The front-line antituberculosis drug isoniazid (INH) and the related drug ethionamide (ETH) are prodrugs that upon activation inhibit the synthesis of mycolic acids, leading to bactericidal activity. Coresistance to INH and ETH can be mediated by dominant mutations in the target gene inhA, encoding an enoyl-ACP reductase, or by recessive mutations in ndh, encoding a type II NADH dehydrogenase (NdhII). To address the mechanism of resistance mediated by the latter, we have isolated novel ndh mutants of Mycobacterium smegmatis and Mycobacterium bovis BCG. The M. smegmatis ndh mutants were highly resistant to INH and ETH, while the M. bovis BCG mutants had low-level resistance to INH and ETH. All mutants had defects in NdhII activity resulting in an increase in intracellular NADH/NAD ؉ ratios. Increasing NADH levels were shown to protect InhA against inhibition by the INH-NAD adduct formed upon INH activation. We conclude that ndh mutations mediate a novel mechanism of resistance by increasing the NADH cellular concentration, which competitively inhibits the binding of INH-NAD or ETH-NAD adduct to InhA.Despite the declaration by the World Health Organization 10 years ago that tuberculosis (TB) was a global health emergency, the problem has worsened primarily due to the growing human immunodeficiency virus epidemic and the emergence of drug resistance (9, 12). Although TB can be cured by a regimen of several drugs for at least 6 months, the emergence of drug-resistant and multidrug-resistant TB has created new challenges to control and defeat the disease. According to the World Health Organization, drug-resistant Mycobacterium tuberculosis strains are found in at least 72 countries at a rate ranging from 3 to 41% (53). Understanding the mechanisms by which drug resistance occurs is important in order to quickly identify drug-resistant strains to treat the patients adequately. Moreover, knowledge of resistance mechanisms leads to the understanding of the mode of drug action and to the development of strategies to overcome drug resistance.Isoniazid (INH) (17,22,27,41,43,57). The recessive nature of the katG mutations, i.e., the restoration of catalase peroxidase activity and INH susceptibility when replaced or complemented with the wild-type gene, is consistent with the fact that KatG activates INH (20,56,57) to generate a hypothetical isonicotinic acyl radical that reacts with NAD and forms an INH-NAD adduct (45). This adduct binds to and inhibits InhA (29,40,44,45,52), resulting in mycolic acid biosynthesis inhibition (49) and cell death (49, 51). In contrast, overexpressed inhA alleles or alleles causing amino acid substitutions within the structural gene have been shown to confer INH resistance in a dominant fashion, i.e., conferring INH resistance when the mutant alleles replace or complement the wild-type gene (1,26,51). Mutations in INH r clinical isolates of M. tuberculosis have been mapped to the promoter region or the structural gene of inhA (1,2,17,22,27,31,38,41,43). The dominant nature of these mutations w...

The x-ray crystal structures of five triclosan analogs, in addition to that of the isoniazid-NAD adduct, are described in relation to their integral role in the design of potent inhibitors of the malarial enzyme Plasmodium falciparum enoyl acyl carrier protein reductase (PfENR). Many of the novel 5-substituted analogs exhibit low micromolar potency against in vitro cultures of drug-resistant and drug-sensitive strains of the P. falciparum parasite and inhibit purified PfENR enzyme with IC 50 values of <200 nM. This study has significantly expanded the knowledge base with regard to the structure-activity relationship of triclosan while affording gains against cultured parasites and purified PfENR enzyme. In contrast to a recent report in the literature, these results demonstrate the ability to improve the in vitro potency of triclosan significantly by replacing the suboptimal 5-chloro group with larger hydrophobic moieties. The biological and x-ray crystallographic data thus demonstrate the flexibility of the active site and point to future rounds of optimization to improve compound potency against purified enzyme and intracellular Plasmodium parasites.