Crystal structure of the Mycobacterium tuberculosis VirS regulator reveals its interaction with the lead compound SMARt751.

Ethionamide (ETO) is a prodrug that is primarily used as a second-line agent in the treatment of tuberculosis. Among the bacterial ETO activators, the monooxygenase MymA has been recently identified, and its expression is regulated by the mycobacterial regulator VirS. The discovery of VirS ligands that can enhance mymA expression and thereby increase the antimycobacterial efficacy of ETO, has led to the development of a novel therapeutic strategy against tuberculosis. This strategy involves the selection of preclinical candidates, including SMARt751. We report the first crystal structure of the AraC-like regulator VirS, in complex with SMARt751, refined at 1.69 Å resolution. Crystals were obtained via an in situ proteolysis method in the requisite presence of SMARt751. The elucidated structure corresponds to the ligand-binding domain of VirS, adopting an α/ß fold with structural similarities to H-NOX domains. Within the VirS structure, SMARt751 is situated in a completely enclosed hydrophobic cavity, where it forms hydrogen bonds with Asn11 and Asn149 as well as van der Waals contacts with various hydrophobic amino acids. Comprehensive structural comparisons within the AraC family of transcriptional regulators are conducted and analyzed to figure out the effects of the SMARt751 binding on the regulatory activity of VirS.

Loss-of-function mutations in ndh do not confer delamanid, ethionamide, isoniazid, or pretomanid resistance in Mycobacterium tuberculosis.

Results from clinical strains and knockouts of the H37Rv and CDC1551 laboratory strains demonstrated that ndh (Rv1854c) is not a resistance-conferring gene for isoniazid, ethionamide, delamanid, or pretomanid in Mycobacterium tuberculosis. This difference in the susceptibility to NAD-adduct-forming drugs compared with other mycobacteria may be driven by differences in the absolute intrabacterial NADH concentration.

Structural analysis of the interaction between spiroisoxazoline SMARt-420 and the Mycobacterium tuberculosis repressor EthR2.

Inhibition of transcriptional regulators of bacterial pathogens with the aim of reprogramming their metabolism to modify their antibiotic susceptibility constitutes a promising therapeutic strategy. One example is the bio-activation of the anti-tubercular pro-drug ethionamide, which activity could be enhanced by inhibiting the transcriptional repressor EthR. Recently, we discovered that inhibition of a second transcriptional repressor, EthR2, leads to the awakening of a new ethionamide bio-activation pathway. The x-ray structure of EthR2 was solved at 2.3 Å resolution in complex with a compound called SMARt-420 (Small Molecule Aborting Resistance). Detailed comparison and structural analysis revealed interesting insights for the upcoming structure-based design of EthR2 inhibitors as an alternative to revert ethionamide resistance in Mycobacterium tuberculosis.

New active leads for tuberculosis booster drugs by structure-based drug discovery.

The transcriptional repressor EthR from Mycobacterium tuberculosis, a member of the TetR family of prokaryotic homo-dimeric transcription factors, controls the expression of the mycobacterial mono-oxygenase EthA. EthA is responsible for the bio-activation of the second-line tuberculosis pro-drug ethionamide, and consequently EthR inhibitors boost drug efficacy. Here, we present a comprehensive in silico structure-based screening protocol that led to the identification of a number of novel scaffolds of EthR inhibitors in subsequent biophysical screening by thermal shift assay. Growth inhibition assays demonstrated that five of the twenty biophysical hits were capable of boosting ethionamide activity in vitro, with the best novel scaffold displaying an EC50 of 34 µM. In addition, the co-crystal structures of EthR with four new ligands at resolution ranging from 2.1 to 1.4 Å confirm the binding and inactivation mode, and will enable future lead development.

A fragment merging approach towards the development of small molecule inhibitors of Mycobacterium tuberculosis EthR for use as ethionamide boosters.

With the ever-increasing instances of resistance to frontline TB drugs there is the need to develop novel strategies to fight the worldwide TB epidemic. Boosting the effect of the existing second-line antibiotic ethionamide by inhibiting the mycobacterial transcriptional repressor protein EthR is an attractive therapeutic strategy. Herein we report the use of a fragment based drug discovery approach for the structure-guided systematic merging of two fragment molecules, each binding twice to the hydrophobic cavity of EthR from M. tuberculosis. These together fill the entire binding pocket of EthR. We elaborated these fragment hits and developed small molecule inhibitors which have a 100-fold improvement of potency in vitro over the initial fragments.

