Attenuation of Mycobacterium tuberculosis by disruption of a mas-like gene or a chalcone synthase-like gene,which causes deficiency in dimycocerosyl phthiocerol synthesis

Tuberculosis is one of the leading preventable causes of death. Emergence of drug-resistant tuberculosis makes the discovery of new targets for antimycobacterial drugs critical. The unique mycobacterial cell wall lipids are known to play an important role in pathogenesis, and therefore the genes responsible for their biosynthesis offer potential new targets. To assess the possible role of some of the genes potentially involved in cell wall lipid synthesis, we disrupted a mas-like gene, msl7, and a chalcone synthase-like gene, pks10, with phage-mediated delivery of the disruption construct, in which the target gene was disrupted by replacement of an internal segment with the hygromycin resistance gene (hyg). Gene disruption by allelic exchange in the case of each disruptant was confirmed by PCR and Southern blot analyses. Neither msl7 nor pks10 mutants could produce dimycocerosyl phthiocerol, although both could produce mycocerosic acids. Thus, it is concluded that these gene products are involved in the biosynthesis of phthiocerol. Both mutants were found to be attenuated in a murine model, supporting the hypothesis that dimycocerosyl phthiocerol is a virulence factor and thus the many steps involved in its biosynthesis offer potential novel targets for antimycobacterial therapy.     

New tuberculosis drugs on the horizon

Tuberculosis (TB) remains a major global health concern whose control has been exacerbated by HIV and the emergence of multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains of Mycobacterium tuberculosis. The demand for new and faster acting TB drugs is thus greater than ever. In the past decade intensive efforts have been made to discover new leads for TB drug development using both target-based and cell-based approaches. Here, we describe the most promising anti-tubercular drug candidates that are in clinical development and introduce some nitro-aromatic compounds that inhibit a new target, DprE1, an essential enzyme involved in a crucial step in mycobacterial cell wall biosynthesis.     

New classes of alanine racemase inhibitors identified by high-throughput screening show antimicrobial activity against Mycobacterium tuberculosis

Background

In an effort to discover new drugs to treat tuberculosis (TB) we chose alanine racemase as the target of our drug discovery efforts. In Mycobacterium tuberculosis, the causative agent of TB, alanine racemase plays an essential role in cell wall synthesis as it racemizes L-alanine into D-alanine, a key building block in the biosynthesis of peptidoglycan. Good antimicrobial effects have been achieved by inhibition of this enzyme with suicide substrates, but the clinical utility of this class of inhibitors is limited due to their lack of target specificity and toxicity. Therefore, inhibitors that are not substrate analogs and that act through different mechanisms of enzyme inhibition are necessary for therapeutic development for this drug target.

Methodology/Principal Findings

To obtain non-substrate alanine racemase inhibitors, we developed a high-throughput screening platform and screened 53,000 small molecule compounds for enzyme-specific inhibitors. We examined the ‘hits’ for structural novelty, antimicrobial activity against M. tuberculosis, general cellular cytotoxicity, and mechanism of enzyme inhibition. We identified seventeen novel non-substrate alanine racemase inhibitors that are structurally different than any currently known enzyme inhibitors. Seven of these are active against M. tuberculosis and minimally cytotoxic against mammalian cells.

Conclusions/Significance

This study highlights the feasibility of obtaining novel alanine racemase inhibitor lead compounds by high-throughput screening for development of new anti-TB agents.     

Comparative Genomics Analysis of Mycobacterium ulcerans for the Identification of Putative Essential Genes and Therapeutic Candidates

Mycobacterium ulcerans, the causative agent of Buruli ulcer, is the third most common mycobacterial disease after tuberculosis and leprosy. The present treatment options are limited and emergence of treatment resistant isolates represents a serious concern and a need for better therapeutics. Conventional drug discovery methods are time consuming and labor-intensive. Unfortunately, the slow growing nature of M. ulcerans in experimental conditions is also a barrier for drug discovery and development. In contrast, recent advancements in complete genome sequencing, in combination with cheminformatics and computational biology, represent an attractive alternative approach for the identification of therapeutic candidates worthy of experimental research. A computational, comparative genomics workflow was defined for the identification of novel therapeutic candidates against M. ulcerans, with the aim that a selected target should be essential to the pathogen, and have no homology in the human host. Initially, a total of 424 genes were predicted as essential from the M. ulcerans genome, via homology searching of essential genome content from 20 different bacteria. Metabolic pathway analysis showed that the most essential genes are associated with carbohydrate and amino acid metabolism. Among these, 236 proteins were identified as non-host and essential, and could serve as potential drug and vaccine candidates. Several drug target prioritization parameters including druggability were also calculated. Enzymes from several pathways are discussed as potential drug targets, including those from cell wall synthesis, thiamine biosynthesis, protein biosynthesis, and histidine biosynthesis. It is expected that our data will facilitate selection of M. ulcerans proteins for successful entry into drug design pipelines.     

