New therapeutic strategies for Mycobacterium abscessus pulmonary diseases - untapping the mycolic acid pathway.

INTRODUCTION: Treatment options against Mycobacterium abscessus infections are very limited. New compounds are needed to cure M. abscessus pulmonary diseases. While the mycolic acid biosynthetic pathway has been largely exploited for the treatment of tuberculosis, this metabolic process has been overlooked in M. abscessus, although it offers many potential drug targets for the treatment of this opportunistic pathogen. AREAS COVERED: Herein, the authors review the role of the MmpL3 membrane protein and the enoyl-ACP reductase InhA involved in the transport and synthesis of mycolic acids, respectively. They discuss their importance as two major vulnerable drug targets in M. abscessus and report the activity of MmpL3 and InhA inhibitors. In particular, they focus on NITD-916, a direct InhA inhibitor against M. abscessus, particularly warranted in the context of multidrug resistance. EXPERT OPINION: There is an increasing body of evidence validating the mycolic acid pathway as an attractive drug target to be further exploited for M. abscessus lung disease treatments. The NITD-916 studies provide a proof-of-concept that direct inhibitors of InhA are efficient in vitro, in macrophages and in zebrafish. Future work is now required to improve the activity and pharmacological properties of these inhibitors and their evaluation in pre-clinical models.

Structure-Based Optimization of Coumestan Derivatives as Polyketide Synthase 13-Thioesterase(Pks13-TE) Inhibitors with Improved hERG Profiles for Mycobacterium tuberculosis Treatment.

Pks13 was identified as a key enzyme involved in the final step of mycolic acid biosynthesis. We previously identified antitubercular coumestans that targeted Pks13-TE, and these compounds exhibited high potency both in vitro and in vivo. However, lead compound 8 presented potential safety concerns because it inhibits the hERG potassium channel in electrophysiology patch-clamp assays (IC50 = 0.52 µM). By comparing the Pks13-TE-compound 8 complex and the ligand-binding pocket of the hERG ion channel, fluoro-substituted and oxazine-containing coumestans were designed and synthesized. Fluoro-substituted compound 23 and oxazine-containing coumestan 32 showed excellent antitubercular activity against both drug-susceptible and drug-resistant Mtb strains (MIC = 0.0039-0.0078 µg/mL) and exhibited limited hERG inhibition (IC50 ≥ 25 µM). Moreover, 32 exhibited improved metabolic stability relative to parent compound 8 while showing favorable bioavailability in mouse models via serum inhibition titration assays.

Structure-based drug design, synthesis and screening of MmaA1 inhibitors as novel anti-TB agents.

Tuberculosis is one of the leading causes of death across the world. The treatment regimens for tuberculosis are well established, but still the control of the disease faces many challenges such as lengthy treatment protocols, drug resistance and toxicity. In the present work, mycolic acid methyl transferase (MmaA1), a protein involved in the maturation of mycolic acids in the biochemical pathway of the Mycobacterium, was studied for novel drug discovery. The homology model of the MmaA1 protein was built and validated by using computational techniques. The MmaA1 protein has 286 amino acid residues consisting of 10 α-helices and 7 ß-sheets. The active site of the MmaA1 protein was identified using CASTp, SiteMap and PatchDock. Virtual screening studies were performed with two small molecule ligand databases: Asinex synergy and Diverse_Elite_Gold_Platinum databases having a total of 43,446 molecules and generated 1,30,814 conformers against the predicted and validated active site of the MmaA1 protein. Binding analysis showed that the residues ASP 19, PHE 22, TRP 30, TYR 32, TRP 74 and ALA 77 of MmaA1 protein have consistent interactions with the ligands. The hit ligands were further filtered by in silico ADME properties to eliminate potentially toxic molecules. Of the top 10 molecules, 3-(2-morpholinoacetamido)-N-(1,4-dihydro-4-oxoquinazolin-6-yl) benzamide was synthesised and screened for in vitro anti-TB activity against Mtb H37Rv using MABA assay. The compound and its intermediates exhibited good in vitro anti-TB activity which can be taken up for future lead optimisation studies. Structure based virtual screening study was performed using a validated homology model against small molecules from two virtual compound libraries. Synthesised the lead compound 3-(2-morpholinoacetamido)-N-(1,4-dihydro-4-oxoquinazolin-6-yl)benzamide obtained from virtual screening. In vitro activity against Mtb H37Rv has given a promising result.

