The benzothiazinone lead compound, BTZ043, kills Mycobacterium tuberculosis by inhibiting the essential flavo-enzyme DprE1, decaprenylphosphoryl-beta-D-ribose 2-epimerase. Here, we synthesized a new series of piperazine-containing benzothiazinones (PBTZ) and show that, like BTZ043, the preclinical candidate PBTZ169 binds covalently to DprE1. The crystal structure of the DprE1-PBTZ169 complex reveals formation of a semimercaptal adduct with Cys387 in the active site and explains the irreversible inactivation of the enzyme. Compared to BTZ043, PBTZ169 has improved potency, safety and efficacy in zebrafish and mouse models of tuberculosis (TB). When combined with other TB drugs, PBTZ169 showed additive activity against M. tuberculosis in vitro except with bedaquiline (BDQ) where synergy was observed. A new regimen comprising PBTZ169, BDQ and pyrazinamide was found to be more efficacious than the standard three drug treatment in a murine model of chronic disease. PBTZ169 is thus an attractive drug candidate to treat TB in humans.Subject Categories Microbiology, Virology & Host Pathogen Interaction; Pharmacology & Drug Discovery

We report the discovery of a series of new drug leads that have potent activity against Mycobacterium tuberculosis as well as against other bacteria, fungi, and a malaria parasite. The compounds are analogues of the new tuberculosis (TB) drug SQ109 (1), which has been reported to act by inhibiting a transporter called MmpL3, involved in cell wall biosynthesis. We show that 1 and the new compounds also target enzymes involved in menaquinone biosynthesis and electron transport, inhibiting respiration and ATP biosynthesis, and are uncouplers, collapsing the pH gradient and membrane potential used to power transporters. The result of such multitarget inhibition is potent inhibition of TB cell growth, as well as very low rates of spontaneous drug resistance. Several targets are absent in humans but are present in other bacteria, as well as in malaria parasites, whose growth is also inhibited.

The expectation that genomics would result in new therapeutic interventions for infectious diseases remains unfulfilled. In the post-genomic era, the decade immediately following the availability of the genome sequence of Mycobacterium tuberculosis, tuberculosis (TB) drug discovery relied heavily on the target-based approach but this proved unsuccessful leading to a return to whole cell screening. Genomics underpinned screening by providing knowledge and many enabling technologies, most importantly whole genome resequencing to find resistance mutations and targets, and this resulted in a selection of leads and new TB drug candidates that are reviewed here. Unexpectedly, many new targets were found to be ‘promiscuous’ as they were inhibited by a variety of different compounds. In the post-post-genomics era, more advanced technologies have been implemented and these include high-content screening, screening for inhibitors of latency, the use of conditional knock-down mutants for validated targets and siRNA screens. In addition, immunomodulation and pharmacological manipulation of host functions are being explored in an attempt to widen our therapeutic options.

Clofazimine (CZM) is an antileprosy drug that was recently repurposed for treatment of multidrug-resistant tuberculosis. In Mycobacterium tuberculosis, CZM appears to act as a prodrug, which is reduced by NADH dehydrogenase (NDH-2), to release reactive oxygen species upon reoxidation by O 2 . CZM presumably competes with menaquinone (MK-4), a key cofactor in the mycobacterial electron transfer chain, for its reduction by NDH-2. We studied the effect of MK-4 supplementation on the activity of CZM against M. tuberculosis and found direct competition between CZM and MK-4 for the cidal effect of CZM, against nonreplicating and actively growing bacteria, as MK-4 supplementation blocked the drug's activity against nonreplicating bacteria. We demonstrated that CZM, like bedaquiline, is synergistic in vitro with benzothiazinones such as 2-piperazino-benzothiazinone 169 (PBTZ169), and this synergy also occurs against nonreplicating bacteria. The synergy between CZM and PBTZ169 was lost in an MK-4-rich medium, indicating that MK-4 is the probable link between their activities. The efficacy of the dual combination of CZM and PBTZ169 was tested in vivo, where a great reduction in bacterial load was obtained in a murine model of chronic tuberculosis. Taken together, these data confirm the potential of CZM in association with PBTZ169 as the basis for a new regimen against drug-resistant strains of M. tuberculosis. With approximately 9 million incident cases of tuberculosis (TB) worldwide and around 1.5 million deaths in 2012, Mycobacterium tuberculosis infection is one of the most important causes of death from a single infectious agent (1). The spread of multidrug-resistant TB (MDR-TB), namely, with resistance to isoniazid and rifampin, poses additional challenges to treatment with currently available anti-TB drugs. The situation is exacerbated by the increasing emergence of extensively drug-resistant (XDR) strains of M. tuberculosis, which cause diseases essentially untreatable with existing compounds. It is nowadays widely acknowledged that we need to develop new antibiotic combinations for TB and that these new regimens should be tested together at the preclinical stage, rather than testing a series of single drugs separately, in order to fill the TB drug development pipeline more efficiently (2-4).Some of the compounds in advanced clinical trials for TB are molecules that were originally used to treat other infectious diseases and have been repurposed for TB. Among the repurposed molecules, clofazimine (CZM), a riminophenazine originally developed as a drug to treat TB but overlooked for decades, has been used as a standard component of the treatment of leprosy for 50 years. It was recently repurposed for managing MDR-TB cases, notably following the results of the so-called Bangladesh study, which demonstrated that a CZM-containing regimen can cure such resistant cases in 9 to 12 months (5). Grosset et al. demonstrated the substantial benefit of adding CZM to second-line regimens in mice infected with isoniazid-resistant...

