DprE2 is a molecular target of the anti-tubercular nitroimidazole compounds pretomanid and delamanid.

Mycobacterium tuberculosis is one of the global leading causes of death due to a single infectious agent. Pretomanid and delamanid are new antitubercular agents that have progressed through the drug discovery pipeline. These compounds are bicyclic nitroimidazoles that act as pro-drugs, requiring activation by a mycobacterial enzyme; however, the precise mechanisms of action of the active metabolite(s) are unclear. Here, we identify a molecular target of activated pretomanid and delamanid: the DprE2 subunit of decaprenylphosphoribose-2'-epimerase, an enzyme required for the synthesis of cell wall arabinogalactan. We also provide evidence for an NAD-adduct as the active metabolite of pretomanid. Our results highlight DprE2 as a potential antimycobacterial target and provide a foundation for future exploration into the active metabolites and clinical development of pretomanid and delamanid.

Assay development and inhibition of the Mt-DprE2 essential reductase from Mycobacterium tuberculosis.

DprE2 is an essential enzyme in the synthesis of decaprenylphosphoryl-ß-d-arabinofuranose (DPA) and subsequently arabinogalactan, and is a significant new drug target for M. tuberculosis. Two compounds from the GSK-177 box set, GSK301A and GSK032A, were identified through Mt-DprE2-target overexpression studies. The Mt-DprE1-DprE2 complex was co-purified and a new in vitro DprE2 assay developed, based on the oxidation of the reduced nicotinamide adenine dinucleotide cofactor of DprE2 (NADH/NADPH). The Mt-DprE1-DprE2 complex showed interesting kinetics in both the DprE1 resazurin-based assay, where Mt-DprE2 was found to enhance Mt-DprE1 activity and reduce substrate inhibition; and also in the DprE2 assay, which similarly exhibited substrate inhibition and a difference in kinetics of the two potential cofactors, NADH and NADPH. Although, no inhibition was observed in the DprE2 assay by the two GSK set compounds, spontaneous mutant generation indicated a possible explanation in the form of a pro-drug activation pathway, involving fgd1 and fbiC.

NSC19723, a Thiacetazone-Like Benzaldehyde Thiosemicarbazone Improves the Efficacy of TB Drugs In Vitro and In Vivo.

The complexity and duration of tuberculosis (TB) treatment contributes to the emergence of drug resistant tuberculosis (DR-TB) and drug-associated side effects. Alternate chemotherapeutic agents are needed to shorten the time and improve efficacy of current treatment. In this study, we have assessed the antitubercular activity of NSC19723, a benzaldehyde thiosemicarbazone molecule. NSC19723 is structurally similar to thiacetazone (TAC), a second-line anti-TB drug used to treat individuals with DR-TB. NSC19723 displayed better MIC values than TAC against Mycobacterium tuberculosis and Mycobacterium bovis BCG. In our checkerboard experiments, NSC19723 displayed better profiles than TAC in combination with known first-line and recently approved drugs. Mechanistic studies revealed that NSC19723 inhibits mycolic acid biosynthesis by targeting the HadABC complex. Computational studies revealed that the binding pocket of HadAB is similarly occupied by NSC19723 and TAC. NSC19723 also improved the efficacy of isoniazid in macrophages and mouse models of infection. Cumulatively, we have identified a benzaldehyde thiosemicarbazone scaffold that improved the activity of TB drugs in liquid cultures, macrophages, and mice. IMPORTANCE Mycobacterium tuberculosis, the causative agent of TB is among the leading causes of death among infectious diseases in humans. This situation has worsened due to the failure of BCG vaccines and the increased number of cases with HIV-TB coinfections and drug-resistant strains. Another challenge in the field is the lengthy duration of therapy for drug-sensitive and -resistant TB. Here, we have deciphered the mechanism of action of NSC19723, benzaldehyde thiosemicarbazone. We show that NSC19723 targets HadABC complex and inhibits mycolic acid biosynthesis. We also show that NSC19723 enhances the activity of known drugs in liquid cultures, macrophages, and mice. We have also performed molecular docking studies to identify the interacting residues of HadAB with NSC19723. Taken together, we demonstrate that NSC19723, a benzaldehyde thiosemicarbazone, has better antitubercular activity than thiacetazone.

GSK2556286 Is a Novel Antitubercular Drug Candidate Effective In Vivo with the Potential To Shorten Tuberculosis Treatment.

