Compartmentalization of galactan biosynthesis in mycobacteria.

Galactan polymer is a prominent component of the mycobacterial cell wall core. Its biogenesis starts at the cytoplasmic side of the plasma membrane by a build-up of the linker disaccharide [rhamnosyl (Rha) - N-acetyl-glucosaminyl (GlcNAc) phosphate] on the decaprenyl-phosphate carrier. This decaprenyl-P-P-GlcNAc-Rha intermediate is extended by two bifunctional galactosyl transferases, GlfT1 and GlfT2, and then it is translocated to the periplasmic space by an ABC transporter Wzm-Wzt. The cell wall core synthesis is finalized by the action of an array of arabinosyl transferases, mycolyl transferases, and ligases that catalyze an attachment of the arabinogalactan polymer to peptidoglycan through the linker region. Based on visualization of the GlfT2 enzyme fused with fluorescent tags it was proposed that galactan polymerization takes place in a specific compartment of the mycobacterial cell envelope, the intracellular membrane domain, representing pure plasma membrane free of cell wall components (previously denoted as the "PMf" domain), which localizes to the polar region of mycobacteria. In this work, we examined the activity of the galactan-producing cellular machine in the cell-wall containing cell envelope fraction and in the cell wall-free plasma membrane fraction prepared from Mycobacterium smegmatis by the enzyme assays using radioactively labeled substrate UDP-[14C]-galactose as a tracer. We found that despite a high abundance of GlfT2 in both of these fractions as confirmed by their thorough proteomic analyses, galactan is produced only in the reaction mixtures containing the cell wall components. Our findings open the discussion about the distribution of GlfT2 and the regulation of its activity in mycobacteria.

An ABC transporter Wzm-Wzt catalyzes translocation of lipid-linked galactan across the plasma membrane in mycobacteria.

Mycobacterium tuberculosis, one of the deadliest pathogens in human history, is distinguished by a unique, multilayered cell wall, which offers the bacterium a high level of protection from the attacks of the host immune system. The primary structure of the cell wall core, composed of covalently linked peptidoglycan, branched heteropolysaccharide arabinogalactan, and mycolic acids, is well known, and numerous enzymes involved in the biosynthesis of its components are characterized. The cell wall biogenesis takes place at both cytoplasmic and periplasmic faces of the plasma membrane, and only recently some of the specific transport systems translocating the metabolic intermediates between these two compartments have been characterized [M. Jackson, C. M. Stevens, L. Zhang, H. I. Zgurskaya, M. Niederweis, Chem. Rev., 10.1021/acs.chemrev.0c00869 (2020)]. In this work, we use CRISPR interference methodology in Mycobacterium smegmatis to functionally characterize an ATP-binding cassette (ABC) transporter involved in the translocation of galactan precursors across the plasma membrane. We show that genetic knockdown of the transmembrane subunit of the transporter results in severe morphological changes and the accumulation of an aberrantly long galactan precursor. Based on similarities with structures and functions of specific O-antigen ABC transporters of gram-negative bacteria [C. Whitfield, D. M. Williams, S. D. Kelly, J. Biol. Chem. 295, 10593-10609 (2020)], we propose a model for coupled synthesis and export of the galactan polymer precursor in mycobacteria.

The Veterinary Anti-Parasitic Selamectin Is a Novel Inhibitor of the Mycobacterium tuberculosis DprE1 Enzyme.

Avermectins are macrocyclic lactones with anthelmintic activity. Recently, they were found to be effective against Mycobacterium tuberculosis, which accounts for one third of the worldwide deaths from antimicrobial resistance. However, their anti-mycobacterial mode of action remains to be elucidated. The activity of selamectin was determined against a panel of M. tuberculosis mutants. Two strains carrying mutations in DprE1, the decaprenylphosphoryl-ß-D-ribose oxidase involved in the synthesis of mycobacterial arabinogalactan, were more susceptible to selamectin. Biochemical assays against the Mycobacterium smegmatis DprE1 protein confirmed this finding, and docking studies predicted a binding site in a loop that included Leu275. Sequence alignment revealed variants in this position among mycobacterial species, with the size and hydrophobicity of the residue correlating with their MIC values; M. smegmatis DprE1 variants carrying these point mutations validated the docking predictions. However, the correlation was not confirmed when M. smegmatis mutant strains were constructed and MIC phenotypic assays performed. Likewise, metabolic labeling of selamectin-treated M. smegmatis and M. tuberculosis cells with 14C-labeled acetate did not reveal the expected lipid profile associated with DprE1 inhibition. Together, our results confirm the in vitro interactions of selamectin and DprE1 but suggest that selamectin could be a multi-target anti-mycobacterial compound.

