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Tuberculosis, caused by Mycobacterium tuberculosis, claims ∼1.5 million lives annually. Effective chemotherapy is essential to control TB, however the emergence of drug-resistant strains of TB have seriously threatened global attempts to control and eradicate this deadly pathogen. Trehalose recycling viathe LpqY-SugABC importer is essential for the virulence and survival of Mtband inhibiting or hijacking this transport system is an attractive approach for the development of novel anti-tubercular and diagnostic agents. Therefore, we interrogated the drug-like compounds in the open-source Medicines for Malaria Pathogen Box and successfully identified seven compounds from the TB, kinetoplastids and reference compound disease sets that recognise LpqY. The molecules have diverse chemical scaffolds, are not specific trehalose analogues, and may be used as novel templates to facilitate the development of therapeutics that kill Mtbwith a novel mechanism of action viathe mycobacterial trehalose LpqY-SugABC transport system.

Securing novel, safe, and effective medicines to treat Mycobacterium tuberculosisremains an elusive goal, particularly influenced by the largely impervious Mtbenvelope that limits exposure and thus efficacy of inhibitors at their cellular and periplasmic targets. The impact of physicochemical properties on pharmacokinetic parameters that govern oral absorption and exposure at sites of infection is considered alongside how these properties influence penetration of the Mtbenvelope, with the likely influence of transporter proteins. The findings are discussed to benchmark current drugs and the emerging pipeline, whilst considering tactics for future rational and targeted design strategies, based around emerging data on Mtbtransporters and their structures and functions.

Mycobacterium tuberculosis(Mtb) is the causative agent of tuberculosis (TB) and has evolved an incredible ability to survive latently within the human host for decades. The Mtbpathogen encodes for a low number of ATP-binding cassette (ABC) importers for the acquisition of carbohydrates that may reflect the nutrient poor environment within the host macrophages. MtbUgpB (Rv2833c) is the substrate binding domain of the UgpABCE transporter that recognizes glycerophosphocholine (GPC), indicating that this transporter has a role in recycling glycerophospholipid metabolites. By using a combination of saturation transfer difference (STD) NMR and X-ray crystallography, we report the structural analysis of MtbUgpB complexed with GPC and have identified that MtbUgpB not only recognizes GPC but is also promiscuous for a broad range of glycerophosphodiesters. Complementary biochemical analyses and site-directed mutagenesis precisely define the molecular basis and specificity of glycerophosphodiester recognition. Our results provide critical insights into the structural and functional role of the MtbUgpB transporter and reveal that the specificity of this ABC-transporter is not limited to GPC, therefore optimizing the ability of Mtbto scavenge scarce nutrients and essential glycerophospholipid metabolites via a single transporter during intracellular infection.

Because of the emergence of antimicrobial resistance to traditional small-molecule drugs, cationic antimicrobial polymers are appealing targets. Mycobacterium tuberculosisis a particular problem, with multi- and total drug resistance spreading and more than a billion latent infections globally. This study reports nanoparticles bearing variable densities of poly(dimethylaminoethyl methacrylate) and the unexpected and distinct mechanisms of action this multivalent presentation imparts against Escherichia coliversus Mycobacterium smegmatis(model of M. tuberculosis), leading to killing or growth inhibition, respectively. A convergent “grafting to” synthetic strategy was used to assemble a 50-member nanoparticle library, and using a high-throughput screen identified that only the smallest (2 nm) particles were stable in both saline and complex cell media. Compared with the linear polymers, the nanoparticles displayed two- and eight-fold enhancements in antimicrobial activity against M. smegmatisand E. coli, respectively. Mechanistic studies demonstrated that the antimicrobial particles were bactericidal against E. colidue to rapid disruption of the cell membranes. Conversely, against M. smegmatisthe particles did not lyse the cell membrane but rather had a bacteriostatic effect. These results demonstrate that to develop new polymeric antituberculars the widely assumed, broad spectrum, membrane-disrupting mechanism of polycations must be re-evaluated. It is clear that synthetic nanomaterials can engage in more complex interactions with mycobacteria, which we hypothesize is due to the unique cell envelope at the surface of these bacteria.

