Targeting Metabolic Vulnerabilities: Valinomycin Augments the Potency of Bioenergetic Inhibitors in Combatting Drug-Resistant and Dormant Mycobacterium tuberculosis

Tuberculosis (TB) treatment is hampered by monotherapy limitations and phenotypic drug tolerance, citing the need for effective drug combinations. The mycobacterial electron transport chain (ETC), crucial for oxidative phosphorylation and ATP production in dormant Mycobacterium tuberculosis ( Mtb ), is a key target. This study investigates combining established bioenergetic inhibitors-bedaquiline (BDQ), telacebec (Q203), and clofazimine (CFZ) with the potassium ionophore valinomycin, which disrupts the proton motive force ( pmf ). We demonstrated that valinomycin significantly potentiated the anti-TB activity of these inhibitors against both replicating and nutrient-starved non-replicating Mtb . Checkerboard assays revealed synergistic activity with BDQ and additive effects with Q203 and CFZ, correlated with a two to three-fold reduction in ATP IC ₅₀ values. Critically, valinomycin converted the bacteriostatic activity of the inhibitors at sub-MIC concentration into bactericidal killing in time-dependent killing assay, achieving sterilization. This lethal synergy for the combinations was also observed in a THP-1 macrophage intracellular model for TB. Respiration assays confirmed that the combinations collectively halted oxygen consumption. We conclude that concurrently targeting specific ETC on Mtb ’s bioenergetics. This strategy, particularly the BDQ/valinomycin synergy, represents a promising cornerstone for developing novel sterilizing regimens to shorten TB therapy and overcoming drug tolerance.