Diarylcoumarins inhibit mycolic acid biosynthesis and kill Mycobacterium tuberculosis by targeting FadD32.

Infection with the bacterial pathogen Mycobacterium tuberculosis imposes an enormous burden on global public health. New antibiotics are urgently needed to combat the global tuberculosis pandemic; however, the development of new small molecules is hindered by a lack of validated drug targets. Here, we describe the identification of a 4,6-diaryl-5,7-dimethyl coumarin series that kills M. tuberculosis by inhibiting fatty acid degradation protein D32 (FadD32), an enzyme that is required for biosynthesis of cell-wall mycolic acids. These substituted coumarin inhibitors directly inhibit the acyl-acyl carrier protein synthetase activity of FadD32. They effectively block bacterial replication both in vitro and in animal models of tuberculosis, validating FadD32 as a target for antibiotic development that works in the same pathway as the established antibiotic isoniazid. Targeting new steps in well-validated biosynthetic pathways in antitubercular therapy is a powerful strategy that removes much of the usual uncertainty surrounding new targets and in vivo clinical efficacy, while circumventing existing resistance to established targets.

Synthesis and structure-activity relationships of phenyl-substituted coumarins with anti-tubercular activity that target FadD32.

In an effort to develop new and potent agents for therapy against tuberculosis, a high-throughput screen was performed against Mycobacterium tuberculosis strain H37Rv. Two 6-aryl-5,7-dimethyl-4-phenylcoumarin compounds 1a and 1b were found with modest activity. A series of coumarin derivatives were synthesized to improve potency and to investigate the structure-activity relationship of the series. Among them, compounds 1o and 2d showed improved activity with IC90 of 2 µM and 0.5 µM, respectively. Further optimization provided compound 3b with better physiochemical properties with IC90 0.4 µM which had activity in a mouse model of infection. The role of the conformation of the 4- and 6-aryl substituents is also described.

Baeyer-Villiger Monooxygenases EthA and MymA Are Required for Activation of Replicating and Non-replicating Mycobacterium tuberculosis Inhibitors.

Successful treatment of Mycobacterium tuberculosis infection typically requires a complex regimen administered over at least 6 months. Interestingly, many of the antibiotics used to treat M. tuberculosis are prodrugs that require intracellular activation. Here, we describe three small molecules, active against both replicating and non-replicating M. tuberculosis, that require activation by Baeyer-Villiger monooxygenases (BVMOs). Two molecules require BVMO EthA (Rv3854c) for activation and the third molecule requires the BVMO MymA (Rv3083). While EthA is known to activate the antitubercular drug ethionamide, this is the first description of MymA as an activating enzyme of a prodrug. Furthermore, we found that MymA also plays a role in activating ethionamide, with loss of MymA function resulting in ethionamide-resistant M. tuberculosis. These findings suggest overlap in function and specificity of the BVMOs in M. tuberculosis.

Identification of novel inhibitors of nonreplicating Mycobacterium tuberculosis using a carbon starvation model.

During Mycobacterium tuberculosis infection, a population of bacteria is thought to exist in a nonreplicating state, refractory to antibiotics, which may contribute to the need for prolonged antibiotic therapy. The identification of inhibitors of the nonreplicating state provides tools that can be used to probe this hypothesis and the physiology of this state. The development of such inhibitors also has the potential to shorten the duration of antibiotic therapy required. Here we describe the development of a novel nonreplicating assay amenable to high-throughput chemical screening coupled with secondary assays that use carbon starvation as the in vitro model. Together these assays identify compounds with activity against replicating and nonreplicating M. tuberculosis as well as compounds that inhibit the transition from nonreplicating to replicating stages of growth. Using these assays we successfully screened over 300,000 compounds and identified 786 inhibitors of nonreplicating M. tuberculosis In order to understand the relationship among different nonreplicating models, we tested 52 of these molecules in a hypoxia model, and four different chemical scaffolds in a stochastic persister model, and a streptomycin-dependent model. We found that compounds display varying levels of activity in different models for the nonreplicating state, suggesting important differences in bacterial physiology between models. Therefore, chemical tools identified in this assay may be useful for determining the relevance of different nonreplicating in vitro models to in vivo M. tuberculosis infection. Given our current limited understanding, molecules that are active across multiple models may represent more promising candidates for further development.

The first synthesis of an epidiselenodiketopiperazine.

Epidithiodiketopiperazines (ETPs) are natural products (e.g., gliotoxin) with varied and important biological activity, which often is attributed to the redox properties of the disulfide moiety. As such, analogs with altered redox properties and similar structural characteristics would be of value to biological investigations. The use of an ETP as the point of departure in the first synthesis of an epidiselenodiketopiperazine (ESeP) and its activity against Mycobacterium tuberculosis (MTB) is reported.

Identification of novel inhibitors of M. tuberculosis growth using whole cell based high-throughput screening.

Despite the urgent need for new antitubercular drugs, few are on the horizon. To combat the problem of emerging drug resistance, structurally unique chemical entities that inhibit new targets will be required. Here we describe our investigations using whole cell screening of a diverse collection of small molecules as a methodology for identifying novel inhibitors that target new pathways for Mycobacterium tuberculosis drug discovery. We find that conducting primary screens using model mycobacterial species may limit the potential for identifying new inhibitors with efficacy against M. tuberculosis. In addition, we confirm the importance of developing in vitro assay conditions that are reflective of in vivo biology for maximizing the proportion of hits from whole cell screening that are likely to have activity in vivo. Finally, we describe the identification and characterization of two novel inhibitors that target steps in M. tuberculosis cell wall biosynthesis. The first is a novel benzimidazole that targets mycobacterial membrane protein large 3 (MmpL3), a proposed transporter for cell wall mycolic acids. The second is a nitro-triazole that inhibits decaprenylphosphoryl-ß-D-ribose 2'-epimerase (DprE1), an epimerase required for cell wall biosynthesis. These proteins are both among the small number of new targets that have been identified by forward chemical genetics using resistance generation coupled with genome sequencing. This suggests that methodologies currently employed for screening and target identification may lead to a bias in target discovery and that alternative methods should be explored.