As a result of a high-throughput compound screening campaign using Mycobacterium tuberculosis-infected macrophages, a new drug candidate for the treatment of tuberculosis has been identified. GSK2556286 inhibits growth within human macrophages (50% inhibitory concentration [IC50] = 0.07 µM), is active against extracellular bacteria in cholesterol-containing culture medium, and exhibits no cross-resistance with known antitubercular drugs. In addition, it has shown efficacy in different mouse models of tuberculosis (TB) and has an adequate safety profile in two preclinical species. These features indicate a compound with a novel mode of action, although still not fully defined, that is effective against both multidrug-resistant (MDR) or extensively drug-resistant (XDR) and drug-sensitive (DS) M. tuberculosis with the potential to shorten the duration of treatment in novel combination drug regimens. (This study has been registered at ClinicalTrials.gov under identifier NCT04472897).

Synthesis and recycling of the mycobacterial cell envelope.

Mycobacterium tuberculosis (Mtb), the causative agent of the disease tuberculosis, is a recognised global health concern. The efficacy of the current treatment regime is under threat due to the emergence of antibiotic resistance, directing an urgent requirement for the discovery of new anti-tubercular agents and drug targets. The mycobacterial cell wall is a well-validated drug target for Mtb and is composed of three adaptive macromolecular structures, peptidoglycan, arabinogalactan and mycolic acids, an array of complex lipids and carbohydrates. The majority of the enzymes involved in cell wall synthesis have been established, whilst studies directed towards the mechanisms of remodelling and recycling have been neglected. This review briefly describes mycobacterial cell wall synthesis, and focuses on aspects of remodelling and recycling, thus highlighting opportunities for future research.

Two-Way Regulation of MmpL3 Expression Identifies and Validates Inhibitors of MmpL3 Function in Mycobacterium tuberculosis.

MmpL3, an essential mycolate transporter in the inner membrane of Mycobacterium tuberculosis (Mtb), has been identified as a target of multiple, chemically diverse antitubercular drugs. However, several of these molecules seem to have secondary targets and inhibit bacterial growth by more than one mechanism. Here, we describe a cell-based assay that utilizes two-way regulation of MmpL3 expression to readily identify MmpL3-specific inhibitors. We successfully used this assay to identify a novel guanidine-based MmpL3 inhibitor from a library of 220 compounds that inhibit growth of Mtb by largely unknown mechanisms. We furthermore identified inhibitors of cytochrome bc1-aa3 oxidase as one class of off-target hits in whole-cell screens for MmpL3 inhibitors and report a novel sulfanylacetamide as a potential QcrB inhibitor.

Anti-tubercular derivatives of rhein require activation by the monoglyceride lipase Rv0183.

The emergence and perseverance of drug resistant strains of Mycobacterium tuberculosis (Mtb) ensures that drug discovery efforts remain at the forefront of tuberculosis research. There are numerous different approaches that can be employed to lead to the discovery of anti-tubercular agents. In this work, we endeavored to optimize the anthraquinone chemical scaffold of a known drug, rhein, converting it from a compound with negligible activity against Mtb, to a series of compounds with potent activity. Two compounds exhibited low toxicity and good liver microsome stability and were further progressed in attempts to identify the biological target. Whole genome sequencing of resistant isolates revealed inactivating mutations in a monoglyceride lipase. Over-expression trials and an enzyme assay confirmed that the designed compounds are prodrugs, activated by the monoglyceride lipase. We propose that rhein is the active moiety of the novel compounds, which requires chemical modifications to enable access to the cell through the extensive cell wall structure. This work demonstrates that re-engineering of existing antimicrobial agents is a valid method in the development of new anti-tubercular compounds.

Mycobacterial drug discovery.

Mycobacterium tuberculosis is the causative pathogen of the pulmonary disease tuberculosis. Despite the availability of effective treatment programs, there is a global pursuit of new anti-tubercular agents to respond to the developing threat of drug resistance, in addition to reducing the extensive duration of chemotherapy and any associated toxicity. The route to mycobacterial drug discovery can be considered from two directions: target-to-drug and drug-to-target. The former approach uses conventional methods including biochemical assays along with innovative computational screens, but is yet to yield any drug candidates to the clinic, with a high attrition rate owing to lack of whole cell activity. In the latter approach, compound libraries are screened for efficacy against the bacilli or model organisms, ensuring whole cell activity, but here subsequent target identification is the rate-limiting step. Advances in a variety of scientific fields have enabled the amalgamation of aspects of both approaches in the development of novel drug discovery tools, which are now primed to accelerate the discovery of novel hits and leads with known targets and whole cell activity. This review discusses these traditional and innovative techniques, which are widely used in the quest for new anti-tubercular compounds.