Mycobacterial Epoxide Hydrolase EphD Is Inhibited by Urea and Thiourea Derivatives.

The genome of the human intracellular pathogen Mycobacterium tuberculosis encodes an unusually large number of epoxide hydrolases, which are thought to be involved in lipid metabolism and detoxification reactions needed to endure the hostile environment of host macrophages. These enzymes therefore represent suitable targets for compounds such as urea derivatives, which are known inhibitors of soluble epoxide hydrolases. In this work, we studied in vitro the effect of the thiourea drug isoxyl on six epoxide hydrolases of M. tuberculosis using a fatty acid substrate. We show that one of the proteins inhibited by isoxyl is EphD, an enzyme involved in the metabolism of mycolic acids, key components of the mycobacterial cell wall. By analyzing mycolic acid profiles, we demonstrate the inhibition of EphD epoxide hydrolase activity by isoxyl and two other urea-based inhibitors, thiacetazone and AU1235, inside the mycobacterial cell.

Impact of the epoxide hydrolase EphD on the metabolism of mycolic acids in mycobacteria.

Mycolic acids are the hallmark of the cell envelope in mycobacteria, which include the important human pathogens Mycobacterium tuberculosis and Mycobacterium leprae Mycolic acids are very long C60-C90 α-alkyl ß-hydroxy fatty acids having a variety of functional groups on their hydrocarbon chain that define several mycolate types. Mycobacteria also produce an unusually large number of putative epoxide hydrolases, but the physiological functions of these enzymes are still unclear. Here, we report that the mycobacterial epoxide hydrolase EphD is involved in mycolic acid metabolism. We found that orthologs of EphD from M. tuberculosis and M. smegmatis are functional epoxide hydrolases, cleaving a lipophilic substrate, 9,10-cis-epoxystearic acid, in vitro and forming a vicinal diol. The results of EphD overproduction in M. smegmatis and M. bovis BCG Δhma strains producing epoxymycolic acids indicated that EphD is involved in the metabolism of these forms of mycolates in both fast- and slow-growing mycobacteria. Moreover, using MALDI-TOF-MS and 1H NMR spectroscopy of mycolic acids and lipids isolated from EphD-overproducing M. smegmatis, we identified new oxygenated mycolic acid species that accumulated during epoxymycolate depletion. Disruption of the ephD gene in M. tuberculosis specifically impaired the synthesis of ketomycolates and caused accumulation of their precursor, hydroxymycolate, indicating either direct or indirect involvement of EphD in ketomycolate biosynthesis. Our results clearly indicate that EphD plays a role in metabolism of oxygenated mycolic acids in mycobacteria.

Identification of aminopyrimidine-sulfonamides as potent modulators of Wag31-mediated cell elongation in mycobacteria.

There is an urgent need to discover new anti-tubercular agents with novel mechanisms of action in order to tackle the scourge of drug-resistant tuberculosis. Here, we report the identification of such a molecule - an AminoPYrimidine-Sulfonamide (APYS1) that has potent, bactericidal activity against M. tuberculosis. Mutations in APYS1-resistant M. tuberculosis mapped exclusively to wag31, a gene that encodes a scaffolding protein thought to orchestrate cell elongation. Recombineering confirmed that a Gln201Arg mutation in Wag31 was sufficient to cause resistance to APYS1, however, neither overexpression nor conditional depletion of wag31 impacted M. tuberculosis susceptibility to this compound. In contrast, expression of the wildtype allele of wag31 in APYS1-resistant M. tuberculosis was dominant and restored susceptibility to APYS1 to wildtype levels. Time-lapse imaging and scanning electron microscopy revealed that APYS1 caused gross malformation of the old pole of M. tuberculosis, with eventual lysis. These effects resembled the morphological changes observed following transcriptional silencing of wag31 in M. tuberculosis. These data show that Wag31 is likely not the direct target of APYS1, but the striking phenotypic similarity between APYS1 exposure and genetic depletion of Wag31 in M. tuberculosis suggests that APYS1 might indirectly affect Wag31 through an as yet unknown mechanism.