Synthesis of mannopyranose disaccharides as photoaffinity probes for mannosyltransferases in Mycobacterium tuberculosis

Mannosyltransferases play a crucial role in mycobacterial cell-wall biosynthesis and are potential new drug targets for the treatment of tuberculosis. Herein, we describe the synthesis of alpha-(1-->2)- and alpha-(1-->6)-linked mannopyranosyl disaccharides possessing a 5-azidonaphthlene-1-sulfonamidoethyl group as photoaffinity probes for active-site labeling studies of mannosyltransferases in Mycobacterium tuberculosis.     

Identification of arylamine N -acetyltransferase inhibitors as an approach towards novel anti-tuberculars

New anti-tubercular drugs and drug targets are urgently needed to reduce the time for treatment and also to identify agents that will be effective against Mycobacterium tuberculosis persisting intracellularly. Mycobacteria have a unique cell wall. Deletion of the gene for arylamine N-acetyltransferase (NAT) decreases mycobacterial cell wall lipids, particularly the distinctive mycolates, and also increases antibiotic susceptibility and killing within macrophage of Mycobacterium bovis BCG. The nat gene and its associated gene cluster are almost identical in sequence in M. bovis BCG and M. tuberculosis. The gene cluster is essential for intracellular survival of mycobacteria. We have therefore used pure NAT protein for high-throughput screening to identify several classes of small molecules that inhibit NAT activity. Here, we characterize one class of such molecules— triazoles—in relation to its effects on the target enzyme and on both M. bovis BCG and M. tuberculosis. The most potent triazole mimics the effects of deletion of the nat gene on growth, lipid disruption and intracellular survival. We also present the structure-activity relationship between NAT inhibition and effects on mycobacterial growth, and use ligand-protein analysis to give further insight into the structure-activity relationships. We conclude that screening a chemical library with NAT protein yields compounds that have high potential as anti-tubercular agents and that the inhibitors will allow further exploration of the biochemical pathway in which NAT is involved.     

Novel targets for the future development of antibacterial agents

Recent advances in DNA sequencing technology have made it possible to elucidate the entire genomes of pathogenic bacteria, and advancements in bioinformatic tools have driven comparative studies of these genome sequences. These evaluations are dramatically increasing our ability to make valid considerations of the limitations and advantages of particular targets based on their predicted spectrum and selectivity. In addition, developments in gene knockout technologies amenable to pathogenic organisms have enabled new genes and gene products critical to bacterial growth and pathogenicity to be uncovered at an unprecedented rate. Specific target examples in the areas of cell wall biosynthesis, aromatic amino acid biosynthesis, cell division, two component signal transduction, fatty acid biosynthesis, isopreniod biosynthesis and tRNA synthetases illustrate how aspects of the above capabilities are impacting on the discovery and characterization of novel antibacterial targets. An example of a novel inhibitor of bacterial fatty acid biosynthesis discovered from high throughput screening processes is described, along with its subsequent chemical optimization. Furthermore, the application and importance of technologies for tracking the mode of antibacterial action of these novel inhibitors is discussed.     

An ultraefficient affinity-based high-throughout screening process: application to bacterial cell wall biosynthesis enzyme MurF

The authors describe the discovery of a new class of inhibitors to an essential Streptococcus pneumoniae cell wall biosyn-thesis enzyme, MurF, by a novel affinity screening method. The strategy involved screening very large mixtures of diverse small organic molecules against the protein target on the basis of equilibrium binding, followed by iterative ultrafiltration steps and ligand identification by mass spectrometry. Hits from any affinity-based screening method often can be relatively nonselective ligands, sometimes referred to as "nuisance" or "promiscuous" compounds. Ligands selective in their binding affinity for the MurF target were readily identified through electronic subtraction of an empirically determined subset of promiscuous compounds in the library without subsequent selectivity panels. The complete strategy for discovery and identification of novel specific ligands can be applied to all soluble protein targets and a wide variety of ligand libraries.     