Chemical Genetic Interaction Profiling Reveals Determinants of Intrinsic Antibiotic Resistance in Mycobacterium tuberculosis.

Chemotherapy for tuberculosis (TB) is lengthy and could benefit from synergistic adjuvant therapeutics that enhance current and novel drug regimens. To identify genetic determinants of intrinsic antibiotic susceptibility in Mycobacterium tuberculosis, we applied a chemical genetic interaction (CGI) profiling approach. We screened a saturated transposon mutant library and identified mutants that exhibit altered fitness in the presence of partially inhibitory concentrations of rifampin, ethambutol, isoniazid, vancomycin, and meropenem, antibiotics with diverse mechanisms of action. This screen identified the M. tuberculosis cell envelope to be a major determinant of antibiotic susceptibility but did not yield mutants whose increase in susceptibility was due to transposon insertions in genes encoding efflux pumps. Intrinsic antibiotic resistance determinants affecting resistance to multiple antibiotics included the peptidoglycan-arabinogalactan ligase Lcp1, the mycolic acid synthase MmaA4, the protein translocase SecA2, the mannosyltransferase PimE, the cell envelope-associated protease CaeA/Hip1, and FecB, a putative iron dicitrate-binding protein. Characterization of a deletion mutant confirmed FecB to be involved in the intrinsic resistance to every antibiotic analyzed. In contrast to its predicted function, FecB was dispensable for growth in low-iron medium and instead functioned as a critical mediator of envelope integrity.

Targeting Mycolic Acid Transport by Indole-2-carboxamides for the Treatment of Mycobacterium abscessus Infections.

Mycobacterium abscessus is a fast-growing, multidrug-resistant organism that has emerged as a clinically significant pathogen in cystic fibrosis (CF) patients. The intrinsic resistance of M. abscessus to most commonly available antibiotics seriously restricts chemotherapeutic options. Herein, we report the potent activity of a series of indolecarboxamides against M. abscessus. The lead compounds, 6 and 12, exhibited strong activity in vitro against a wide panel of M. abscessus isolates and in infected macrophages. High resistance levels to the indolecarboxamides appear to be associated with an A309P mutation in the mycolic acid transporter MmpL3. Biochemical analyses demonstrated that while de novo mycolic acid synthesis remained unaffected, the indolecarboxamides strongly inhibited the transport of trehalose monomycolate, resulting in the loss of trehalose dimycolate production and abrogating mycolylation of arabinogalactan. Our data introduce a hereto unexploited chemical structure class active against M. abscessus infections with promising translational development possibilities for the treatment of CF patients.

In silico structure-based drug screening of novel antimycobacterial pharmacophores by DOCK-GOLD tandem screening.

BACKGROUND: Enzymes responsible for cell wall development in Mycobacterium tuberculosis are considered as potential targets of anti-tuberculosis (TB) agents. Mycobacterial cyclopropane mycolic acid synthase 1 (CmaA1) is essential for mycobacterial survival because of its critical role in synthesizing mycolic acids. MATERIALS AND METHODS: We screened compounds that were capable of interacting with the mycobacterial CmaA1 active site using a virtual compound library with an in silico structure-based drug screening (SBDS). Following the selection of such compounds, their antimycobacterial activity was examined. RESULTS: With the in silico SBDS, for which we also used DOCK-GOLD programs and screening methods that utilized the structural similarity between the selected active compounds, we identified two compounds with potent inhibitory effects on mycobacterial growth. The antimycobacterial effect of the compounds was comparable to that of isoniazid, which is used as a first-line anti-TB drug. CONCLUSION: The compounds identified through SBDS were expected to be a novel class of anti-TB pharmacophores.

New approaches to target the mycolic acid biosynthesis pathway for the development of tuberculosis therapeutics.