Unconventional surface plasmon resonance signals reveal quantitative inhibition of transcriptional repressor EthR by synthetic ligands.

EthR is a mycobacterial repressor that limits the bioactivation of ethionamide, a commonly used anti-tuberculosis second-line drug. Several efforts have been deployed to identify EthR inhibitors abolishing the DNA-binding activity of the repressor. This led to the demonstration that stimulating the bioactivation of Eth through EthR inhibition could be an alternative way to fight Mycobacterium tuberculosis. We propose a new surface plasmon resonance (SPR) methodology to study the affinity between inhibitors and EthR. Interestingly, the binding between inhibitors and immobilized EthR produced a dose-dependent negative SPR signal. We demonstrate that this signal reveals the affinity of small molecules for the repressor. The affinity constants (K(D)) correlate with their capacity to inhibit the binding of EthR to DNA. We hypothesize that conformational changes in EthR during ligand interaction could be responsible for this SPR signal. Practically, this unconventional result opens perspectives onto the development of an SPR assay that would at the same time reveal structural changes in the target upon binding with an inhibitor and the binding constant of this interaction.

Structural activation of the transcriptional repressor EthR from Mycobacterium tuberculosis by single amino acid change mimicking natural and synthetic ligands.

Ethionamide is an antituberculous drug for the treatment of multidrug-resistant Mycobacterium tuberculosis. This antibiotic requires activation by the monooxygenase EthA to exert its activity. Production of EthA is controlled by the transcriptional repressor EthR, a member of the TetR family. The sensitivity of M. tuberculosis to ethionamide can be artificially enhanced using synthetic ligands of EthR that allosterically inactivate its DNA-binding activity. Comparison of several structures of EthR co-crystallized with various ligands suggested that the structural reorganization of EthR resulting in its inactivation is controlled by a limited portion of the ligand-binding-pocket. In silico simulation predicted that mutation G106W may mimic ligands. X-ray crystallography of variant G106W indeed revealed a protein structurally similar to ligand-bound EthR. Surface plasmon resonance experiments established that this variant is unable to bind DNA, while thermal shift studies demonstrated that mutation G106W stabilizes EthR as strongly as ligands. Proton NMR of the methyl regions showed a lesser contribution of exchange broadening upon ligand binding, and the same quenched dynamics was observed in apo-variant G106W. Altogether, we here show that the area surrounding Gly106 constitutes the molecular switch involved in the conformational reorganization of EthR. These results also shed light on the mechanistic of ligand-induced allosterism controlling the DNA binding properties of TetR family repressors.

Both Nitro Groups Are Essential for High Antitubercular Activity of 3,5-Dinitrobenzylsulfanyl Tetrazoles and 1,3,4-Oxadiazoles through the Deazaflavin-Dependent Nitroreductase Activation Pathway.

3,5-Dinitrobenzylsulfanyl tetrazoles and 1,3,4-oxadiazoles, previously identified as having high in vitro activities against both replicating and nonreplicating mycobacteria and favorable cytotoxicity and genotoxicity profiles were investigated. First we demonstrated that these compounds act in a deazaflavin-dependent nitroreduction pathway and thus require a nitro group for their activity. Second, we confirmed the necessity of both nitro groups for antimycobacterial activity through extensive structure-activity relationship studies using 32 structural types of analogues, each in a five-membered series. Only the analogues with shifted nitro groups, namely, 2,5-dinitrobenzylsulfanyl oxadiazoles and tetrazoles, maintained high antimycobacterial activity but in this case mainly as a result of DprE1 inhibition. However, these analogues also showed increased toxicity to the mammalian cell line. Thus, both nitro groups in 3,5-dinitrobenzylsulfanyl-containing antimycobacterial agents remain essential for their high efficacy, and further efforts should be directed at finding ways to address the possible toxicity and solubility issues, for example, by targeted delivery.

Structural and docking studies of potent ethionamide boosters.