The Mycobacterium tuberculosis FAS-II condensing enzymes: their role in mycolic acid biosynthesis, acid-fastness, pathogenesis and in future drug development

Mycolic acids are very long-chain fatty acids representing essential components of the mycobacterial cell wall. Considering their importance, characterization of key enzymes participating in mycolic acid biosynthesis not only allows an understanding of their role in the physiology of mycobacteria, but also might lead to the identification of new drug targets. Mycolates are synthesized by at least two discrete elongation systems, the type I and type II fatty acid synthases (FAS-I and FAS-II respectively). Among the FAS-II components, the condensing enzymes that catalyse the formation of carbon-carbon bonds have received considerable interest. Four condensases participate in initiation (mtFabH), elongation (KasA and KasB) and termination (Pks13) steps, leading to full-length mycolates. We present the recent biochemical and structural data for these important enzymes. Special emphasis is given to their role in growth, intracellular survival, biofilm formation, as well as in the physiopathology of tuberculosis. Recent studies demonstrated that phosphorylation of these enzymes by mycobacterial kinases affects their activities. We propose here a model in which kinases that sense environmental changes can phosphorylate the condensing enzymes, thus representing a novel mechanism of regulating mycolic acid biosynthesis. Finally, we discuss the attractiveness of these enzymes as valid targets for future antituberculosis drug development.     

Identification of a novel arabinofuranosyltransferase (AftA) involved in cell wall arabinan biosynthesis in Mycobacterium tuberculosis

The cell wall mycolyl-arabinogalactan-peptidoglycan complex is essential in mycobacterial species, such as Mycobacterium tuberculosis, and is the target of several anti-tubercular drugs. For instance, ethambutol targets arabinogalactan biosynthesis through inhibition of the arabinofuranosyltransferases Mt-EmbA and Mt-EmbB. Following a detailed bioinformatics analysis of genes surrounding the conserved emb locus, we present the identification and characterization of a novel arabinofuranosyltransferase AftA (Rv3792). The enzyme catalyzes the addition of the first key arabinofuranosyl residue from the sugar donor beta-D-arabinofuranosyl-1-monophosphoryldecaprenol to the galactan domain of the cell wall, thus "priming" the galactan for further elaboration by the arabinofuranosyltransferases. Because aftA is an essential gene in M. tuberculosis, we deleted its orthologue in Corynebacterium glutamicum to produce a slow growing but viable mutant. Analysis of its cell wall revealed the complete absence of arabinose resulting in a truncated cell wall structure possessing only a galactan core with a concomitant loss of cell wall-bound mycolates. Complementation of the mutant was fully restored to the wild type phenotype by Cg-aftA. In addition, by developing an in vitro assay using recombinant Escherichia coli expressing Mt-aftA and use of cell wall galactan as an acceptor, we demonstrated the transfer of arabinose from beta-D-arabinofuranosyl-1-monophosphoryldecaprenol to galactan, and unlike the Mt-Emb proteins, Mt-AftA was not inhibited by ethambutol. This newly discovered glycosyltransferase represents an attractive drug target for further exploitation by chemotherapeutic intervention.     

Novel inhibitors of Mycobacterium tuberculosis dTDP-6-deoxy-L-lyxo-4-hexulose reductase (RmlD) identified by virtual screening

The complex and highly impermeable cell wall of Mycobacterium tuberculosis (Mtb) is largely responsible for the ability of the mycobacterium to resist the action of chemical therapeutics. An L-rhamnosyl residue, which occupies an important anchoring position in the Mtb cell wall, is an attractive target for novel anti-tuberculosis drugs. In this work, we report a virtual screening (VS) study targeting Mtb dTDP-deoxy-L-lyxo-4-hexulose reductase (RmlD), the last enzyme in the L-rhamnosyl synthesis pathway. Through two rounds of VS, we have identified four RmlD inhibitors with half inhibitory concentrations of 0.9-25 μM, and whole-cell minimum inhibitory concentrations of 20-200 μg/ml. Compared with our previous high throughput screening targeting another enzyme involved in L-rhamnosyl synthesis, virtual screening produced higher hit rates, supporting the use of computational methods in future anti-tuberculosis drug discovery efforts.     