Mechanochemical Synthesis and Biological Evaluation of Novel Isoniazid Derivatives with Potent Antitubercular Activity.

A series of isoniazid derivatives bearing a phenolic or heteroaromatic coupled frame were obtained by mechanochemical means. Their pH stability and their structural (conformer/isomer) analysis were checked. The activity of prepared derivatives against Mycobacterium tuberculosis cell growth was evaluated. Some compounds such as phenolic hydrazine 1a and almost all heteroaromatic ones, especially 2, 5 and 7, are more active than isoniazid, and their activity against some M. tuberculosis MDR clinical isolates was determined. Compounds 1a and 7 present a selectivity index >1400 evaluated on MRC5 human fibroblast cells. The mechanism of action of selected hydrazones was demonstrated to block mycolic acid synthesis due to InhA inhibition inside the mycobacterial cell.

The phosphatidyl-myo-inositol mannosyltransferase PimA is essential for Mycobacterium tuberculosis growth in vitro and in vivo.

The cell envelope of Mycobacterium tuberculosis contains glycans and lipids of peculiar structure that play prominent roles in the biology and pathogenesis of tuberculosis. Consequently, the chemical structure and biosynthesis of the cell wall have been intensively investigated in order to identify novel drug targets. Here, we validate that the function of phosphatidyl-myo-inositol mannosyltransferase PimA is vital for M. tuberculosis in vitro and in vivo. PimA initiates the biosynthesis of phosphatidyl-myo-inositol mannosides by transferring a mannosyl residue from GDP-Man to phosphatidyl-myo-inositol on the cytoplasmic side of the plasma membrane. To prove the essential nature of pimA in M. tuberculosis, we constructed a pimA conditional mutant by using the TetR-Pip off system and showed that downregulation of PimA expression causes bactericidality in batch cultures. Consistent with the biochemical reaction catalyzed by PimA, this phenotype was associated with markedly reduced levels of phosphatidyl-myo-inositol dimannosides, essential structural components of the mycobacterial cell envelope. In addition, the requirement of PimA for viability was clearly demonstrated during macrophage infection and in two different mouse models of infection, where a dramatic decrease in viable counts was observed upon silencing of the gene. Notably, depletion of PimA resulted in complete clearance of the mouse lungs during both the acute and chronic phases of infection. Altogether, the experimental data highlight the importance of the phosphatidyl-myo-inositol mannoside biosynthetic pathway for M. tuberculosis and confirm that PimA is a novel target for future drug discovery programs.

An ethA-ethR-deficient Mycobacterium bovis BCG mutant displays increased adherence to mammalian cells and greater persistence in vivo, which correlate with altered mycolic acid composition.

Tuberculosis remains a major worldwide epidemic because of its sole etiological agent, Mycobacterium tuberculosis. Ethionamide (ETH) is one of the major antitubercular drugs used to treat infections with multidrug-resistant M. tuberculosis strains. ETH is a prodrug that requires activation within the mycobacterial cell; its bioactivation involves the ethA-ethR locus, which encodes the monooxygenase EthA, while EthR is a transcriptional regulator that binds to the intergenic promoter region of the ethA-ethR locus. While most studies have focused on the role of EthA-EthR in ETH bioactivation, its physiological role in mycobacteria has remained elusive, although a role in bacterial cell detoxification has been proposed. Moreover, the importance of EthA-EthR in vivo has never been reported on. Here we constructed and characterized an EthA-EthR-deficient mutant of Mycobacterium bovis BCG. Our results indicate that absence of the ethA-ethR locus led to greater persistence of M. bovis BCG in the mouse model of mycobacterial infection, which correlated with greater adherence to mammalian cells. Furthermore, analysis of cell wall lipid composition by thin-layer chromatography and mass spectrometry revealed differences between the ethA-ethR KO mutant and the parental strain in the relative amounts of α- and keto-mycolates. Therefore, we propose here that M. bovis BCG ethA-ethR is involved in the cell wall-bound mycolate profile, which impacts mycobacterial adherence properties and in vivo persistence. This study thus provides some experimental clues to the possible physiological role of ethA-ethR and proposes that this locus is a novel factor involved in the modulation of mycobacterial virulence.

