerging role of ARSs in antimicrobial immunity. In contrast to their ability to defend against infection, host ARSs are inevitably co-opted by viruses for survival and propagation. We therefore provide a brief overview of the communication between viruses and the ARS system. Finally, we discuss encouraging new approaches to develop ARSs as therapeutics for infectious diseases.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Antibacterianos/metabolismo , Antivirais/metabolismo , Doenças Transmissíveis/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Aminoacil-tRNA Sintetases/antagonistas & inibidores , Animais , Antibacterianos/administração & dosagem , Antivirais/administração & dosagem , Doenças Transmissíveis/tratamento farmacológico , Sistemas de Liberação de Medicamentos/tendências , Humanos , Testes de Sensibilidade Microbiana/métodos , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
20.
Eur J Med Chem ; 148: 384-396, 2018 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-29477072

RESUMO

Aminoacyl-tRNA synthetases (aaRSs) are enzymes that precisely attach an amino acid to its cognate tRNA. This process, which is essential for protein translation, is considered a viable target for the development of novel antimicrobial agents, provided species selective inhibitors can be identified. Aminoacyl-sulfamoyl adenosines (aaSAs) are potent orthologue specific aaRS inhibitors that demonstrate nanomolar affinities in vitro but have limited uptake. Following up on our previous work on substitution of the base moiety, we evaluated the effect of the N3-position of the adenine by synthesizing the corresponding 3-deazaadenosine analogues (aaS3DAs). A typical organism has 20 different aaRS, which can be split into two distinct structural classes. We therefore coupled six different amino acids, equally targeting the two enzyme classes, via the sulfamate bridge to 3-deazaadenosine. Upon evaluation of the inhibitory potency of the obtained analogues, a clear class bias was noticed, with loss of activity for the aaS3DA analogues targeting class II enzymes when compared to the equivalent aaSA. Evaluation of the available crystallographic structures point to the presence of a conserved water molecule which could have importance for base recognition within class II enzymes, a property that can be explored in future drug design efforts.


Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Anti-Infecciosos/química , Tubercidina/química , Aminoácidos/química , Desenho de Fármacos , Proteínas de Escherichia coli , Ácidos Sulfônicos/química , Tubercidina/farmacologia
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