Simultaneous Inhibition of Cytochrome <i>bd</i> Oxidase and ATP Synthase for Complete Eradication of <i>Mycobacterium tuberculosis</i>

The clinical utility of bedaquiline (BDQ), a first-in-class ATP synthase inhibitor and cornerstone of modern multidrug-resistant tuberculosis (MDR-TB) therapy, is compromised by a delayed onset of bactericidal killing and the emergence of resistance. This tolerance is linked to <i>Mycobacterium tuberculosis</i> metabolic heterogeneity and functional redundancy within its electron transport chain (ETC), notably the activity of the cytochrome <i>bd</i> oxidase (Cyt<i>-bd</i>). We hypothesized that cotargeting the cytochrome <i>bd</i> oxidase and the F₁F₀-ATP synthase would induce potent, synergistic killing by completely disrupting bacterial energetics. Our results demonstrate that this combination drives rapid, synergistic bactericidal activity within 09 days and complete clearance within 12 days. Mechanistic studies revealed that Cyt-<i>bd</i> inhibitor ND-011992 blocks the respiratory redundancy that permits survival under BDQ pressure, leading to catastrophic ATP depletion and a substantial arrest of oxygen consumption. This potentiation was equally effective against nonreplicating, hypoxic bacilli and eradicated 90% of intracellular bacteria in a macrophage model. These findings provide critical proof-of-concept that targeting compensatory respiratory pathways can overcome phenotypic drug tolerance. Thus, this validates Cyt<i>-bd</i> as a high-value target and outlines a potent, sterilizing combination regimen capable of countering the heterogeneity that sabotages current TB therapies.