Tuberculosis remains the second only to HIV as the leading cause of death by infectious disease worldwide, and was responsible for 1.4 million deaths globally in 2011. One of the essential drugs of the second-line antitubercular regimen is the prodrug ethionamide, introduced in the 1960s. Ethionamide is primarily used in cases of multi-drug resistant (MDR) and extensively drug resistant (XDR) TB due to severe adverse side effects. As a prodrug, ethionamide is bioactivated by EthA, a mono-oxygenase whose activity is repressed by EthR, a member of the TetR family of regulators. Previous studies have established that inhibition of EthR improves ethionamide potency. We report here the crystal structures of three EthR inhibitors at 0.8 Šresolution (3-oxo-3-{4-[3-(thiophen-2-yl)-1,2,4-oxadiazol-5-yl]piperidin-1-yl}propanenitrile (BDM31343), 4,4,4-trifluoro-1-{4-[3-(6-methoxy-1,3-benzothiazol-2-yl)-1,2,4-oxadiazol-5-yl]piperidin-1-yl}butanone (BDM41325) and 5,5,5-trifluoro-1-{4-[3-(4-methanesulfonylphenyl)-1,2,4-oxadiazol-5-yl]piperidin-1-yl}pentanone (BDM41907)), and the docking studies undertaken to investigate possible binding modes. The results revealed two distinct orientations of the three compounds in the binding channel, a direct consequence of the promiscuous nature of the largely lipophilic binding site.

Exploring the Antitubercular Activity of Anthranilic Acid Derivatives: From MabA (FabG1) Inhibition to Intrabacterial Acidification.

Mycobacterium tuberculosis, the pathogen that causes tuberculosis, is responsible for the death of 1.5 million people each year and the number of bacteria resistant to the standard regimen is constantly increasing. This highlights the need to discover molecules that act on new M. tuberculosis targets. Mycolic acids, which are very long-chain fatty acids essential for M. tuberculosis viability, are synthesized by two types of fatty acid synthase (FAS) systems. MabA (FabG1) is an essential enzyme belonging to the FAS-II cycle. We have recently reported the discovery of anthranilic acids as MabA inhibitors. Here, the structure-activity relationships around the anthranilic acid core, the binding of a fluorinated analog to MabA by NMR experiments, the physico-chemical properties and the antimycobacterial activity of these inhibitors were explored. Further investigation of the mechanism of action in bacterio showed that these compounds affect other targets than MabA in mycobacterial cells and that their antituberculous activity is due to the carboxylic acid moiety which induces intrabacterial acidification.

The small-molecule SMARt751 reverses Mycobacterium tuberculosis resistance to ethionamide in acute and chronic mouse models of tuberculosis.

The sensitivity of Mycobacterium tuberculosis, the pathogen that causes tuberculosis (TB), to antibiotic prodrugs is dependent on the efficacy of the activation process that transforms the prodrugs into their active antibacterial moieties. Various oxidases of M. tuberculosis have the potential to activate the prodrug ethionamide. Here, we used medicinal chemistry coupled with a phenotypic assay to select the N-acylated 4-phenylpiperidine compound series. The lead compound, SMARt751, interacted with the transcriptional regulator VirS of M. tuberculosis, which regulates the mymA operon encoding a monooxygenase that activates ethionamide. SMARt751 boosted the efficacy of ethionamide in vitro and in mouse models of acute and chronic TB. SMARt751 also restored full efficacy of ethionamide in mice infected with M. tuberculosis strains carrying mutations in the ethA gene, which cause ethionamide resistance in the clinic. SMARt751 was shown to be safe in tests conducted in vitro and in vivo. A model extrapolating animal pharmacokinetic and pharmacodynamic parameters to humans predicted that as little as 25 mg of SMARt751 daily would allow a fourfold reduction in the dose of ethionamide administered while retaining the same efficacy and reducing side effects.

Fragment-Based Optimized EthR Inhibitors with in Vivo Ethionamide Boosting Activity.

