Thioamide drugs, ethionamide (ETH) and prothionamide (PTH), are clinically effective in the treatment of Mycobacterium tuberculosis, M. leprae, and M. avium complex infections. Although generally considered second-line drugs for tuberculosis, their use has increased considerably as the number of multidrug resistant and extensively drug resistant tuberculosis cases continues to rise. Despite the widespread use of thioamide drugs to treat tuberculosis and leprosy, their precise mechanisms of action remain unknown. Using a cell-based activation method, we now have definitive evidence that both thioamides form covalent adducts with nicotinamide adenine dinucleotide (NAD) and that these adducts are tight-binding inhibitors of M. tuberculosis and M. leprae InhA. The crystal structures of the inhibited M. leprae and M. tuberculosis InhA complexes provide the molecular details of target–drug interactions. The purified ETH-NAD and PTH-NAD adducts both showed nanomolar Kis against M. tuberculosis and M. leprae InhA. Knowledge of the precise structures and mechanisms of action of these drugs provides insights into designing new drugs that can overcome drug resistance.

Mycobacterium tuberculosis enoyl-acyl-ACP reductase (InhA) has been demonstrated to be the primary target of isoniazid (INH). Recently, it was postulated that M. tuberculosis dihydrofolate reductase (DHFR) is also a target of INH, based on the findings that a 4R-INH-NADP adduct synthesized from INH by a nonenzymatic approach showed strong inhibition of DHFR in vitro , and overexpression of M. tuberculosis dfrA in M. smegmatis conferred a 2-fold increase of resistance to INH. In the present study, a plasmid expressing M. tuberculosis dfrA was transformed into M. smegmatis and M. tuberculosis strains, respectively. The transformant strains were tested for their resistance to INH. Compared to the wild-type strains, overexpression of dfrA in M. smegmatis and M. tuberculosis did not confer any resistance to INH based on the MIC values. Similar negative results were obtained with 14 other overexpressed proteins that have been proposed to bind some form of INH-NAD(P) adduct. An Escherichia coli cell-based system was designed that allowed coexpression of both M. tuberculosis katG and dfrA genes in the presence of INH. The DHFR protein isolated from the experimental sample was not found bound with any INH-NADP adduct by enzyme inhibition assay and mass spectroscopic analysis. We also used whole-genome sequencing to determine whether polymorphisms in dfrA could be detected in six INH-resistant clinical isolates known to lack mutations in inhA and katG , but no such mutations were found. The dfrA overexpression experiments, together with the biochemical and sequencing studies, conclusively demonstrate that DHFR is not a target relevant to the antitubercular activity of INH.

The remarkable survival ability of Mycobacterium tuberculosis in infected hosts is related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate expression of these lipids in response to environmental changes are unknown. Here we demonstrate that the enoyl-ACP reductase activity of InhA, an essential enzyme of the mycolic acid biosynthetic pathway and the primary target of the anti-tubercular drug isoniazid, is controlled via phosphorylation. Thr-266 is the unique kinase phosphoacceptor, both in vitro and in vivo. The physiological relevance of Thr-266 phosphorylation was demonstrated using inhA phosphoablative (T266A) or phosphomimetic (T266D/E) mutants. Enoyl reductase activity was severely impaired in the mimetic mutants in vitro, as a consequence of a reduced binding affinity to NADH. Importantly, introduction of inhA_T266D/E failed to complement growth and mycolic acid defects of an inhA-thermosensitive Mycobacterium smegmatis strain, in a similar manner to what is observed following isoniazid treatment. This study suggests that phosphorylation of InhA may represent an unusual mechanism that allows M. tuberculosis to regulate its mycolic acid content, thus offering a new approach to future anti-tuberculosis drug development.