Mycolic acids are the major lipid components of the unique mycobacterial cell wall responsible for the protection of the tuberculosis bacilli from many outside threats. Mycolic acids are synthesized in the cytoplasm and transported to the outer membrane as trehalose- containing glycolipids before being esterified to the arabinogalactan portion of the cell wall and outer membrane glycolipids. The large size of these unique fatty acids is a result of a huge metabolic investment that has been evolutionarily conserved, indicating the importance of these lipids to the mycobacterial cellular survival. There are many key enzymes involved in the mycolic acid biosynthetic pathway, including fatty acid synthesis (KasA, KasB, MabA, InhA, HadABC), mycolic acid modifying enzymes (SAM-dependent methyltransferases, aNAT), fatty acid activating and condensing enzymes (FadD32, Acc, Pks13), transporters (MmpL3) and tranferases (Antigen 85A-C) all of which are excellent potential drug targets. Not surprisingly, in recent years many new compounds have been reported to inhibit specific portions of this pathway, discovered through both phenotypic screening and target enzyme screening. In this review, we analyze the new and emerging inhibitors of this pathway discovered in the post-genomic era of tuberculosis drug discovery, several of which show great promise as selective tuberculosis therapeutics.

Current knowledge on mycolic acids in Corynebacterium glutamicum and their relevance for biotechnological processes.

Corynebacterium glutamicum is the world's largest producer of glutamate and lysine. Industrial glutamate overproduction is induced by empirical processes, such as biotin limitation, supplementation with specific surfactants or addition of sublethal concentration of certain antibiotics to the culture media. Although Gram-positive bacteria, C. glutamicum and related bacterial species and genera contain, in addition to the plasma membrane, an outer permeability membrane similar to that of Gram-negative microorganisms. As the amino acids have to cross both membranes, their integrity, composition and fluidity influence the export process. While the precise mechanism of the export of the amino acids by C. glutamicum is not fully understood, the excretion of amino acids through the inner membrane involved at least a major export system mechanosensitive channel MscS family (MscCG) encoded by NCgl1221. As the various industrial treatments have been shown to affect the lipid content of the bacterial cell, it is strongly believed that defects in the hallmark of the outer membrane, 2-alkyl, 3-hydroxylated long-chain fatty acids (mycolic acids), could be key factors in the glutamate overproduction. This review aims at giving an overview of the current knowledge on mycolic acids structure, biosynthesis and transfer in C. glutamicum and their relevance for amino acid biotechnological production.

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.

Exocarpic acid inhibits mycolic acid biosynthesis in Mycobacterium tuberculosis.

Exocarpic acid (13 E-octadecene-9,11-diynoic acid) from Exocarpos latifolius R.Br. (Santalaceae) was previously shown to have specific antimycobacterial activity. Microarray data suggested inhibition of fatty acid metabolism as a potential mode of action. Experiments designed to elucidate the mechanism of action showed that exocarpic acid was effective at inhibition of mycolic acid biosynthesis and did not act by dissipating the proton gradient in treated M. tuberculosis. Amide derivatives of exocarpic acid displayed similar properties to exocarpic acid, while other polyacetylenic fatty acids varied in their effects on mycolic acid biosynthesis.

Mycolyltransferase from Mycobacterium leprae excludes mycolate-containing glycolipid substrates.

Trehalose dimycolate (TDM) is a major surface-exposed mycolyl glycolipid that contributes to the hydrophobic cell wall architecture of mycobacteria. Nevertheless, because of its potent adjuvant functions, pathogenic mycobacteria appear to have evolved an evasive maneuver to down-regulate TDM expression within the host. We have shown previously that Mycobacterium tuberculosis (M.tb) and Mycobacterium avium (M.av), replace TDM with glucose monomycolate (GMM) by borrowing host-derived glucose as an alternative substrate for the FbpA mycolyltransferase. Mycobacterium leprae (M.le), the causative microorganism of human leprosy, is also known to down-regulate TDM expression in infected tissues, but the function of its mycolyltransferases has been poorly analysed. We found that, unlike M.tb and M.av FbpA enzymes, M.av FbpA was unexpectedly inefficient in transferring alpha-branched mycolates, resulting in impaired production of both TDM and GMM. Molecular modelling and mutational analysis indicated that a bulky side chain of leucine at position 130 of M.le FbpA obstructed the intramolecular tunnel that was proposed to accommodate the alpha-branch portion of the substrates. Notably, even after a highly reductive evolution, M.le FbpA remained functional in terms of transferring unbranched acyl chains, suggesting a role that is distinct from that as a mycolyltransferase.

Changes in composition and content of mycolic acids in glutamate-overproducing Corynebacterium glutamicum.