Comparative cell wall core biosynthesis in the mycolated pathogens, Mycobacterium tuberculosis and Corynebacterium diphtheriae

The recent determination of the complete genome sequence of Corynebacterium diphtheriae, the aetiological agent of diphtheria, has allowed a detailed comparison of its physiology with that of its closest sequenced pathogenic relative Mycobacterium tuberculosis. Of major importance to the pathogenicity and resilience of the latter is its particularly complex cell envelope. The corynebacteria share many of the features of this extraordinary structure although to a lesser level of complexity. The cell envelope of M. tuberculosis has provided the molecular targets for several of the major anti-tubercular drugs. Given a backdrop of emerging multi-drug resistant strains of the organism (MDR-TB) and its continuing global threat to human health, the search for novel anti-tubercular agents is of paramount importance. The unique structure of this cell wall and the importance of its integrity to the viability of the organism suggest that the search for novel drug targets within the array of enzymes responsible for its construction may prove fruitful. Although the application of modern bioinformatics techniques to the 'mining' of the M. tuberculosis genome has already increased our knowledge of the biosynthesis and assembly of the mycobacterial cell wall, several issues remain uncertain. Further analysis by comparison with its relatives may bring clarity and aid the early identification of novel cellular targets for new anti-tuberculosis drugs. In order to facilitate this aim, this review intends to illustrate the broad similarities and highlight the structural differences between the two bacterial envelopes and discuss the genetics of their biosynthesis.     

Interpreting Expression Data with Metabolic Flux Models: Predicting Mycobacterium tuberculosis Mycolic Acid Production

Metabolism is central to cell physiology, and metabolic disturbances play a role in numerous disease states. Despite its importance, the ability to study metabolism at a global scale using genomic technologies is limited. In principle, complete genome sequences describe the range of metabolic reactions that are possible for an organism, but cannot quantitatively describe the behaviour of these reactions. We present a novel method for modeling metabolic states using whole cell measurements of gene expression. Our method, which we call E-Flux (as a combination of flux and expression), extends the technique of Flux Balance Analysis by modeling maximum flux constraints as a function of measured gene expression. In contrast to previous methods for metabolically interpreting gene expression data, E-Flux utilizes a model of the underlying metabolic network to directly predict changes in metabolic flux capacity. We applied E-Flux to Mycobacterium tuberculosis, the bacterium that causes tuberculosis (TB). Key components of mycobacterial cell walls are mycolic acids which are targets for several first-line TB drugs. We used E-Flux to predict the impact of 75 different drugs, drug combinations, and nutrient conditions on mycolic acid biosynthesis capacity in M. tuberculosis, using a public compendium of over 400 expression arrays. We tested our method using a model of mycolic acid biosynthesis as well as on a genome-scale model of M. tuberculosis metabolism. Our method correctly predicts seven of the eight known fatty acid inhibitors in this compendium and makes accurate predictions regarding the specificity of these compounds for fatty acid biosynthesis. Our method also predicts a number of additional potential modulators of TB mycolic acid biosynthesis. E-Flux thus provides a promising new approach for algorithmically predicting metabolic state from gene expression data.     

Studies on alpha(1-->5) linked octyl arabinofuranosyl disaccharides for mycobacterial arabinosyl transferase activity

The appearance multi-drug resistant Mycobacterium tuberculosis (MTB) throughout the world has prompted a search for new, safer and more active agents against tuberculosis. Based on studies of the biosynthesis of mycobacterial cell wall polysaccharides, octyl 5-O-(alpha-D-arabinofuranosyl)-alpha-D-arabinofuranoside analogues were synthesized and evaluated as inhibitors for M. tuberculosis and Mycobacterium avium. A cell free assay system has been used for the evaluation of these disaccharides as substrates for mycobacterial arabinosyltransferase activity.     