Killing more than one million people each year, tuberculosis remains the leading cause of death from a single infectious agent. The growing threat of multidrug-resistant strains of Mycobacterium tuberculosis stresses the need for alternative therapies. EthR, a mycobacterial transcriptional regulator, is involved in the control of the bioactivation of the second-line drug ethionamide. We have previously reported the discovery of in vitro nanomolar boosters of ethionamide through fragment-based approaches. In this study, we have further explored the structure-activity and structure-property relationships in this chemical family. By combining structure-based drug design and in vitro evaluation of the compounds, we identified a new oxadiazole compound as the first fragment-based ethionamide booster which proved to be active in vivo, in an acute model of tuberculosis infection.

Discovery of the first Mycobacterium tuberculosis MabA (FabG1) inhibitors through a fragment-based screening.

Mycobacterium tuberculosis (M.tb), the etiologic agent of tuberculosis, remains the leading cause of death from a single infectious agent worldwide. The emergence of drug-resistant M.tb strains stresses the need for drugs acting on new targets. Mycolic acids are very long chain fatty acids playing an essential role in the architecture and permeability of the mycobacterial cell wall. Their biosynthesis involves two fatty acid synthase (FAS) systems. Among the four enzymes (MabA, HadAB/BC, InhA and KasA/B) of the FAS-II cycle, MabA (FabG1) remains the only one for which specific inhibitors have not been reported yet. The development of a new LC-MS/MS based enzymatic assay allowed the screening of a 1280 fragment-library and led to the discovery of the first small molecules that inhibit MabA activity. A fragment from the anthranilic acid series was optimized into more potent inhibitors and their binding to MabA was confirmed by 19F ligand-observed NMR experiments.

A comprehensive analysis of the protein-ligand interactions in crystal structures of Mycobacterium tuberculosis EthR.

The Mycobacterium tuberculosis EthR is a member of the TetR family of repressors, controlling the expression of EthA, a mono-oxygenase responsible for the bioactivation of the prodrug ethionamide. This protein was established as a promising therapeutic target against tuberculosis, allowing, when inhibited by a drug-like molecule, to boost the action of ethionamide. Dozens of EthR crystal structures have been solved in complex with ligands. Herein, we disclose EthR structures in complex with 18 different small molecules and then performed in-depth analysis on the complete set of EthR structures that provides insights on EthR-ligand interactions. The 81 molecules solved in complex with EthR show a large diversity of chemical structures that were split up into several chemical clusters. Two of the most striking common points of EthR-ligand interactions are the quasi-omnipresence of a hydrogen bond bridging compounds with Asn179 and the high occurrence of π-π interactions involving Phe110. A systematic analysis of the protein-ligand contacts identified eight hot spot residues that defined the basic structural features governing the binding mode of small molecules to EthR. Implications for the design of new potent inhibitors are discussed.

A fragment-based approach towards the discovery of N-substituted tropinones as inhibitors of Mycobacterium tuberculosis transcriptional regulator EthR2.

Tuberculosis (TB) caused by the pathogen Mycobacterium tuberculosis, represents one of the most challenging threat to public health worldwide, and with the increasing resistance to approved TB drugs, it is needed to develop new strategies to address this issue. Ethionamide is one of the most widely used drugs for the treatment of multidrug-resistant TB. It is a prodrug that requires activation by mycobacterial monooxygenases to inhibit the enoyl-ACP reductase InhA, which is involved in mycolic acid biosynthesis. Very recently, we identified that inhibition of a transcriptional repressor, termed EthR2, derepresses a new bioactivation pathway that results in the boosting of ethionamide activation. Herein, we describe the identification of potent EthR2 inhibitors using fragment-based screening and structure-based optimization. A target-based screening of a fragment library using thermal shift assay followed by X-ray crystallography identified 5 hits. Rapid optimization of the tropinone chemical series led to compounds with improved in vitro potency.

Intrinsic Antibacterial Activity of Nanoparticles Made of ß-Cyclodextrins Potentiates Their Effect as Drug Nanocarriers against Tuberculosis.