Overproduction of glutamate by Corynebacterium glutamicum is induced by biotin limitation or by the supplementation of specific detergents, sublethal amounts of penicillin, or cerulenin. But, it remains unclear why these different treatments, which have different sites of primary action, produce similar effects. In this study, it was found that the cellular content of mycolic acids--characteristic constituents of Corynebacterineae that are synthesized from fatty acids and form a cell surface layer--decreased under all conditions that induced glutamate overproduction. Furthermore, short mycolic acids increased under conditions of biotin limitation and cerulenin supplementation. These results suggest that different treatments produce the same effect that causes defects in the mycolic acid layer. This is perhaps one of the key factors in overproduction of glutamate by C. glutamicum.

Unique mechanism of action of the thiourea drug isoxyl on Mycobacterium tuberculosis.

The thiourea isoxyl (thiocarlide; 4,4'-diisoamyloxydiphenylthiourea) is known to be an effective anti-tuberculosis drug, active against a range of multidrug-resistant strains of Mycobacterium tuberculosis and has been used clinically. Little was known of its mode of action. We now demonstrate that isoxyl results in a dose-dependent decrease in the synthesis of oleic and, consequently, tuberculostearic acid in M. tuberculosis with complete inhibition at 3 microg/ml. Synthesis of mycolic acid was also affected. The anti-bacterial effect of isoxyl was partially reversed by supplementing growth medium with oleic acid. The specificity of this inhibition pointed to a Delta9-stearoyl desaturase as the drug target. Development of a cell-free assay for Delta9-desaturase activity allowed direct demonstration of the inhibition of oleic acid synthesis by isoxyl. Interestingly, sterculic acid, a known inhibitor of Delta9-desaturases, emulated the effect of isoxyl on oleic acid synthesis but did not affect mycolic acid synthesis, demonstrating the lack of a relationship between the two effects of the drug. The three putative fatty acid desaturases in the M. tuberculosis genome, desA1, desA2, and desA3, were cloned and expressed in Mycobacterium bovis BCG. Cell-free assays and whole cell labeling demonstrated increased Delta9-desaturase activity and oleic acid synthesis only in the desA3-overexpressing strain and an increase in the minimal inhibitory concentration for isoxyl, indicating that DesA3 is the target of the drug. These results validate membrane-bound Delta9-desaturase, DesA3, as a new therapeutic target, and the thioureas as anti-tuberculosis drugs worthy of further development.

Use of gas chromatographic fatty acid and mycolic acid cleavage product determination to differentiate among Mycobacterium genavense, Mycobacterium fortuitum, Mycobacterium simiae, and Mycobacterium tuberculosis.

Three Mycobacterium genavense strains and three American Type Culture Collection reference strains each of Mycobacterium fortuitum, Mycobacterium simiae, and Mycobacterium tuberculosis were subcultured onto Mycobacteria 7H11 agar (Difco Laboratories, Detroit, Mich.) supplemented with mycobactin J (Allied Laboratories, Fayette, Mo.). After 4 weeks of incubation at 37 degrees C in 10% CO2, the cultures were analyzed by gas-liquid chromatography (GLC) for their fatty acids and mycolic acid cleavage products. M. fortuitum was clearly differentiated from M. genavense by the presence of the specific marker 2-methyloctadecenoic acid in M. fortuitum and by the ratio of tetracosanoic acid to hexacosanoic acid. This ratio was <1 for M. genavense and >3 for M. fortuitum. M. fortuitum also contained docosanoic acid, which was not detected in M. genavense. M. genavense, M. simiae, and M. tuberculosis, which have similar GLC profiles, were also differentiated from each other by the presence of either cis-10-hexadecenoic acid or cis-11-hexadecenoic acid and by tetradecanoic acid content.

Direct identification of Mycobacterium species in Bactec 7H12B medium by gas-liquid chromatography.

Twenty-nine Mycobacterium reference strains representing 10 species and 60 mycobacterial cultures isolated from sputum specimens were studied. These cultures were grown in Bactec 7H12B medium (Becton Dickinson and Co., Paramus, N.J.) supplemented with oleic acid-albumin-dextrose-catalase enrichment broth (Becton Dickinson and Co., Cockeysville, Md.). The cultures were analyzed by gas-liquid chromatography for their fatty acids, secondary alcohols, and mycolic acid cleavage products. All of the clinical isolates could be identified by comparing their gas-liquid chromatography profiles with those of the reference strains. The data indicate that this method significantly shortens the turnaround time and could be used for the early detection and identification of mycobacterial species.