Time-Dependent Inhibition of Leucyl-tRNA-Synthetase (LeuRS): Insight into Target Vulnerability

Abstract

The postantibiotic effect (PAE) is the persistent suppression of microbial growth following the removal of antimicrobial therapy. In general, antibiotics that generate a PAE are dosed less frequently, and thus, the PAE has important implications for dosing regimens. PAEs can arise through several mechanisms, including the extended occupancy of the drug target following drug elimination, and the correlation between drug-target residence time and PAE provides insight into target vulnerability. To assess the vulnerability of Escherichia coli leucyl-tRNA synthetase (ecLeuRS), which is an essential enzyme in protein synthesis, the time-dependent inhibition of the enzyme was studied by the benzoxaborole class of compounds that inhibit LeuRS by forming a stable LeuRS-tRNA^Leu-benzoxaborole adduct. Preincubation of epetraborole with ecLeuRS resulted in a decrease in the IC₅₀ value for enzyme inhibition from 38 to 3 nM, consistent with the slow formation of the final enzyme–inhibitor complex, and similar shifts in IC₅₀ were observed for three other benzoxaboroles. The benzoxaboroles generated short PAEs (<1 h) in E. coli, however, the PAE values of AN3334 and epetraborole increased from 0.88 to 1.70–3 h when a sub-MIC concentration of the aminoglycoside tobramycin was included in the media. pSILAC revealed that the synthesis rate of ecLeuRS was reduced 1.6-fold in the presence of sub-MIC tobramycin, reinforcing the role that protein turnover plays in target vulnerability.