scholarly journals Discovery and Characterization of Chemical Compounds That Inhibit the Function of Aspartyl-tRNA Synthetase from Pseudomonas aeruginosa

2017 ◽  
Vol 23 (3) ◽  
pp. 294-301 ◽  
Author(s):  
Araceli Corona ◽  
Stephanie O. Palmer ◽  
Regina Zamacona ◽  
Benjamin Mendez ◽  
Frank B. Dean ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Aspartic Acid ◽  
Cell Cultures ◽  
Human Cell ◽  
Efflux Pump ◽  
Chemical Compounds ◽  
Opportunistic Pathogen ◽  
Trna Synthetase ◽  
Bacterial Strains ◽  
Human Cell Cultures

Pseudomonas aeruginosa, an opportunistic pathogen, is highly susceptible to developing resistance to multiple antibiotics. The gene encoding aspartyl-tRNA synthetase (AspRS) from P. aeruginosa was cloned and the resulting protein characterized. AspRS was kinetically evaluated, and the KM values for aspartic acid, ATP, and tRNA were 170, 495, and 0.5 μM, respectively. AspRS was developed into a screening platform using scintillation proximity assay (SPA) technology and used to screen 1690 chemical compounds, resulting in the identification of two inhibitory compounds, BT02A02 and BT02C05. The minimum inhibitory concentrations (MICs) were determined against nine clinically relevant bacterial strains, including efflux pump mutant and hypersensitive strains of P. aeruginosa. The compounds displayed broad-spectrum antibacterial activity and inhibited growth of the efflux and hypersensitive strains with MICs of 16 μg/mL. Growth of wild-type strains were unaffected, indicating that efflux was likely responsible for this lack of activity. BT02A02 did not inhibit growth of human cell cultures at any concentration. However, BT02C05 did inhibit human cell cultures with a cytotoxicity concentration (CC50) of 61.6 μg/mL. The compounds did not compete with either aspartic acid or ATP for binding AspRS, indicating that the mechanism of action of the compound occurs outside the active site of aminoacylation.

scholarly journals Identification and Characterization of Chemical Compounds that Inhibit Leucyl-tRNA Synthetase from Pseudomonas aeruginosa

2020 ◽  
Vol 17 (1) ◽  
pp. 119-130 ◽  
Author(s):  
Regina Zamacona ◽  
Pamela N. Chavero ◽  
Eduardo Medellin ◽  
Yanmei Hu ◽  
Casey A. Hughes ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Human Cell ◽  
Bacterial Infections ◽  
Chemical Compounds ◽  
Human Cell Line ◽  
Trna Synthetase ◽  
Multi Drug Resistance ◽  
Bacterial Strains ◽  
Gene Encoding ◽  
Screening Platform

Background: Pseudomonas aeruginosa is an opportunistic multi-drug resistance pathogen implicated as the causative agent in a high-percentage of nosocomial and community acquired bacterial infections. The gene encoding leucyl-tRNA synthetase (LeuRS) from P. aeruginosa was overexpressed in Escherichia coli and the resulting protein was characterized. Methods: LeuRS was kinetically evaluated and the KM values for interactions with leucine, ATP and tRNA were 6.5, 330, and 3.0 μM, respectively. LeuRS was developed into a screening platform using scintillation proximity assay (SPA) technology and used to screen over 2000 synthetic and natural chemical compounds. Results: The initial screen resulted in the identification of two inhibitory compounds, BT03C09 and BT03E07. IC50s against LeuRS observed for BT03C09 and BT03E07 were 23 and 15 μM, respectively. The minimum inhibitory concentrations (MIC) were determined against nine clinically relevant bacterial strains. In time-kill kinetic analysis, BT03C09 was observed to inhibit bacterial growth in a bacteriostatic manner, while BT03E07 acted as a bactericidal agent. Neither compound competed with leucine or ATP for binding LeuRS. Limited inhibition was observed in aminoacylation assays with the human mitochondrial form of LeuRS, however when tested in cultures of human cell line, BT03C09 was toxic at all concentration whereas BT03E07 only showed toxic effects at elevated concentrations. Conclusion: Two compounds were identified as inhibitors of LeuRS in a screen of over 2000 natural and synthetic compounds. After characterization one compound (BT03E07) exhibited broad spectrum antibacterial activity while maintaining low toxicity against human mitochondrial LeuRS as well as against human cell cultures.

scholarly journals Lysyl-tRNA Synthetase from Pseudomonas aeruginosa: Characterization and Identification of Inhibitory Compounds