Multi-drug-resistant tuberculosis (TB) is a major public health problem, concerning about half a million cases each year. Patients hardly adhere to the current strict treatment consisting of more than 10 000 tablets over a 2-year period. There is a clear need for efficient and better formulated medications. We have previously shown that nanoparticles made of cross-linked poly-ß-cyclodextrins (pßCD) are efficient vehicles for pulmonary delivery of powerful combinations of anti-TB drugs. Here, we report that in addition to being efficient drug carriers, pßCD nanoparticles are endowed with intrinsic antibacterial properties. Empty pßCD nanoparticles are able to impair Mycobacterium tuberculosis (Mtb) establishment after pulmonary administration in mice. pßCD hamper colonization of macrophages by Mtb by interfering with lipid rafts, without inducing toxicity. Moreover, pßCD provoke macrophage apoptosis, leading to depletion of infected cells, thus creating a lung microenvironment detrimental to Mtb persistence. Taken together, our results suggest that pßCD nanoparticles loaded or not with antibiotics have an antibacterial action on their own and could be used as a carrier in drug regimen formulations effective against TB.

Efficient analoging around ethionamide to explore thioamides bioactivation pathways triggered by boosters in Mycobacterium tuberculosis.

Ethionamide is a key antibiotic prodrug of the second-line chemotherapy regimen to treat tuberculosis. It targets the biosynthesis of mycolic acids thanks to a mycobacterial bioactivation carried out by the Baeyer-Villiger monooxygenase EthA, under the control of a transcriptional repressor called EthR. Recently, the drug-like molecule SMARt-420, which triggers a new transcriptional regulator called EthR2, allowed the derepression a cryptic alternative bioactivation pathway of ethionamide. In order to study the bioactivation of a collection of thioisonicotinamides through the two bioactivation pathways, we developed a new two-step chemical pathway that led to the efficient synthesis of eighteen ethionamide analogues. Measurements of the antimycobacterial activity of these derivatives, used alone and in combination with boosters BDM41906 or SMARt-420, suggest that the two different bioactivation pathways proceed via the same mechanism, which implies the formation of similar metabolites. In addition, an electrochemical study of the aliphatic thioisonicotinamide analogues was undertaken to see whether their oxidation potential correlates with their antitubercular activity measured in the presence or in the absence of the two boosters.

Cyclodextrin-based nanocarriers containing a synergic drug combination: A potential formulation for pulmonary administration of antitubercular drugs.

Tuberculosis (TB) remains a major global health problem. The use of ethionamide (ETH), a main second line drug, is associated to severe toxic side-effects due to its low therapeutic index. In this challenging context, "booster" molecules have been synthetized to increase the efficacy of ETH. However, the administration of ETH/booster pair is mostly hampered by the low solubility of these drugs and the tendency of ETH to crystallize. Here, ETH and a poorly water-soluble booster, so-called BDM43266, were simultaneously loaded in polymeric ß-cyclodextrin nanoparticles (pßCyD NPs) following a "green" protocol. The interaction of ETH and BDM43266 with pßCyD NPs was investigated by complementary techniques. Remarkably, the inclusion of ETH and BDM43266 pßCyD NPs led to an increase of their apparent solubility in water of 10- and 90-fold, respectively. Competition studies of ETH and BDM43266 for the CyD cavities of pßCyD NPs corroborated the fact that the drugs did not compete with each other, confirming the possibility to simultaneously co-incorporate them in NPs. The drug-loaded NP suspensions could be filtered through 0.22µm filters. Finally, the drug-loaded NPs were passed through a Microsprayer® to evaluate the feasibility to administer pßCyD NPs by pulmonary route. Each spray delivered a constant amount of both drugs and the NPs were totally recovered after passage through the Microsprayer®. These promising results pave the way for a future use of pßCyD NPs for the pulmonary delivery of the ETH/BDM43266 pair.

Fragment-Sized EthR Inhibitors Exhibit Exceptionally Strong Ethionamide Boosting Effect in Whole-Cell Mycobacterium tuberculosis Assays.

Small-molecule inhibitors of the mycobacterial transcriptional repressor EthR have previously been shown to act as boosters of the second-line antituberculosis drug ethionamide. Fragment-based drug discovery approaches have been used in the past to make highly potent EthR inhibitors with ethionamide boosting activity both in vitro and ex vivo. Herein, we report the development of fragment-sized EthR ligands with nanomolar minimum effective concentration values for boosting the ethionamide activity in Mycobacterium tuberculosis whole-cell assays.

Reversion of antibiotic resistance in Mycobacterium tuberculosis by spiroisoxazoline SMARt-420.

Antibiotic resistance is one of the biggest threats to human health globally. Alarmingly, multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis 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 M. tuberculosis, 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.