Inhibition of mycolic acid transport across the Mycobacterium tuberculosis plasma membrane.

New chemotherapeutics active against multidrug-resistant Mycobacterium tuberculosis are urgently needed. We report on the identification of an adamantyl urea compound that shows potent bactericidal activity against M. tuberculosis and a unique mode of action, namely the abolition of the translocation of mycolic acids from the cytoplasm, where they are synthesized to the periplasmic side of the plasma membrane and are in turn transferred onto cell wall arabinogalactan or used in the formation of virulence-associated, outer membrane, trehalose-containing glycolipids. Whole-genome sequencing of spontaneous-resistant mutants of M. tuberculosis selected in vitro followed by genetic validation experiments revealed that our prototype inhibitor targets the inner membrane transporter MmpL3. Conditional gene expression of mmpL3 in mycobacteria and analysis of inhibitor-treated cells validate MmpL3 as essential for mycobacterial growth and support the involvement of this transporter in the translocation of trehalose monomycolate across the plasma membrane.

Purification and characterization of the acyltransferase involved in biosynthesis of the major mycobacterial cell envelope glycolipid--monoacylated phosphatidylinositol dimannoside.

Phosphatidylinositol mannosides are essential structural components of the mycobacterial cell envelope. They are implicated in host-pathogen interactions during infection and serve as a basis for biosynthesis of other unique molecules with immunomodulatory properties - mycobacterial lipopolysaccharides lipoarabinomannan and lipomannan. Acyltransferase Rv2611 is involved in one of the initial steps in the assembly of these molecules in Mycobacterium tuberculosis - the attachment of an acyl group to position-6 of the 2-linked mannosyl residue of the phosphatidylinositol mannoside anchor. Although the function of this enzyme was annotated 10 years ago, it has never been completely biochemically characterized due to lack of the pure protein. We have successfully overexpressed and purified MSMEG_2934, the ortholog of Rv2611c from the non-pathogenic model organism Mycobacteriumsmegmatis mc(2)155 using mycobacterial pJAM2 expression system, which allowed confirmation of its in vitro acyltransferase activity, and establishment of its substrate specificity.

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.

Dynamic microfluidic single-cell screening identifies pheno-tuning compounds to potentiate tuberculosis therapy.

Drug-recalcitrant infections are a leading global-health concern. Bacterial cells benefit from phenotypic variation, which can suggest effective antimicrobial strategies. However, probing phenotypic variation entails spatiotemporal analysis of individual cells that is technically challenging, and hard to integrate into drug discovery. In this work, we develop a multi-condition microfluidic platform suitable for imaging two-dimensional growth of bacterial cells during transitions between separate environmental conditions. With this platform, we implement a dynamic single-cell screening for pheno-tuning compounds, which induce a phenotypic change and decrease cell-to-cell variation, aiming to undermine the entire bacterial population and make it more vulnerable to other drugs. We apply this strategy to mycobacteria, as tuberculosis poses a major public-health threat. Our lead compound impairs Mycobacterium tuberculosis via a peculiar mode of action and enhances other anti-tubercular drugs. This work proves that harnessing phenotypic variation represents a successful approach to tackle pathogens that are increasingly difficult to treat.

A small multidrug resistance-like transporter involved in the arabinosylation of arabinogalactan and lipoarabinomannan in mycobacteria.