Treatment of latent tuberculosis infection remains an important goal of global TB eradication. To this end, targets that are essential for intracellular survival of Mycobacterium tuberculosisare particularly attractive. Arylamine N-acetyltransferase (NAT) represents such a target as it is, along with the enzymes encoded by the associated gene cluster, essential for mycobacterial survival inside macrophages and involved in cholesterol degradation. Cholesterol is likely to be the fuel for M. tuberculosisinside macrophages. Deleting the nat gene and inhibiting the NAT enzyme prevents survival of the microorganism in macrophages and induces cell wall alterations, rendering the mycobacterium sensitive to antibiotics to which it is normally resistant. To date, NAT from M. marinum(MMNAT) is considered the best available model for NAT from M. tuberculosis (TBNAT). The enzyme catalyses the acetylation and propionylation of arylamines and hydrazines. Hydralazine is a good acetyl and propionyl acceptor for both MMNAT and TBNAT. The MMNAT structure has been solved to 2.1 Å resolution following crystallisation in the presence of hydralazine and is compared to available NAT structures. From the mode of ligand binding, features of the binding pocket can be identified, which point to a novel mechanism for the acetylation reaction that results in a 3-methyltriazolo[3,4-a]phthalazine ring compound as product.

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 tuberculosispersisting 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 bovisBCG. The natgene and its associated gene cluster are almost identical in sequence in M. bovisBCG 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. bovisBCG and M. tuberculosis. The most potent triazole mimics the effects of deletion of the natgene 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.

Polymorphic Human arylamine N-acetyltransferase (NAT2) inactivates the anti-tubercular drug isoniazid by acetyltransfer from acetylCoA. There are active NAT proteins encoded by homologous genes in mycobacteria including M. tuberculosis, M. bovis BCG, M. smegmatis and M. marinum. Crystallographic structures of NATs from M. smegmatis and M. marinum, as native enzymes and with isoniazid bound share a similar fold with the first NAT structure, Salmonella typhimurium NAT. There are three approximately equal domains and an active site essential catalytic triad of cysteine, histidine and aspartate in the first two domains. An acetyl group from acetylCoA is transferred to cysteine and then to the acetyl acceptor e.g. isoniazid. M. marinum NAT binds CoA in a more open mode compared with CoA binding to human NAT2. The structure of mycobacterial NAT may promote its role in synthesis of cell wall lipids, identified through gene deletion studies. NAT protein is essential for survival of M. bovis BCG in macrophage as are the proteins encoded by other genes in the same gene cluster (hsaA-D). HsaA-D degrade cholesterol, essential for mycobacterial survival inside macrophage. Nat expression remains to be fully understood but is co-ordinated with hsaA-D and other stress response genes in mycobacteria. Amide synthase genes in the streptomyces are also nat homologues. The amide synthases are predicted to catalyse intramolecular amide bond formation and creation of cyclic molecules, e.g. geldanamycin. Lack of conservation of the CoA binding cleft residues of M. marinum NAT suggests the amide synthase reaction mechanism does not involve a soluble CoA intermediate during amide formation and ring closure.

Arylamine N-acetyltransferases (NATs), known as drug- and carcinogen-metabolising enzymes, have had historic roles in cellular metabolism, carcinogenesis and pharmacogenetics, including epidemiological studies of disease susceptibility. NAT research in the past 5 years builds on that history and additionally paves the way for establishing the following new concepts in biology and opportunities in drug discovery: i) NAT polymorphisms can be used as tools in molecular anthropology to study human evolution; ii) tracing NAT protein synthesis and degradation within cells is providing insight into protein folding in cell biology; iii) studies on control of NATgene expression may help to understand the increase in the human NAT isoenzyme, NAT1, in breast cancer; iv) a NAT homologue in mycobacteria plays an essential role in cell-wall synthesis and mycobacterial survival inside host macrophage, thus identifying a novel biochemical pathway; v) transgenic mice, with genetic modifications of all Natgenes, provide in vivotools for drug metabolism; and vi) structures of NAT isoenzymes provide essential in silicotools for drug discovery.

The crystal structure of the mycobacterial DprE1 reveals how the TB drug benzothiazinone BTZ043 blocks this microbial enzyme target.