Benzothiazinones (BTZ) are a new class of drug candidates to combat tuberculosis that inhibit decaprenyl-phosphoribose epimerase (DprE1), an essential enzyme involved in arabinan biosynthesis. Using the checkerboard method and cell viability assays, we have studied the interaction profiles of BTZ043, the current lead compound, with several antituberculosis drugs or drug candidates against Mycobacterium tuberculosis strain H37Rv, namely, rifampin, isoniazid, ethambutol, TMC207, PA-824, moxifloxacin, meropenem with or without clavulanate, and SQ-109. No antagonism was found between BTZ043 and the tested compounds, and most of the interactions were purely additive. Data from two different approaches clearly indicate that BTZ043 acts synergistically with TMC207, with a fractional inhibitory concentration index of 0.5. TMC207 at a quarter of the MIC (20 ng/ml) used in combination with BTZ043 (1/4 MIC, 0.375 ng/ml) had a stronger bactericidal effect on M. tuberculosis than TMC207 alone at a concentration of 80 ng/ml. This synergy was not observed when the combination was tested on a BTZ-resistant M. tuberculosis mutant, suggesting that DprE1 inhibition is the basis for the interaction. This finding excludes the possibility of synergy occurring through an off-target mechanism. We therefore hypothesize that sub-MICs of BTZ043 weaken the bacterial cell wall and allow improved penetration of TMC207 to its target. Synergy between two new antimycobacterial compounds, such as TMC207 and BTZ043, with novel targets, offers an attractive foundation for a new tuberculosis regimen. W ith 8.8 million incident cases of tuberculosis (TB) worldwide and around 1.5 million deaths in 2010, Mycobacterium tuberculosis infection is one of the most important causes of death from an infectious disease (14). The spread of multidrug-resistant TB (MDR-TB), namely, resistance to both isoniazid (INH) and rifampin (RIF), poses additional challenges to treatment with currently available anti-TB drugs. The situation is exacerbated by the increasing emergence of extensively drug-resistant (XDR) strains of M. tuberculosis, which cause diseases essentially untreatable with existing compounds. Greater effort is required to find more efficacious combinations of molecules in order to meet the desired goals of killing both active and persistent tubercle bacilli while decreasing treatment duration. The TB drug development pipeline now comprises several candidates that are in clinical trials or soon will be (2, 12). The recommendations of the Global TB Alliance for Drug Development are to aim for a completely new TB chemotherapy with innovative molecules in combination in order to decrease the risk of emergence of drug resistance (3). Here, benzothiazinones (BTZ) are an extremely potent class of novel antimycobacterials that act by blocking the synthesis of decaprenyl-phospho-arabinose, the precursor of the arabinans in the mycobacterial cell wall (5). BTZ043, the current lead compound of this class, displays similar activity against all clinical isolates of...

