Abstract
The global escalation of multidrug-resistant tuberculosis (MDR-TB) demands identification of novel therapeutic targets. Here, we establish Vitamin B12-independent methionine synthase E 'MetE' (Rv1133c) as a crucial drug target and validate tobramycin as its inhibitor. Through a multi-faceted approach, including virtual screening and MD simulations, we identified interactions between the tobramycin-MetE complex, which were experimentally validated. Through Isothermal calorimetry we found tobramycin binds to MetE with high affinity (Ka⁓10-8M). Subsequent biophysical characterization revealed an interesting mechanism of action: tobramycin binding induces conformational perturbations of the MetE enzyme, as confirmed by changes in intrinsic fluorescence, denaturation patterns and circular dichroism. Under In-vitro conditions tobramycin demonstrated potent anti-mycobacterial activity at concentrations nontoxic to human macrophage cells. We hypothesize that this inhibition involves classical 30S ribosomal binding and MetE disruption. Most significantly, we discovered a powerful synergistic effect between tobramycin and the first-line anti-TB drug, rifampicin. Together, these findings establish a robust preclinical foundation for repurposing tobramycin against management of tuberculosis.