The side effects associated with tuberculosis therapy bring with them the risk of noncompliance and subsequent drug resistance. Increasing the therapeutic index of antituberculosis drugs should thus improve treatment effectiveness. Several antituberculosis compounds require in situ metabolic activation to become inhibitory. Various thiocarbamide-containing drugs, including ethionamide, are activated by the mycobacterial monooxygenase EthA, the production of which is controlled by the transcriptional repressor EthR. Here we identify drug-like inhibitors of EthR that boost the bioactivation of ethionamide. Compounds designed and screened for their capacity to inhibit EthR-DNA interaction were co-crystallized with EthR. We exploited the three-dimensional structures of the complexes for the synthesis of improved analogs that boosted the ethionamide potency in culture more than tenfold. In Mycobacterium tuberculosis-infected mice, one of these analogs, BDM31343, enabled a substantially reduced dose of ethionamide to lessen the mycobacterial load as efficiently as the conventional higher-dose treatment. This provides proof of concept that inhibiting EthR improves the therapeutic index of thiocarbamide derivatives, which should prompt reconsideration of their use as first-line drugs.

Antibiotic resistance is one of the biggest threats to human health globally. Alarmingly, multidrug-resistant and extensively drug-resistant have now spread worldwide. Some key antituberculosis antibiotics are prodrugs, for which resistance mechanisms are mainly driven by mutations in the bacterial enzymatic pathway required for their bioactivation. We have developed drug-like molecules that activate a cryptic alternative bioactivation pathway of ethionamide in, circumventing the classic activation pathway in which resistance mutations have now been observed. The first-of-its-kind molecule, named SMARt-420 (Small Molecule Aborting Resistance), not only fully reverses ethionamide-acquired resistance and clears ethionamide-resistant infection in mice, it also increases the basal sensitivity of bacteria to ethionamide.

We report in this article an extensive structure-activity relationships (SAR) study with 58 thiophen-2-yl-1,2,4-oxadiazoles as inhibitors of EthR, a transcriptional regulator controling ethionamide bioactivation in Mycobacterium tuberculosis. We explored the replacement of two key fragments of the starting lead BDM31343. We investigated the potency of all analogues to boost subactive doses of ethionamide on a phenotypic assay involving M. tuberculosis infected macrophages and then ascertained the mode of action of the most active compounds using a functional target-based surface plasmon resonance assay. This process revealed that introduction of 4,4,4-trifluorobutyryl chain instead of cyanoacetyl group was crucial for intracellular activity. Replacement of 1,4-piperidyl by (R)-1,3-pyrrolidyl scaffold did not enhance activity but led to improved pharmacokinetic properties. Furthermore, the crystal structures of ligand-EthR complexes were consistent with the observed SAR. In conclusion, we identified EthR inhibitors that boost antibacterial activity of ethionamide with nanomolar potency while improving solubility and metabolic stability.

Tuberculosis is a major disease causing every year 1.8 million deaths worldwide and represents the leading cause of mortality resulting from a bacterial infection. Introduction in the 60's of first-line drug regimen resulted in the control of the disease and TB was perceived as defeating. However, since the progression of HIV leading to co-infection with AIDS and the emergence of drug resistant strains, the need of new anti-tuberculosis drugs was not overstated. However in the past 40 years any new molecule did succeed in reaching the market. Today, the pipeline of potential new treatments has been fulfilled with several compounds in clinical trials or preclinical development with promising activities against sensitive and resistant Mycobacterium tuberculosis strains. Compounds as gatifloxacin, moxifloxacin, metronidazole or linezolid already used against other bacterial infections are currently evaluated in clinical phases 2 or 3 for treating tuberculosis. In addition, analogues of known TB drugs (PA-824, OPC-67683, PNU-100480, AZD5847, SQ609, SQ109, DC-159a) and new chemical entities (TMC207, BTZ043, DNB1, BDM31343) are under development. In this review, we report the chemical synthesis, mode of action when known, in vitro and in vivo activities and clinical data of all current small molecules targeting tuberculosis.