The biosynthesis of the major cell envelope glycoconjugates of Mycobacterium tuberculosis is topologically split across the plasma membrane, yet nothing is known of the transporters required for the translocation of lipid-linked sugar donors and oligosaccharide intermediates from the cytoplasmic to the periplasmic side of the membrane in mycobacteria. One of the mechanisms used by prokaryotes to translocate lipid-linked phosphate sugars across the plasma membrane relies on translocases that share resemblance with small multidrug resistance transporters. The presence of an small multidrug resistance-like gene, Rv3789, located immediately upstream from dprE1/dprE2 responsible for the formation of decaprenyl-monophosphoryl-ß-D-arabinose (DPA) in the genome of M. tuberculosis led us to investigate its potential involvement in the formation of the major arabinosylated glycopolymers, lipoarabinomannan (LAM) and arabinogalactan (AG). Disruption of the ortholog of Rv3789 in Mycobacterium smegmatis resulted in a reduction of the arabinose content of both AG and LAM that accompanied the accumulation of DPA in the mutant cells. Interestingly, AG and LAM synthesis was restored in the mutant not only upon expression of Rv3789 but also upon that of the undecaprenyl phosphate aminoarabinose flippase arnE/F genes from Escherichia coli. A bacterial two-hybrid system further indicated that Rv3789 interacts in vivo with the galactosyltransferase that initiates the elongation of the galactan domain of AG. Biochemical and genetic evidence is thus consistent with Rv3789 belonging to an AG biosynthetic complex, where its role is to reorient DPA to the periplasm, allowing this arabinose donor to then be used in the buildup of the arabinan domains of AG and LAM.

A common mechanism of inhibition of the Mycobacterium tuberculosis mycolic acid biosynthetic pathway by isoxyl and thiacetazone.

Isoxyl (ISO) and thiacetazone (TAC), two prodrugs once used in the clinical treatment of tuberculosis, have long been thought to abolish Mycobacterium tuberculosis (M. tuberculosis) growth through the inhibition of mycolic acid biosynthesis, but their respective targets in this pathway have remained elusive. Here we show that treating M. tuberculosis with ISO or TAC results in both cases in the accumulation of 3-hydroxy C(18), C(20), and C(22) fatty acids, suggestive of an inhibition of the dehydratase step of the fatty-acid synthase type II elongation cycle. Consistently, overexpression of the essential hadABC genes encoding the (3R)-hydroxyacyl-acyl carrier protein dehydratases resulted in more than a 16- and 80-fold increase in the resistance of M. tuberculosis to ISO and TAC, respectively. A missense mutation in the hadA gene of spontaneous ISO- and TAC-resistant mutants was sufficient to confer upon M. tuberculosis high level resistance to both drugs. Other mutations found in hypersusceptible or resistant M. tuberculosis and Mycobacterium kansasii isolates mapped to hadC. Mutations affecting the non-essential mycolic acid methyltransferases MmaA4 and MmaA2 were also found in M. tuberculosis spontaneous ISO- and TAC-resistant mutants. That MmaA4, at least, participates in the activation of the two prodrugs as proposed earlier is not supported by our biochemical evidence. Instead and in light of the known interactions of both MmaA4 and MmaA2 with HadAB and HadBC, we propose that mutations affecting these enzymes may impact the binding of ISO and TAC to the dehydratases.

Novel pyrimidine-1,3,4-oxadiazole hybrids and their precursors as potential antimycobacterial agents.

Background: Molecular hybridization and isostery are proven approaches in medicinal chemistry, and as such we used them to design novel compounds that we investigated as potential antimycobacterials to combat drug-resistant strains. Methods & results: Prepared N-alkyl-2-(pyrimidine-5-carbonyl)hydrazine-1-carboxamides were cyclized to N-alkyl-5-(pyrimidin-5-yl)-1,3,4-oxadiazol-2-amines along with their analogues. A total of 48 compounds were tested against Mycobacterium tuberculosis H37Rv, Mycobacterium avium and Mycobacterium kansasii, with oxadiazoles and C8-C12 alkyls being the most effective from a concentration of 2 µM. Multidrug-resistant strains were inhibited at same concentrations as the susceptible strain. For the most potent N-dodecyl-5-(pyrimidin-5-yl)-1,3,4-oxadiazol-2-amine, the mechanism of action related to cell wall biosynthesis was investigated. Conclusion: Pyrimidine-1,3,4-oxadiazole hybrids are unique antimycobacterial agents inhibiting mainly M. tuberculosis strains without cross-resistance to current drugs and are thus promising drug candidates.

Antimycobacterial pyridine carboxamides: From design to in vivo activity.