2019 ◽  
Vol 25 (1) ◽  
pp. 57-69
Author(s):  
Samantha Balboa ◽  
Yanmei Hu ◽  
Frank B. Dean ◽  
James M. Bullard
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Opportunistic Pathogen ◽  
Trna Synthetase ◽  
Gram Negative Bacteria ◽  
Synthetic Compound ◽  
Compound Libraries ◽  
Mechanism Of Inhibition ◽  
Inhibitory Compounds ◽  
Human Cell Cultures ◽  
Substrate Competition

Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial infections and has highly developed systems for acquiring resistance against numerous antibiotics. The gene ( lysS) encoding P. aeruginosa lysyl-tRNA synthetase (LysRS) was cloned and overexpressed, and the resulting protein was purified to 98% homogeneity. LysRS was kinetically evaluated, and the Km values for the interaction with lysine, adenosine triphosphate (ATP), and tRNALys were determined to be 45.5, 627, and 3.3 µM, respectively. The kcatobs values were calculated to be 13, 22.8, and 0.35 s−1, resulting in kcatobs/ KM values of 0.29, 0.036, and 0.11 s−1µM−1, respectively. Using scintillation proximity assay technology, natural product and synthetic compound libraries were screened to identify inhibitors of function of the enzyme. Three compounds (BM01D09, BT06F11, and BT08F04) were identified with inhibitory activity against LysRS. The IC50 values were 17, 30, and 27 µM for each compound, respectively. The minimum inhibitory concentrations were determined against a panel of clinically important pathogens. All three compounds were observed to inhibit the growth of gram-positive organisms with a bacteriostatic mode of action. However, two compounds (BT06F11 and BT08F04) were bactericidal against cultures of gram-negative bacteria. When tested against human cell cultures, BT06F11 was not toxic at any concentration tested, and BM01D09 was toxic only at elevated levels. However, BT08F04 displayed a CC50 of 61 µg/mL. In studies of the mechanism of inhibition, BM01D09 inhibited LysRS activity by competing with ATP for binding, and BT08F04 was competitive with ATP and uncompetitive with the amino acid. BT06F11 inhibited LysRS activity by a mechanism other than substrate competition.

Automatic nitrous oxide synchronization of mitotic human cell cultures

1987 ◽  
Vol 165 (1) ◽  
pp. 56-58 ◽  
Author(s):  
C.S. Downes ◽  
D.M. Unwin ◽  
R.G.W. Northfield ◽  
M.J. Berry
Keyword(s):  
Nitrous Oxide ◽  
Cell Cultures ◽  
Human Cell ◽  
Human Cell Cultures

scholarly journals The Genomic Basis of Rapid Adaptation to Antibiotic Combination Therapy in Pseudomonas aeruginosa

2020 ◽  
Author(s):  
Camilo Barbosa ◽  
Niels Mahrt ◽  
Julia Bunk ◽  
Matthias Graßer ◽  
Philip Rosenstiel ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Combination Therapy ◽  
Bacterial Resistance ◽  
Efflux Pump ◽  
Regulatory Gene ◽  
Opportunistic Pathogen ◽  
Regulatory Function ◽  
Central Target ◽  
Resistance Evolution ◽  
Intergenic Regions

Abstract Combination therapy is a common antibiotic treatment strategy that aims at minimizing the risk of resistance evolution in several infectious diseases. Nonetheless, evidence supporting its efficacy against the nosocomial opportunistic pathogen Pseudomonas aeruginosa remains elusive. Identification of the possible evolutionary paths to resistance in multidrug environments can help to explain treatment outcome. For this purpose, we here performed whole-genome sequencing of 127 previously evolved populations of P. aeruginosa adapted to sublethal doses of distinct antibiotic combinations and corresponding single-drug treatments, and experimentally characterized several of the identified variants. We found that alterations in the regulation of efflux pumps are the most favored mechanism of resistance, regardless of the environment. Unexpectedly, we repeatedly identified intergenic variants in the adapted populations, often with no additional mutations and usually associated with genes involved in efflux pump expression, possibly indicating a regulatory function of the intergenic regions. The experimental analysis of these variants demonstrated that the intergenic changes caused similar increases in resistance against single and multidrug treatments as those seen for efflux regulatory gene mutants. Surprisingly, we could find no substantial fitness costs for a majority of these variants, most likely enhancing their competitiveness toward sensitive cells, even in antibiotic-free environments. We conclude that the regulation of efflux is a central target of antibiotic-mediated selection in P. aeruginosa and that, importantly, changes in intergenic regions may represent a usually neglected alternative process underlying bacterial resistance evolution, which clearly deserves further attention in the future.

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