We are interested in understanding the mode of action and mechanisms of resistance of new anti-tubercular agents. We have previously identified novel chemical series which have potent and rapid bactericidal activity against Mycobacterium tuberculosis. We determined that the 8-hydroxyquinoline (8HQ) and aminothiazole (AmT) series both act as copper ionophores, but that the downstream consequences differ. 8HQs led to increased intracellular copper and the production of reactive oxygen species (ROS) which exert a broad toxic effect. AmTs also induce production of ROS, but this is transient and is not linked to their killing effect. Instead, we hypothesized that the accumulation of intracellular copper leads to inhibition of enolase, a metalloenzyme. We demonstrated that enolase activity was inhibited by copper, suggesting a mechanism of action by inhibition of glycolysis. This was supported by the observation that ATP levels were reduced after exposure to AmT compounds. We isolated resistant mutants to both series and identified a common mechanism of resistance which involves mutation in the Esx-3 type VII secretion system (T7SS). This system is required for the uptake of iron and we propose that disruption of metal ion homeostasis occurs after compound exposure. Our preliminary data suggests that the mutants have altered permeability leading to reduced accumulation of compounds which would explain why mutants are also resistant to a wide variety of chemical scaffolds. This mechanism of resistance may have clinical relevance as mutations in the Esx-3 system are seen in clinical isolates. Further work to understand the mechanism of resistance is underway.