A structure-guided fragment-based approach was used to target the lipophilic allosteric binding site of Mycobacterium tuberculosis EthR. This elongated channel has many hydrophobic residues lining the binding site, with few opportunities for hydrogen bonding. We demonstrate that a fragment-based approach involving the inclusion of flexible fragments in the library leads to an efficient exploration of chemical space, that fragment binding can lead to an extension of the cavity, and that fragments are able to identify hydrogen-bonding opportunities in this hydrophobic environment that are not exploited in Nature. In the present paper, we report the identification of a 1 μM affinity ligand obtained by structure-guided fragment linking.

dBenzothiazinones (BTZs) are a class of compounds found to be extremely potent against both drug-susceptible and drug-resistant Mycobacterium tuberculosis strains. The potency of BTZs is explained by their specificity for their target decaprenylphosphoryl-D-ribose oxidase (DprE1), in particular by covalent binding of the activated form of the compound to the critical cysteine 387 residue of the enzyme. To probe the role of C387, we used promiscuous site-directed mutagenesis to introduce other codons at this position into dprE1 of M. tuberculosis. The resultant viable BTZ-resistant mutants were characterized in vitro, ex vivo, and biochemically to gain insight into the effects of these mutations on DprE1 function and on M. tuberculosis. Five different mutations (C387G, C387A, C387S, C387N, and C387T) conferred various levels of resistance to BTZ and exhibited different phenotypes. The C387G and C387N mutations resulted in a lower growth rate of the mycobacterium on solid medium, which could be attributed to the significant decrease in the catalytic efficiency of the DprE1 enzyme. All five mutations rendered the mycobacterium less cytotoxic to macrophages. Finally, differences in the potencies of covalent and noncovalent DprE1 inhibitors in the presence of C387 mutations were revealed by enzymatic assays. As expected from the mechanism of action, the covalent inhibitor PBTZ169 only partially inhibited the mutant DprE1 enzymes compared to the near-complete inhibition with a noncovalent DprE1 inhibitor, Ty38c. This study emphasizes the importance of the C387 residue for DprE1 activity and for the killing action of covalent inhibitors such as BTZs and other recently identified nitroaromatic inhibitors. Mycobacterium tuberculosis is the etiological agent of tuberculosis (TB), an infectious disease which is a leading cause of death worldwide and poses a major threat to global health. The World Health Organization estimates that in 2014, 9.6 million people contracted TB, and 1.5 million people died (1). In addition, the emergence and worldwide spread of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) are alarming. With MDR-TB strains being resistant to the frontline drugs isoniazid and rifampin and XDR-TB strains being resistant to frontline and additionally second-line drugs, there is an urgent need for new drugs for TB.1,3-Benzothiazin-4-ones (BTZs) were discovered in 2009, with the lead compound BTZ043 having high potency (MIC of 1 ng/l) against M. tuberculosis strain H37Rv (2) and demonstrating efficacy against MDR and XDR clinical isolates (3). Piperazine-containing BTZ (PBTZ) derivatives were then designed with improved pharmacological properties (4), and the optimized lead compound PBTZ169 is currently in clinical trials (5).Genetic analysis of resistant mutants and enzymology have identified the target of BTZs as decaprenylphosphoryl-␤-D-ribose oxidase (DprE1), an essential flavoenzyme in M. tuberculosis involved in cell wall synthesis (2). DprE1 acts in concert with DprE2 to catalyze ...

Targeting dormant Mycobacterium tuberculosis represents a challenge to antituberculosis drug discovery programs. We previously reported and validated the use of the streptomycin (STR)-dependent M. tuberculosis 18b strain as a tool for assessing drug potency against nonreplicating bacteria both in vitro and in vivo. In this study, we generated a luminescent 18b strain, named 18b-Lux, by transforming the bacteria with a vector expressing the luxCDABE operon from Photorhabdus luminescens. Luciferase expression was demonstrated under replicating conditions, and, more importantly, luminescence levels significantly above background were detected following STR removal. The sensitivity of STR-starved 18b-Lux to approved and candidate antituberculosis therapeutic agents was evaluated by means of a luciferase assay in a 96-well format. Results mirrored the data obtained with the standard resazurin reduction microplate assay, and the luminescence readout allowed time course assessments of drug efficacy in vitro. Specifically, we proved that bedaquiline, the rifamycins, and sutezolid displayed time-dependent activity against dormant bacteria, while pyrazinamide and SQ109 showed bactericidal effects at the highest concentrations tested. Overall, we established the optimal conditions for an inexpensive, simple, and very sensitive assay with great potential for future applications.