The Role of a Novel Auxiliary Pocket in Bacterial Phenylalanyl-tRNA Synthetase Druggability

Abibi, Ayome; Ferguson, Andrew D.; Fleming, Paul R.; Gao, Ning; Hajec, Laurel I.; Hu, Jun; Laganas, Valerie A.; McKinney, David C.; McLeod, Sarah M.; Prince, D. Bryan; Shapiro, Adam B.; Buurman, Ed T.

doi:10.1074/jbc.m114.574061

Cited by 23 publications

(25 citation statements)

References 52 publications

(61 reference statements)

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“…coli IleRS with over 8000-fold more strength that rat IleRS despite only differing in two amino acids at the catalityc site (Nakama et al 2001). The antiprotozoal halofuginone works in a similar fashion, mimicking both proline and tRNA Pro in the active site (Zhou, 2013) as is the case with phenyl-thiazolylurea-sulfonamides, that occupies the tRNA binding pocket of PheRS, inhibiting its activity (Abibi et al 2014). On the other hand, agents such as pentamidine and purpuromycin have been described to inhibit different synthetases via nonspecific tRNA binding mechanisms (Kirillov et al 1997;Sun and Zhang 2008).…”

Section: Drug Design Against Aarssmentioning

confidence: 99%

Aminoacyl-tRNA synthetases

Rubio

Ibba

2020

RNA

207

151

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The aminoacyl-tRNA synthetases are an essential and universally distributed family of enzymes that plays a critical role in protein synthesis, pairing tRNAs with their cognate amino acids for decoding mRNAs according to the genetic code. Synthetases help to ensure accurate translation of the genetic code by using both highly accurate cognate substrate recognition and stringent proofreading of noncognate products. While alterations in the quality control mechanisms of synthetases are generally detrimental to cellular viability, recent studies suggest that in some instances such changes facilitate adaption to stress conditions. Beyond their central role in translation, synthetases are also emerging as key players in an increasing number of other cellular processes, with far-reaching consequences in health and disease. The biochemical versatility of the synthetases has also proven pivotal in efforts to expand the genetic code, further emphasizing the wide-ranging roles of the aminoacyl-tRNA synthetase family in synthetic and natural biology.

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Section: Drug Design Against Aarssmentioning

confidence: 99%

Aminoacyl-tRNA synthetases

Rubio

Ibba

2020

RNA

207

151

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“…Interestingly, BM03E08 showed bactericidal activity against Gram-negative bacteria, whereas bacteriostatic activity was observed against Gram-positive bacteria. This phenomenon has also been observed with the use of phenyl-thiazolylurea-sulfonamides, a PheRS inhibitor, in which the stringent response was induced by administration of the compound in certain Gram-positive bacteria; however, in susceptible Gram-negative bacteria a bactericidal effect was observed (45,46). Previously, we developed the 16-membered macrolides, spiramycin and tylosin, as controls for the E. coli A/T system in which they exhibited IC 50 s of 0.022 and 0.038 M, respectively.…”

Section: Discussionmentioning

confidence: 92%

Discovery and Analysis of Natural-Product Compounds Inhibiting Protein Synthesis in Pseudomonas aeruginosa

