Mechanism of the Dual Action Self-Potentiating Antitubercular Drug Morphazinamide

Abstract Pyrazinamide (PZA) is a cornerstone of first-line antitubercular drug therapy and is unique in its ability to kill nongrowing populations of Mycobacterium tuberculosis through disruption of coenzyme A synthesis. Unlike other drugs, PZA action is conditional and requires potentiation by host-relevant environmental stressors, such as low pH and nutrient limitation. Despite its pivotal role in tuberculosis therapy, the durability of this crucial drug is challenged by the emergent spread of drug-resistance. To advance drug discovery efforts, we characterized the activity of a more potent PZA analog, morphazinamide (MZA). Here, we demonstrate that like PZA, MZA acts in part through impairment of coenzyme A synthesis. Unexpectedly, we find that, in contrast to PZA, MZA does not require potentiation and maintains bactericidal activity against PZA-resistant strains due to an additional mechanism involving aldehyde release. Further, we find that the principal mechanism for resistance to the aldehyde component is through promoter mutations that increase expression of the mycothiol oxidoreductase MscR. Our findings reveal a dual action synergistic mechanism of MZA that results in a faster kill rate and a higher barrier to resistance. These observations provide new insights for discovery of improved therapeutic approaches for addressing the growing problem of drug-resistant tuberculosis. Significance Statement Pyrazinamide is the only antitubercular drug of its kind, capable of targeting persistent Mycobacterium tuberculosis through disruption of the coenzyme A biosynthetic pathway. With the emergent spread of drug-resistant tuberculosis, it is imperative to identify more effective next-generation drugs. In this study, we characterized the mechanism of action of a more potent analog of pyrazinamide, morphazinamide. We demonstrate that like pyrazinamide, morphazinamide impairs coenzyme A metabolism. In contrast to pyrazinamide, we find that morphazinamide has an additional aldehyde-dependent mechanism that mediates potent bactericidal activity against both pyrazinamide-susceptible and pyrazinamide-resistant strains of M. tuberculosis . These findings open up new opportunities for the development of next-generation antitubercular drugs to tackle the increasing challenge of drug-resistant tuberculosis.