Development of a whole-cell high-throughput phenotypic screen to identify inhibitors of mycobacterial amino acid biosynthesis.

Anti-tubercular drug discovery continues to be dominated by whole-cell high-throughput screening campaigns, enabling the rapid discovery of new inhibitory chemical scaffolds. Target-based screening is a popular approach to direct inhibitor discovery with a specified mode of action, eliminating the discovery of anti-tubercular agents against unsuitable targets. Herein, a screening method has been developed using Mycobacterium bovis BCG to identify inhibitors of amino acid biosynthesis. The methodology was initially optimized using the known branched-chain amino acid biosynthetic inhibitors metsulfuron-methyl (MSM) and sulfometuron-methyl (SMM), and subsequently, whole genome sequencing of resistant mutants and the use of over-expressor strains confirming their mode of action. The GlaxoSmithKline compound library of small molecule inhibitors with known activity against Mycobacterium tuberculosis was then used to validate the screen. In this paper, we have shown that media supplementation with amino acids can rescue M bovis BCG from known amino acid synthesis inhibitors, MSM and SMM, in a pathway specific manner. The therapeutic potential of amino acid biosynthesis inhibitors emphasizes the importance of this innovative screen, enabling the discovery of compounds targeting a multitude of related essential biochemical pathways, without limiting drug discovery toward a single target.

Mycobacterial cell wall biosynthesis: a multifaceted antibiotic target.

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), is recognized as a global health emergency as promoted by the World Health Organization. Over 1 million deaths per year, along with the emergence of multi- and extensively-drug resistant strains of Mtb, have triggered intensive research into the pathogenicity and biochemistry of this microorganism, guiding the development of anti-TB chemotherapeutic agents. The essential mycobacterial cell wall, sharing some common features with all bacteria, represents an apparent 'Achilles heel' that has been targeted by TB chemotherapy since the advent of TB treatment. This complex structure composed of three distinct layers, peptidoglycan, arabinogalactan and mycolic acids, is vital in supporting cell growth, virulence and providing a barrier to antibiotics. The fundamental nature of cell wall synthesis and assembly has rendered the mycobacterial cell wall as the most widely exploited target of anti-TB drugs. This review provides an overview of the biosynthesis of the prominent cell wall components, highlighting the inhibitory mechanisms of existing clinical drugs and illustrating the potential of other unexploited enzymes as future drug targets.

Inhibiting mycobacterial tryptophan synthase by targeting the inter-subunit interface.

Drug discovery efforts against the pathogen Mycobacterium tuberculosis (Mtb) have been advanced through phenotypic screens of extensive compound libraries. Such a screen revealed sulfolane 1 and indoline-5-sulfonamides 2 and 3 as potent inhibitors of mycobacterial growth. Optimization in the sulfolane series led to compound 4, which has proven activity in an in vivo murine model of Mtb infection. Here we identify the target and mode of inhibition of these compounds based on whole genome sequencing of spontaneous resistant mutants, which identified mutations locating to the essential α- and ß-subunits of tryptophan synthase. Over-expression studies confirmed tryptophan synthase as the biological target. Biochemical techniques probed the mechanism of inhibition, revealing the mutant enzyme complex incurs a fitness cost but does not prevent inhibitor binding. Mapping of the resistance conferring mutations onto a low-resolution crystal structure of Mtb tryptophan synthase showed they locate to the interface between the α- and ß-subunits. The discovery of anti-tubercular agents inhibiting tryptophan synthase highlights the therapeutic potential of this enzyme and draws attention to the prospect of other amino acid biosynthetic pathways as future Mtb drug targets.

Modular Synthesis of Diverse Natural Product-Like Macrocycles: Discovery of Hits with Antimycobacterial Activity.

A modular synthetic approach was developed in which variation of the triplets of building blocks used enabled systematic variation of the macrocyclic scaffolds prepared. The approach was demonstrated in the synthesis of 17 diverse natural product-like macrocyclic scaffolds of varied (12-20-membered) ring size. The biological relevance of the chemical space explored was demonstrated through the discovery of a series of macrocycles with significant antimycobacterial activity.

