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.
Characterization and structure determination of prolyl‐tRNA synthetase from
Pseudomonas aeruginosa
and development as a screening platform