Keniry

Palmer

et al. 2016

Antimicrob Agents Chemother

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bBacterial protein synthesis is the target for numerous natural and synthetic antibacterial agents. We have developed a poly(U) mRNA-directed aminoacylation/translation (A/T) protein synthesis system composed of phenylalanyl-tRNA synthetases (PheRS), ribosomes, and ribosomal factors from Pseudomonas aeruginosa. This system has been used for high-throughput screening of a natural-compound library. Assays were developed for each component of the system to ascertain the specific target of inhibitory compounds. In high-throughput screens, 13 compounds were identified that inhibit protein synthesis with 50% inhibitory concentrations ranging from 0.3 to >80 M. MICs were determined for the compounds against the growth of a panel of pathogenic organisms, including Enterococcus faecalis, Escherichia coli, Haemophilus influenzae, Moraxella catarrhalis, P. aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae. Three of the compounds were observed to have broad-spectrum activity and inhibited a hypersensitive strain of P. aeruginosa with MICs of 8 to 16 g/ml. The molecular target of each of the three compounds was determined to be PheRS. One compound was found to be bacteriostatic, and one compound was bactericidal against both Gram-positive and Gram-negative pathogens. The third compound was observed to be bacteriostatic against Gram-positive and bactericidal against Gram-negative bacteria. All three compounds were competitive with the substrate ATP; however, one compound was competitive, one was uncompetitive, and one noncompetitive with the amino acid substrate. Macromolecular synthesis assays confirm the compounds inhibit protein synthesis. The compounds were shown to be more than 25,000-fold less active than the control staurosporine in cytotoxicity MTT testing in human cell lines. Bacterial infections are a continuing major worldwide health problem. Infections can be minor, such as skin rashes and common ear infections in infants, or potentially lethal, such as those in many immunocompromised patients. Pseudomonas aeruginosa is an opportunistic Gram-negative bacterial pathogen and the causative agent in a wide range of infections and is responsible for one-seventh of all nosocomial infections (1, 2). Among clinical isolates of P. aeruginosa, antimicrobial resistance is increasing (3) and has become a major problem in the hospital setting (4). However, the most serious medical problem caused by P. aeruginosa is chronic lung colonization associated with cystic fibrosis patients (5), and it is the leading cause of morbidity and mortality in these patients (6).Antibiotics block cellular processes which are essential for bacterial survival. Many of the current antibiotics, both naturally occurring and synthetic, target protein synthesis as a mechanism of action (for a complete list, see reference 7). Antibiotics that target protein synthesis include the macrolides, clindamycin, chloramphenicol, the aminoglycosides, and the tetracyclines (8-10). Linezolid, one of the newest antibiotics and a protein synthesis inhi...

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“…The architecture of the PheRS enzymes has been extensively described, primarily from structural studies of PheRS from T. thermophilus [18,28-30] and more recently the structure of PheRS from E. coli [31], Staphylococcus haemolyticus [32] and P. aeruginosa [33] has been solved. The amino acid sequence conservation when comparing that of T. thermophilus PheRS with the corresponding enzymes from E. coli , P. aeruginosa , or S. pneumoniae is very similar (Table 1 ).…”

Section: Resultsmentioning

confidence: 99%

High Throughput Screen Identifies Natural Product Inhibitor of Phenylalanyl-tRNA Synthetase from Pseudomonas aeruginosa and Streptococcus pneumoniae

Hu¹,

Palmer²,

Muñoz³

et al. 2015

CDDT

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Pseudomonas aeruginosa and Streptococcus pneumoniae are causative agents in a wide range of infections. Genes encoding proteins corresponding to phenylalanyl-tRNA synthetase (PheRS) were cloned from both bacteria. The two forms of PheRS were kinetically evaluated and the Km’s for P. aeruginosa PheRS with its three substrates, phenylalanine, ATP and tRNAPhe were determined to be 48, 200, and 1.2 μM, respectively, while the Km’s for S. pneumoniae PheRS with respect to phenylalanine, ATP and tRNAPhe were 21, 225 and 0.94 μM, respectively. P. aeruginosa and S. pneumoniae PheRS were used to screen a natural compound library and a single compound was identified that inhibited the function of both enzymes. The compound inhibited P. aeruginosa and S. pneumoniae PheRS with IC50’s of 2.3 and 4.9 μM, respectively. The compound had a Ki of 0.83 and 0.98 μM against P. aeruginosa and S. pneumoniae PheRS, respectively. The minimum inhibitory concentration (MIC) of the compound was determined against a panel of Gram positive and negative bacteria including efflux pump mutants and hyper-sensitive strains. MICs against wild-type P. aeruginosa and S. pneumoniae cells in culture were determined to be 16 and 32 μg/ml, respectively. The mechanism of action of the compound was determined to be competitive with the amino acid, phenylalanine, and uncompetitive with AT P. There was no inhibition of cytoplasmic protein synthesis, however, partial inhibition of the human mitochondrial PheRS was observed.

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The Role of a Novel Auxiliary Pocket in Bacterial Phenylalanyl-tRNA Synthetase Druggability

Cited by 23 publications

References 52 publications

Aminoacyl-tRNA synthetases

Aminoacyl-tRNA synthetases

Discovery and Analysis of Natural-Product Compounds Inhibiting Protein Synthesis in Pseudomonas aeruginosa

High Throughput Screen Identifies Natural Product Inhibitor of Phenylalanyl-tRNA Synthetase from Pseudomonas aeruginosa and Streptococcus pneumoniae

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