Novel inhibitors of Mycobacterium tuberculosis GuaB2 identified by a target based high-throughput phenotypic screen.

High-throughput phenotypic screens have re-emerged as screening tools in antibiotic discovery. The advent of such technologies has rapidly accelerated the identification of 'hit' compounds. A pre-requisite to medicinal chemistry optimisation programmes required to improve the drug-like properties of a 'hit' molecule is identification of its mode of action. Herein, we have combined phenotypic screening with a biased target-specific screen. The inosine monophosphate dehydrogenase (IMPDH) protein GuaB2 has been identified as a drugable target in Mycobacterium tuberculosis, however previously identified compounds lack the desired characteristics necessary for further development into lead-like molecules. This study has identified 7 new chemical series from a high-throughput resistance-based phenotypic screen using Mycobacterium bovis BCG over-expressing GuaB2. Hit compounds were identified in a single shot high-throughput screen, validated by dose response and subjected to further biochemical analysis. The compounds were also assessed using molecular docking experiments, providing a platform for their further optimisation using medicinal chemistry. This work demonstrates the versatility and potential of GuaB2 as an anti-tubercular drug target.

Identification of KasA as the cellular target of an anti-tubercular scaffold.

Phenotypic screens for bactericidal compounds are starting to yield promising hits against tuberculosis. In this regard, whole-genome sequencing of spontaneous resistant mutants generated against an indazole sulfonamide (GSK3011724A) identifies several specific single-nucleotide polymorphisms in the essential Mycobacterium tuberculosis ß-ketoacyl synthase (kas) A gene. Here, this genomic-based target assignment is confirmed by biochemical assays, chemical proteomics and structural resolution of a KasA-GSK3011724A complex by X-ray crystallography. Finally, M. tuberculosis GSK3011724A-resistant mutants increase the in vitro minimum inhibitory concentration and the in vivo 99% effective dose in mice, establishing in vitro and in vivo target engagement. Surprisingly, the lack of target engagement of the related ß-ketoacyl synthases (FabH and KasB) suggests a different mode of inhibition when compared with other Kas inhibitors of fatty acid biosynthesis in bacteria. These results clearly identify KasA as the biological target of GSK3011724A and validate this enzyme for further drug discovery efforts against tuberculosis.

THPP target assignment reveals EchA6 as an essential fatty acid shuttle in mycobacteria.

Phenotypic screens for bactericidal compounds against drug-resistant tuberculosis are beginning to yield novel inhibitors. However, reliable target identification remains challenging. Here, we show that tetrahydropyrazo[1,5-a]pyrimidine-3-carboxamide (THPP) selectively pulls down EchA6 in a stereospecific manner, instead of the previously assigned target Mycobacterium tuberculosis MmpL3. While homologous to mammalian enoyl-coenzyme A (CoA) hydratases, EchA6 is non-catalytic yet essential and binds long-chain acyl-CoAs. THPP inhibitors compete with CoA-binding, suppress mycolic acid synthesis, and are bactericidal in a mouse model of chronic tuberculosis infection. A point mutation, W133A, abrogated THPP-binding and increased both the in vitro minimum inhibitory concentration and the in vivo effective dose 99 in mice. Surprisingly, EchA6 interacts with selected enzymes of fatty acid synthase II (FAS-II) in bacterial two-hybrid assays, suggesting essentiality may be linked to feeding long-chain fatty acids to FAS-II. Finally, our data show that spontaneous resistance-conferring mutations can potentially obscure the actual target or alternative targets of small molecule inhibitors.

Mycobacterial dihydrofolate reductase inhibitors identified using chemogenomic methods and in vitro validation.