A high-throughput, homogeneous, fluorescence resonance energy transfer-based assay for phospho-N-acetylmuramoyl-pentapeptide translocase (MraY)

Peptidoglycan biosynthesis is an essential process in bacteria and is therefore a suitable target for the discovery of new antibacterial drugs. One of the last cytoplasmic steps of peptidoglycan biosynthesis is catalyzed by the integral membrane protein MraY, which attaches soluble UDP-N-acetylmuramoyl-pentapeptide to the membrane-bound acceptor undecaprenyl phosphate. Although several natural product-derived inhibitors of MraY are known, none have the properties necessary to be of clinical use as antibacterial drugs. Here we describe a novel, homogeneous, fluorescence resonance energy transfer-based MraY assay that is suitable for high-throughput screening for novel MraY inhibitors. The assay allows for continuous measurement, or it can be quenched prior to measurement.     

Development of a coupled spectrophotometric assay for GlfT2, a bifunctional mycobacterial galactofuranosyltransferase

As a key constituent of their protective cell wall all mycobacteria produce a large structural component, the mycolyl-arabinogalactan (mAG) complex, which has at its core a galactan moiety of alternating beta-(1-->5) and beta-(1-->6) galactofuranosyl residues. Galactan biosynthesis is essential for mycobacterial viability and thus inhibitors of the enzymes involved in its assembly are potential drugs for the treatment of mycobacterial diseases, including tuberculosis. Only two galactofuranosyltransferases, GlfT1 and GlfT2, are responsible for the biosynthesis of the entire galactan domain of the mAG and we report here the first high-throughput assay for GlfT2. Successful implementation of the assay required the synthesis of multi-milligram amounts of the donor for the enzyme, UDP-Galf, 1, which was achieved using a chemoenzymatic approach. We also describe an improved expression system for GlfT2, which provides a larger amount of active protein for the assay. Kinetic analysis of 1 and a known trisaccharide acceptor for the enzyme, 2, have been carried out and the apparent K(m) and k(cat) values obtained for the latter are in agreement with those obtained using a previously reported radiochemical assay. The assay has been implemented in 384-well microtiter plates, which will facilitate the screening of large numbers of potential GlfT2 inhibitors, with possible utility as novel anti-TB drugs.     

Studies on (beta,1-->5) and (beta,1-->6) linked octyl Gal(f) disaccharides as substrates for mycobacterial galactosyltransferase activity.

The emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis (MTB) and the continuing pandemic of tuberculosis emphasizes the urgent need for the development of new anti-tubercular agents with novel drug targets. The recent structural elucidation of the mycobacterial cell wall highlights a large variety of structurally unique components that may be a basis for new drug development. This publication describes the synthesis, characterization, and screening of several octyl Galf(β,1→5)Galf and octyl Galf(β,1→6)Galf derivatives. A cell-free assay system has been utilized for galactosyltransferase activity using UDP[¹⁴C]Galf as the glycosyl donor, and in vitro inhibitory activity has been determined in a colorimetric broth microdilution assay system against MTB H37Ra and three clinical isolates of Mycobacterium avium complex (MAC). Certain derivatives showed moderate activities against MTB and MAC. The biological evaluation of these disaccharides suggests that more hydrophobic analogues with a blocked reducing end showed better activity as compared to totally deprotected disaccharides that more closely resemble the natural substrates in cell wall biosynthesis.     

Disaccharide analogs as probes for glycosyltransferases in Mycobacterium tuberculosis

Glycosyltransferases (GTs) play a crucial role in mycobacterial cell wall biosynthesis and are necessary for the survival of mycobacteria. Hence, these enzymes are potential new drug targets for the treatment of tuberculosis (TB), especially multiple drug-resistant TB (MDR-TB). Herein, we report the efficient syntheses of Araf(alpha 1-->5)Araf, Galf(beta 1-->5)Galf, and Galf(beta 1-->6)Galf disaccharides possessing a 5-N,N-dimethylaminonaphthalene-1-sulfonamidoethyl (dansyl) unit that were prepared as fluorescent disaccharide acceptors for arabinosyl- and galactosyl-transferases, respectively. Such analogs may offer advantages relative to radiolabeled acceptors or donors for studying the enzymes and for assay development and compound screening. Additionally, analogs possessing a 5-azidonaphthalene-1-sulfonamidoethyl unit were prepared as photoaffinity probes for their potential utility in studying active site labeling of the GTs (arabinosyl and galactosyl) in Mycobacterium tuberculosis (MTB). Beyond their preparation, initial biological testing and kinetic analysis of these disaccharides as acceptors toward glycosyltransferases are also presented.