SUMMARY Identification of unique leads represents a significant challenge in drug discovery. This hurdle is magnified in neglected diseases such as tuberculosis. We have leveraged public high-throughput screening (HTS) data, to experimentally validate virtual screening approach employing Bayesian models built with bioactivity information (single-event model) as well as bioactivity and cytotoxicity information (dual-event model). We virtually screen a commercial library and experimentally confirm actives with hit rates exceeding typical HTS results by 1-2 orders of magnitude. The first dual-event Bayesian model identified compounds with antitubercular whole-cell activity and low mammalian cell cytotoxicity from a published set of antimalarials. The most potent hit exhibits the in vitro activity and in vitro/in vivo safety profile of a drug lead. These Bayesian models offer significant economies in time and cost to drug discovery.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, which kills 1.8 million annually. Mtb RNA polymerase (RNAP) is the target of the first-line antituberculosis drug rifampin (Rif). We report crystal structures of Mtb RNAP, alone and in complex with Rif, at 3.8–4.4 Å resolution. The results identify an Mtb-specific structural module of Mtb RNAP and establish that Rif functions by a steric-occlusion mechanism that prevents extension of RNA. We also report non-Rif-related compounds–Nα-aroyl-N-aryl-phenylalaninamides (AAPs)–that potently and selectively inhibit Mtb RNAP and Mtb growth, and we report crystal structures of Mtb RNAP in complex with AAPs. AAPs bind to a different site on Mtb RNAP than Rif, exhibit no cross-resistance with Rif, function additively when co-administered with Rif, and suppress resistance emergence when co-administered with Rif.

A new drug target-- the "switch region"--has been identified within bacterial RNA polymerase (RNAP), the enzyme that mediates bacterial RNA synthesis. The new target serves as the binding site for compounds that inhibit bacterial RNA synthesis and kill bacteria. Since the new target is present in most bacterial species, compounds that bind to the new target are active against a broad spectrum of bacterial species. Since the new target is different from targets of other antibacterial agents, compounds that bind to the new target are not cross-resistant with other antibacterial agents. Four antibiotocs that function through the new target have been identified: myxopyronin, corallopyronin, ripostatin, and lipiarmycin. This review summarizes the switch region, switch-region inhibitors, and implications for antibacterial drug discovery.

We demonstrate that Mycobacterium tuberculosis grown in vitro is sensitive to glutathione and its derivative S-nitrosoglutathione. Furthermore, our infection studies with J774.1 macrophages indicate that glutathione is essential for the control of the intracellular growth of M. tuberculosis. This study indicates the important role of glutathione in the control of macrophages by M. tuberculosis.Tuberculosis is one of the most prevalent infectious diseases in the world (3). The situation is exacerbated by the emergence of mutlidrug-resistant strains and an ever-growing number of highly susceptible immunocompromised individuals arising from the AIDS pandemic (3). The production of reactive nitrogen intermediates by murine macrophages is considered to be a relatively effective host defense mechanism against Mycobacterium tuberculosis (1,4,7,8,9,13,16).In this study, we show that a virulent laboratory strain of M. tuberculosis, H37Rv, grown in vitro is sensitive to glutathione (GSH) and nitrosoglutathione (GSNO) at physiological concentrations. While GSH at a 5 mM concentration is bacteriostatic to H37Rv ( Fig. 1; Table 1), GSNO at a 5 mM concentration is bactericidal ( Fig. 1; Table 1), as was observed by determining optical density (OD) and numbers of CFU. H37Rv was grown in 7H9 containing albumin dextrose complex (ADC) and Tween 80 to mid-log phase and then diluted to an OD of approximately 0.1. The bacterial suspensions were then treated with 5 mM GSH (Sigma) or 5 mM GSNO (Sigma) or left untreated. The OD was measured every day for 5 days ( Fig. 1). Each day, an aliquot was taken from every sample, diluted 100-fold, and plated on 7H11 plates containing ADC for determination of numbers of CFU (Table 1). All experiments were performed three times in triplicate.The mechanism of action of the antimycobacterial activity of GSH is not certain. One possibility is that the presence of a high concentration of GSH may result in an imbalance in a bacterium containing an alternative thiol for regulating reduction or oxidation activity (i.e., mycothiol). We also consider the hypothesis of Spallholz in this connection (14). According to Spallholz, "GSH is structurally similar to the precursor of the antibiotics produced in fungi in the genera Penicillium and Cephalasporium." Its potential conversion to the penicillinlike derivative glutacillin, a ␤-lactam form of GSH, raises the intriguing question of whether GSH was once a universal penem-like precursor of antibiotics in cells of many life forms (14).

We report that bacterial RNA polymerase (RNAP) is the functional cellular target of the depsipeptide antibiotic salinamide A (Sal), and we report that Sal inhibits RNAP through a novel binding site and mechanism. We show that Sal inhibits RNA synthesis in cells and that mutations that confer Sal-resistance map to RNAP genes. We show that Sal interacts with the RNAP active-center ‘bridge-helix cap’ comprising the ‘bridge-helix N-terminal hinge’, ‘F-loop’, and ‘link region’. We show that Sal inhibits nucleotide addition in transcription initiation and elongation. We present a crystal structure that defines interactions between Sal and RNAP and effects of Sal on RNAP conformation. We propose that Sal functions by binding to the RNAP bridge-helix cap and preventing conformational changes of the bridge-helix N-terminal hinge necessary for nucleotide addition. The results provide a target for antibacterial drug discovery and a reagent to probe conformation and function of the bridge-helix N-terminal hinge.DOI: http://dx.doi.org/10.7554/eLife.02451.001

