Candidate antibacterials are usually identified on the basis of their in vitro activity. However, the apparent inhibitory activity of new leads can be misleading because most culture media do not reproduce an environment relevant to infection in vivo. In this study, while screening for novel anti-tuberculars, we uncovered how carbon metabolism can affect antimicrobial activity. Novel pyrimidine–imidazoles (PIs) were identified in a whole-cell screen against Mycobacterium tuberculosis. Lead optimization generated in vitro potent derivatives with desirable pharmacokinetic properties, yet without in vivo efficacy. Mechanism of action studies linked the PI activity to glycerol metabolism, which is not relevant for M. tuberculosis during infection. PIs induced self-poisoning of M. tuberculosis by promoting the accumulation of glycerol phosphate and rapid ATP depletion. This study underlines the importance of understanding central bacterial metabolism in vivo and of developing predictive in vitro culture conditions as a prerequisite for the rational discovery of new antibiotics.

Pyrazinamide (PZA) is a first-line drug for short-course tuberculosis therapy. Resistance to PZA is usually accompanied by loss of pyrazinamidase (PZase) activity in Mycobacterium tuberculosis. PZase converts PZA to bactericidal pyrazinoic acid, and the loss of PZase activity is associated with PZA resistance. The gene (pncA) encoding the M. tuberculosis PZase has recently been sequenced, and mutations in pncA were previously found in a small number of PZA-resistant M. tuberculosis strains. To further understand the genetic basis of PZA resistance and determine the frequency of PZA-resistant strains having pncA mutations, we analyzed a panel of PZA-resistant clinical isolates and mutants made in vitro. Thirty-three of 38 PZA-resistant clinical isolates had pncA mutations. Among the five strains that did not contain pncA mutations, four were found to be falsely resistant and one was found to be borderline resistant to PZA. The 33 PZA-resistant clinical isolates and 8 mutants made in vitro contained various mutations, including nucleotide substitutions, insertions, or deletions in the pncA gene. The identified mutations were dispersed along the pncA gene, but some degree of clustering of mutations was found at the following regions: Gly132-Thr142, Pro69-Leu85, and Ile5-Asp12. PCR-single-strand conformation polymorphism (SSCP) analysis was shown to be useful for the rapid detection of pncA mutations in the PZA-resistant strains. We conclude that a mutation in the pncA gene is a major mechanism of PZA resistance and that direct sequencing by PCR or SSCP analysis should help to rapidly identify PZA-resistant M. tuberculosis strains.

There is a growing need for new antibiotics. Compounds that target the proton motive force (PMF), uncouplers, represent one possible class of compounds that might be developed because they are already used to treat parasitic infections, and there is interest in their use for the treatment of other diseases, such as diabetes. Here, we tested a series of compounds, most with known antiinfective activity, for uncoupler activity. Many cationic amphiphiles tested positive, and some targeted isoprenoid biosynthesis or affected lipid bilayer structure. As an example, we found that clomiphene, a recently discovered undecaprenyl diphosphate synthase inhibitor active against Staphylococcus aureus, is an uncoupler. Using in silico screening, we then found that the anti-glioblastoma multiforme drug lead vacquinol is an inhibitor of Mycobacterium tuberculosis tuberculosinyl adenosine synthase, as well as being an uncoupler. Because vacquinol is also an inhibitor of M. tuberculosis cell growth, we used similarity searches based on the vacquinol structure, finding analogs with potent (∼0.5-2 μg/mL) activity against M. tuberculosis and S. aureus. Our results give a logical explanation of the observation that most new tuberculosis drug leads discovered by phenotypic screens and genome sequencing are highly lipophilic (logP ∼5.7) bases with membrane targets because such species are expected to partition into hydrophobic membranes, inhibiting membrane proteins, in addition to collapsing the PMF. This multiple targeting is expected to be of importance in overcoming the development of drug resistance because targeting membrane physical properties is expected to be less susceptible to the development of resistance.T here is a need for new antibiotics, due to the increase in drug resistance (1, 2). For example, some studies report that by 2050, absent major improvements in drug discovery and use, more individuals will die from drug-resistant bacterial infections than from cancer, resulting in a cumulative effect on global gross domestic product of as much as 100 trillion dollars (3, 4). To discover new drugs, new targets, leads, concepts, and implementations are needed (5, 6).Currently, one major cause of death from bacterial infections is tuberculosis (TB) (7), where very highly drug-resistant strains have been found (8). Therapy is lengthy, even with drug-sensitive strains, and requires combination therapies with four drugs. Two recent TB drugs/drug leads (9-11) are TMC207 [bedaquiline (1); Sirturo] and SQ109 (2) (Fig. 1). Bedaquiline (1) targets the Mycobacterium tuberculosis ATP synthase (9) whereas SQ109 (2) has been proposed to target MmpL3 (mycobacterial membrane protein large 3), a trehalose monomycolate transporter essential for cell wall biosynthesis (12). SQ109 (2) is a lipophilic base containing an adamantyl "headgroup" connected via an ethylene diamine "linker" to a geranyl (C 10 ) "side chain," and in recent work (13), we synthesized a series of 11 analogs of SQ109 (2) finding that the ethanolamine (3) was more potent th...

