Two Forms of Tyrosyl-tRNA Synthetase from Pseudomonas aeruginosa: Characterization and Discovery of Inhibitory Compounds

Pseudomonas aeruginosa is a multidrug-resistant (MDR) pathogen and a causative agent of both nosocomial and community-acquired infections. The genes (tyrS and tyrZ) encoding both forms of P. aeruginosa tyrosyl-tRNA synthetase (TyrRS-S and TyrRS-Z) were cloned and the resulting proteins purified. TyrRS-S and TyrRS-Z were kinetically evaluated and the K_m values for interaction with Tyr, ATP, and tRNA^Tyr were 172, 204, and 1.5 μM and 29, 496, and 1.9 μM, respectively. The k_cat^obs values for interaction with Tyr, ATP, and tRNA^Tyr were calculated to be 3.8, 1.0, and 0.2 s⁻¹ and 3.1, 3.8, and 1.9 s⁻¹, respectively. Using scintillation proximity assay (SPA) technology, a druglike 2000-compound library was screened to identify inhibitors of the enzymes. Four compounds (BCD37H06, BCD38C11, BCD49D09, and BCD54B04) were identified with inhibitory activity against TyrRS-S. BCD38C11 also inhibited TyrRS-Z. The IC₅₀ values for BCD37H06, BCD38C11, BCD49D09, and BCD54B04 against TyrRS-S were 24, 71, 65, and 50 μM, respectively, while the IC₅₀ value for BCD38C11 against TyrRS-Z was 241 μM. Minimum inhibitory concentrations (MICs) were determined against a panel of clinically important pathogens. All four compounds were observed to inhibit the growth of cultures of both Gram-positive and Gram-negative bacteria organisms with a bacteriostatic mode of action. When tested against human cell cultures, none of the compounds were toxic at concentrations up to 400 μg/mL. In mechanism of inhibition studies, BCD38C11 and BCD49D09 selectively inhibited TyrRS activity by competing with ATP for binding. BCD37H06 and BCD54B04 inhibited TyrRS activity by a mechanism other than substrate competition.