Reactive oxygen and nitrogen intermediates are important antimicrobial defense mechanisms of macrophages and other phagocytic cells. While reactive nitrogen intermediates have been shown to play an important role in tuberculosis control in the murine system, their role in human disease is not clearly established. Glutathione, a tripeptide and antioxidant, is synthesized at high levels by cells during reactive oxygen intermediate and nitrogen intermediate production. Glutathione has been recently shown to play an important role in apoptosis and to regulate antigen-presenting-cell functions. Glutathione also serves as a carrier molecule for nitric oxide, in the form of S-nitrosoglutathione. Previous work from this laboratory has shown that glutathione and S-nitrosoglutathione are directly toxic to mycobacteria. A mutant strain of Mycobacterium bovis BCG, defective in the transport of small peptides such as glutathione, is resistant to the toxic effect of glutathione and S-nitrosoglutathione. Using the peptide transport mutant as a tool, we investigated the role of glutathione and S-nitrosoglutathione in animal and human macrophages in controlling intracellular mycobacterial growth.Tuberculosis remains a leading cause of mortality worldwide due to the ability of Mycobacterium tuberculosis to adapt to a wide range of conditions both inside and outside the human host. The resurgence of tuberculosis worldwide has intensified research efforts directed at host defense and pathogenic mechanisms operative in M. tuberculosis infection. Despite advances in diagnosis and treatment, we still do not know the mechanism by which M. tuberculosis is eliminated by the immune system in healthy subjects (28). A more complete understanding of the roles that each component of the immune system plays in protection against or exacerbation of tuberculosis, as well as of the bacterium's weapons to evade those components, will enhance development of preventive and therapeutic strategies against this enormously successful pathogen (13).The major phagocytic cell involved in protection against M. tuberculosis is the activated macrophage. Macrophages acquire the ability to kill the intracellular pathogen following exposure to cytokines released by antigen-sensitized T lymphocytes (10,28). This event triggers antimicrobial mechanisms such as generation of reactive oxygen intermediates (ROI), generation of reactive nitrogen intermediates (RNI), phagolysosome fusion, etc. (3,12,25,27). This pathway is the essence of cell-mediated immunity to intracellular infection.Glutathione (GSH) is a tripeptide comprised of glutamate, cysteine, and glycine. GSH is present in most cells, where it functions as an antioxidant protecting cells from toxic effects of ROI and RNI (29). In addition to its antioxidant role, GSH plays a vital role in maintenance of cell viability, DNA replication, and thiolation of proteins (9). GSH has also been reported to regulate immune cell functions. GSH regulates the antigen-processing machinery in antigen-presenting cells and al...

Glutathione is a tripeptide and antioxidant, synthesized at high levels by cells during the production of reactive oxygen and nitrogen intermediates. Glutathione also serves as a carrier molecule for nitric oxide in the form of S-nitrosoglutathione. Previous studies from this laboratory have shown that glutathione and Snitrosoglutathione are directly toxic to mycobacteria. Glutathione is not transported into the cells as a tripeptide. Extracellular glutathione is converted to a dipeptide due to the action of transpeptidase, and the dipeptide is then transported into the bacterial cells. The processing of glutathione and S-nitrosoglutathione is brought about by the action of the enzyme ␥-glutamyl transpeptidase. The function of ␥-glutamyl transpeptidase is to cleave glutathione and S-nitrosoglutathione to the dipeptide (Cys-Gly), which is then transported into the bacterium by the multicomponent ABC transporter dipeptide permease. We have created a mutant strain of Mycobacterium tuberculosis lacking this metabolic enzyme. We investigated the sensitivity of this strain to glutathione and S-nitrosoglutathione compared to that of the wild-type bacteria. In addition, we examined the role of glutathione and/or S-nitrosoglutathione in controlling the growth of intracellular M. tuberculosis inside mouse macrophages.Mycobacterium tuberculosis accounts for the largest number of deaths caused by a single human pathogen (5). It is estimated that there will be 80 million new cases and 20 million deaths from tuberculosis in the coming decade (5). The appearance of drug-resistant strains of M. tuberculosis and the human immunodeficiency virus pandemic has exacerbated this situation (5).Glutathione (GSH) is a tripeptide comprised of ␥-glutamate, cysteine, and glycine. It is an antioxidant found in all cells of the body, including macrophages, which are considered major phagocytic cells involved in protection against M. tuberculosis (28, 29). However, mycobacteria do not produce GSH. Instead, they have mycothiols for regulating their reduction or oxidation activities (1, 23).Reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI), generated by phagocytic cells, are thought to play an important role in inhibiting growth of intracellular pathogens (9,13,22). When host cells, such as macrophages, generate ROI and RNI, there will be simultaneous synthesis of GSH for protection against the toxic effects of ROI and RNI. Nitric oxide (NO) has been shown to inhibit the growth of M. tuberculosis in the murine system (7,8,11,19). NO also reacts with GSH to form S-nitrosoglutathione (GSNO). GSNO, an NO donor, can then release NO, leading to the death of the pathogen (12, 24). The stability of NO is increased when it is in complex with GSH. We have previously shown that M. tuberculosis is sensitive to 5 mM GSH and 5 mM GSNO (14,30). The sensitivity of M. tuberculosis to GSNO is due to the bactericidal effects of NO released from the GSNO complex. The mechanism of action of antimycobacterial activity of GSH is uncertain...

Marine sponge samples were collected in Baler, Aurora, Philippines, and extracts were tested for in vitro antituberculosis activity. An orange Agelas sp. sponge yielded the known compound, agelasine F, which inhibited some drug resistant strains of Mycobacterium tuberculosis in vitro at concentrations as low as 3.13 micrograms/ml. Activity against M. tuberculosis residing within macrophages required concentrations of 13-22 micrograms/ml which was below the IC50 for Vero cells (34 micrograms/ml).