Superoxide dismutase (SOD) is a ubiquitous metalloenzyme in aerobic organisms that catalyzes the conversion of superoxide anion to hydrogen peroxide. Mycobacterium tuberculosis is unusual in that it secretes large quantities of iron-cofactored SOD. To determine the role of SOD in pathogenesis, we constructed mutants of M. tuberculosis H37Rv with reduced SOD production. Compared with controls, SOD-diminished isolates were more susceptible to killing by hydrogen peroxide. The isolates were markedly attenuated, exhibiting nearly 100,000-fold fewer bacilli than virulent control strains in the lungs and spleens of C57BL/6 mice 4 wk after intravenous inoculation. In the lung, SOD-attenuated M. tuberculosis induced robust interstitial mononuclear cell infiltration within 24 h and many cells were apoptotic by TUNEL staining, whereas virulent H37Rv exhibited minimal early inflammatory response and only rare interstitial mononuclear cell apoptosis. During prolonged infections, C57BL/6 mice tolerated SOD-attenuated M. tuberculosis better than BCG, exhibiting 68% greater weight gain, quicker eradication of bacilli from the spleen, and less alveolar lung infiltration. These results establish the importance of SOD in the pathogenesis of tuberculosis. Its effect appears to be mediated in part by inhibiting innate host immune responses, including early mononuclear cell infiltration of infected tissues and apoptosis.

During the course of our investigations in the oxazolidinone antibacterial agent area, we have identified a subclass with especially potent in vitro activity against mycobacteria. The salient structural feature of these oxazolidinone analogues, 6 (U-100480), 7 (U-101603), and 8 (U-101244), is their appended thiomorpholine moiety. The rational design, synthesis, and evaluation of the in vitro antimycobacterial activity of these analogues is described. Potent activity against a screening strain of Mycobacterium tuberculosis was demonstrated by 6 and 7 (minimum inhibitory concentrations or MIC's < or = 0.125 micrograms/mL). Oxazolidinones 6 and 8 exhibit MIC90 values of 0.50 micrograms/mL or less against a panel of organisms consisting of five drug-sensitive and five multidrug-resistant strains of M. tuberculosis, with 6 being the most active congener. Potent in vitro activity against other mycobacterial species was also demonstrated by 6. For example, 6 exhibited excellent in vitro activity against multiple clinical isolates of Mycobacterium avium complex (MIC's = 0.5-4 micrograms/mL). Orally administered 6 displays in vivo efficacy against M. tuberculosis and M. avium similar to that of clinical comparators isoniazid and azithromycin, respectively. Consideration of these factors, along with a favorable pharmaco-kinetic and chronic toxicity profile in rats, suggests that 6 (U-100480) is a promising antimycobacterial agent.