Selectivity of the Time-Dependent <i>M. tuberculosis</i> LeuRS Inhibitor Ganfeborole Is Driven by Target Vulnerability

Ganfeborole (GSK3036656) inhibits the <i>Mycobacterium tuberculosis</i> leucyl-tRNA-synthetase (mtLeuRS) and is in Phase 2a clinical trials for the treatment of tuberculosis. Here we show that ganfeborole is a time-dependent inhibitor of mtLeuRS (IC₅₀ 1 nM) and generates a postantibiotic effect of 77 h at 50xMIC (MIC 0.058 μM) with <i>M. tuberculosis</i> H37Rv, indicating that mtLeuRS is a highly vulnerable drug target and supporting the excellent in vivo efficacy of the drug. Ganfeborole is also a potent time-dependent inhibitor of <i>Escherichia coli</i> LeuRS (ecLeuRS, IC₅₀ 2 nM), however no antibacterial activity is observed toward <i>E. coli</i> up to 1 mM ganfeborole despite the observation that less potent ganfeborole analogs have antibacterial activity. To rationalize this observation, we propose that ganfeborole forms a complex with AMP that binds to the ecLeuRS editing site but does not impact aminoacylation. In support, addition of 12.5 μM norvaline generates a ganfeborole MIC of 0.4 μM since ecLeuRS is unable to hydrolyze norvaline-tRNA^Leu. Additionally, mutations that reduce the affinity and residence time of ganfeborole-AMP on ecLeuRS result in antibacterial activity. We propose that the activity of ganfeborole toward <i>M. tuberculosis</i> is because mtLeuRS is a highly vulnerable target so that only low levels of enzyme need to be inhibited by the ganfeborole-tRNA^Leu complex in contrast to ecLeuRS, which we previously demonstrated is a low vulnerability target.