Mycobacterial transcriptional repressor EthR controls the expression of EthA, the bacterial monooxygenase activating ethionamide, and is thus largely responsible for the low sensitivity of the human pathogen Mycobacterium tuberculosis to this antibiotic. We recently reported structure-activity relationships of a series of 1,2,4-oxadiazole EthR inhibitors leading to the discovery of potent ethionamide boosters. Despite high metabolic stability, pharmacokinetic evaluation revealed poor mice exposure; therefore, a second phase of optimization was required. Herein a structure-property relationship study is reported according to the replacement of the two aromatic heterocycles: 2-thienyl and 1,2,4-oxadiazolyl moieties. This work was done using a combination of structure-based drug design and in vitro/ex vivo evaluations of ethionamide boosters on the targeted protein EthR and on the human pathogen Mycobacterium tuberculosis. Thanks to this process, we identified compound 42 (BDM41906), which displays improved efficacy in addition to high exposure to mice after oral administration.

In situ click chemistry has been successfully applied to probe the ligand binding domain of EthR, a mycobacterial transcriptional regulator known to control the sensitivity of Mycobacterium tuberculosis to several antibiotics. Specific protein-templated ligands were generated in situ from one azide and six clusters of 10 acetylenic fragments. Comparative X-ray structures of EthR complexed with either clicked ligand BDM14950 or its azide precursor showed ligand-dependent conformational impacts on the protein architecture. This approach revealed two mobile phenylalanine residues that control the access to a previously hidden hydrophobic pocket that can be further exploited for the development of structurally diverse EthR inhibitors. This report shows that protein-directed in situ chemistry allows medicinal chemists to explore the conformational space of a ligand-binding pocket and is thus a valuable tool to guide drug design in the complex path of hit-to-lead processes.

Tuberculosis (TB) is a leading infectious cause of death worldwide. The use of ethionamide (ETH), a main second line anti-TB drug, is hampered by its severe side effects. Recently discovered “booster” molecules strongly increase the ETH efficacy, opening new perspectives to improve the current clinical outcome of drug-resistant TB. To investigate the simultaneous delivery of ETH and its booster BDM41906 in the lungs, we co-encapsulated these compounds in biodegradable polymeric nanoparticles (NPs), overcoming the bottlenecks inherent to the strong tendency of ETH to crystallize and the limited water solubility of this Booster. The efficacy of the designed formulations was evaluated in TB infected macrophages using an automated confocal high-content screening platform, showing that the drugs maintained their activity after incorporation in NPs. Among tested formulations, “green” β-cyclodextrin (pCD) based NPs displayed the best physico-chemical characteristics and were selected for in vivo studies. The NPs suspension, administered directly into mouse lungs using a Microsprayer®, was proved to be well-tolerated and led to a 3-log decrease of the pulmonary mycobacterial load after 6 administrations as compared to untreated mice. This study paves the way for a future use of pCD NPs for the pulmonary delivery of the [ETH:Booster] pair in TB chemotherapy.

Efflux transporters of the RND family confer resistance to multiple antibiotics in Gram-negative bacteria. Here, we identify and chemically optimize pyridylpiperazine-based compounds that potentiate antibiotic activity in E. coli through inhibition of its primary RND transporter, AcrAB-TolC. Characterisation of resistant E. coli mutants and structural biology analyses indicate that the compounds bind to a unique site on the transmembrane domain of the AcrB L protomer, lined by key catalytic residues involved in proton relay. Molecular dynamics simulations suggest that the inhibitors access this binding pocket from the cytoplasm via a channel exclusively present in the AcrB L protomer. Thus, our work unveils a class of allosteric efflux-pump inhibitors that likely act by preventing the functional catalytic cycle of the RND pump.