Tuberculosis is the number one killer of infectious diseases caused by a single microbe, namely Mycobacterium tuberculosis (Mtb). The success rate of curing this infection is decreasing due to emerging antimicrobial resistance. Therefore, novel treatments are urgently needed. As an attempt to develop new antituberculars effective against both drugs-sensitive and drug-resistant Mtb, we report the synthesis of a novel series inspired by combining fragments from the first-line agents isoniazid and pyrazinamide (series I) and isoniazid with the second-line agent 4-aminosalicylic acid (series II). We identified compound 10c from series II with selective, potent in vitro antimycobacterial activity against both drug-sensitive and drug-resistant Mtb H37Rv strains with no in vitro or in vivo cytotoxicity. In the murine model of tuberculosis, compound 10c caused a statistically significant decrease in colony-forming units (CFU) in spleen. Despite having a 4-aminosalicylic acid fragment in its structure, biochemical studies showed that compound 10c does not directly affect the folate pathway but rather methionine metabolism. In silico simulations indicated the possibility of binding to mycobacterial methionine-tRNA synthetase. Metabolic study in human liver microsomes revealed that compound 10c does not have any known toxic metabolites and has a half-life of 630 min, overcoming the main drawbacks of isoniazid (toxic metabolites) and 4-aminosalicylic acid (short half-life).

Hybridization Approach Toward Novel Antituberculars: Design, Synthesis, and Biological Evaluation of Compounds Combining Pyrazinamide and 4-Aminosalicylic Acid.

Apart from the SARS-CoV-2 virus, tuberculosis remains the leading cause of death from a single infectious agent according to the World Health Organization. As part of our long-term research, we prepared a series of hybrid compounds combining pyrazinamide, a first-line antitubercular agent, and 4-aminosalicylic acid (PAS), a second-line agent. Compound 11 was found to be the most potent, with a broad spectrum of antimycobacterial activity and selectivity toward mycobacterial strains over other pathogens. It also retained its in vitro activity against multiple-drug-resistant mycobacterial strains. Several structural modifications were attempted to improve the in vitro antimycobacterial activity. The δ-lactone form of compound 11 (11') had more potent in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv. Compound 11 was advanced for in vivo studies, where it was proved to be nontoxic in Galleria mellonella and zebrafish models, and it reduced the number of colony-forming units in spleens in the murine model of tuberculosis. Biochemical studies showed that compound 11 targets mycobacterial dihydrofolate reductases (DHFR). An in silico docking study combined with molecular dynamics identified a viable binding mode of compound 11 in mycobacterial DHFR. The lactone 11' opens in human plasma to its parent compound 11 (t1/2 = 21.4 min). Compound 11 was metabolized by human liver fraction by slow hydrolysis of the amidic bond (t1/2 = 187 min) to yield PAS and its starting 6-chloropyrazinoic acid. The long t1/2 of compound 11 overcomes the main drawback of PAS (short t1/2 necessitating frequent administration of high doses of PAS).

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.

Discovery of new diaryl ether inhibitors against Mycobacterium tuberculosis targeting the minor portal of InhA.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) affects 10 million people each year and the emergence of resistant TB augurs for a growing incidence. In the last 60 years, only three new drugs were approved for TB treatment, for which resistances are already emerging. Therefore, there is a crucial need for new chemotherapeutic agents capable of eradicating TB. Enzymes belonging to the type II fatty acid synthase system (FAS-II) are involved in the biosynthesis of mycolic acids, cell envelope components essential for mycobacterial survival. Among them, InhA is the primary target of isoniazid (INH), one of the most effective compounds to treat TB. INH acts as a prodrug requiring activation by the catalase-peroxidase KatG, whose mutations are the major cause for INH resistance. Herein, a new series of direct InhA inhibitors were designed based on a molecular hybridization approach. They exhibit potent inhibitory activities of InhA and, for some of them, good antitubercular activities. Moreover, they display a low toxicity on human cells. A study of the mechanism of action of the most effective molecules shows that they inhibit the biosynthesis of mycolic acids. The X-ray structures of two InhA/NAD+/inhibitor complexes have been obtained showing a binding mode of a part of the molecule in the minor portal, rarely seen in the InhA structures reported so far.