The lack of success in target-based screening approaches to the discovery of antibacterial agents has led to reemergence of phenotypic screening as a successful approach of identifying bioactive, antibacterial compounds. A challenge though with this route is then to identify the molecular target(s) and mechanism of action of the hits. This target identification, or deorphanization step, is often essential in further optimization and validation studies. Direct experimental identification of the molecular target of a screening hit is often complex, precisely because the properties and specificity of the hit are not yet optimized against that target, and so many false positives are often obtained. An alternative is to use computational, predictive, approaches to hypothesize a mechanism of action, which can then be validated in a more directed and efficient manner. Specifically here we present experimental validation of an in silico prediction from a large-scale screen performed against Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. The two potent anti-tubercular compounds studied in this case, belonging to the tetrahydro-1,3,5-triazin-2-amine (THT) family, were predicted and confirmed to be an inhibitor of dihydrofolate reductase (DHFR), a known essential Mtb gene, and already clinically validated as a drug target. Given the large number of similar screening data sets shared amongst the community, this in vitro validation of these target predictions gives weight to computational approaches to establish the mechanism of action (MoA) of novel screening hit.

Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target.

The human pathogen Mycobacterium tuberculosis is the causative agent of pulmonary tuberculosis (TB), a disease with high worldwide mortality rates. Current treatment programs are under significant threat from multi-drug and extensively-drug resistant strains of M. tuberculosis, and it is essential to identify new inhibitors and their targets. We generated spontaneous resistant mutants in Mycobacterium bovis BCG in the presence of 10× the minimum inhibitory concentration (MIC) of compound 1, a previously identified potent inhibitor of mycobacterial growth in culture. Whole genome sequencing of two resistant mutants revealed in one case a single nucleotide polymorphism in the gene aspS at (535)GAC>(535)AAC (D179N), while in the second mutant a single nucleotide polymorphism was identified upstream of the aspS promoter region. We probed whole cell target engagement by overexpressing either M. bovis BCG aspS or Mycobacterium smegmatis aspS, which resulted in a ten-fold and greater than ten-fold increase, respectively, of the MIC against compound 1. To analyse the impact of inhibitor 1 on M. tuberculosis AspS (Mt-AspS) activity we over-expressed, purified and characterised the kinetics of this enzyme using a robust tRNA-independent assay adapted to a high-throughput screening format. Finally, to aid hit-to-lead optimization, the crystal structure of apo M. smegmatis AspS was determined to a resolution of 2.4 Å.

In vitro reconstitution of peptidoglycan assembly from the Gram-positive pathogen Streptococcus pneumoniae.

Understanding the molecular basis of bacterial cell wall assembly is of paramount importance in addressing the threat of increasing antibiotic resistance worldwide. Streptococcus pneumoniae presents a particularly acute problem in this respect, as it is capable of rapid evolution by homologous recombination with related species. Resistant strains selected by treatment with ß-lactams express variants of the target enzymes that do not recognize the drugs but retain their activity in cell wall building, despite the antibiotics being mimics of the natural substrate. Until now, the crucial transpeptidase activity that is inhibited by ß-lactams was not amenable to in vitro investigation with enzymes from Gram-positive organisms, including streptococci, staphylococci, or enterococci pathogens. We report here for the first time the in vitro assembly of peptidoglycan using recombinant penicillin-binding proteins from pneumococcus and the precursor lipid II. The two required enzymatic activities, glycosyl transferase for elongating glycan chains and transpeptidase for cross-linking stem-peptides, were observed. Most importantly, the transpeptidase activity was dependent on the chemical nature of the stem-peptide. Amidation of the second residue glutamate into iso-glutamine by the recently discovered amido-transferase MurT/GatD is required for efficient cross-linking of the peptidoglycan.

Identification of novel imidazo[1,2-a]pyridine inhibitors targeting M. tuberculosis QcrB.

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. Through the use of high throughput whole cell screening of an extensive compound library a number of imidazo[1,2-a]pyridine (IP) compounds were obtained as potent lead molecules active against M. tuberculosis and Mycobacterium bovis BCG. The IP inhibitors (1-4) demonstrated minimum inhibitory concentrations (MICs) in the range of 0.03 to 5 µM against a panel of M. tuberculosis strains. M. bovis BCG spontaneous resistant mutants were generated against IP 1, 3, and 4 at 5× MIC and subsequent whole genome sequencing identified a single nucleotide polymorphism (937)ACC>(937)GCC (T313A) in qcrB, which encodes the b subunit of the electron transport ubiquinol cytochrome C reductase. This mutation also conferred cross-resistance against IP 1, 3 and 4 demonstrating a common target. Gene dosage experiments confirmed M. bovis BCG QcrB as the target where over-expression in M. bovis BCG led to an increase in MIC from 0.5 to >8 µM for IP 3. An acute murine model of TB infection established bacteriostatic activity of the IP series, which await further detailed characterization.