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The identification and development of new anti-tubercular agents are a priority research area. We identified the trifluoromethyl pyrimidinone series of compounds in a whole-cell screen against Mycobacterium tuberculosis . Fifteen primary hits had minimum inhibitory concentrations (MICs) with good potency IC 90 is the concentration at which M. tuberculosis growth is inhibited by 90% (IC 90 < 5 μM). We conducted a structure-activity relationship investigation for this series. We designed and synthesized an additional 44 molecules and tested all analogs for activity against M. tuberculosis and cytotoxicity against the HepG2 cell line. Substitution at the 5-position of the pyrimidinone with a wide range of groups, including branched and straight chain alkyl and benzyl groups, resulted in active molecules. Trifluoromethyl was the preferred group at the 6-position, but phenyl and benzyl groups were tolerated. The 2-pyridyl group was required for activity; substitution on the 5-position of the pyridyl ring was tolerated but not on the 6-position. Active molecules from the series demonstrated low selectivity, with cytotoxicity against eukaryotic cells being an issue. However, there were active and non-cytotoxic molecules; the most promising molecule had an MIC (IC 90 ) of 4.9 μM with no cytotoxicity (IC 50 > 100 μM). The series was inactive against Gram-negative bacteria but showed good activity against Gram-positive bacteria and yeast. A representative molecule from this series showed rapid concentration-dependent bactericidal activity against replicating M. tuberculosis bacilli with ~4 log kill in <7 days. Overall the biological properties were promising, if cytotoxicity could be reduced. There is scope for further medicinal chemistry optimization to improve the properties without major change in structural features., Competing Interests: EH, TM, and PH were employed by Eli Lilly and Company. PO was employed by company Apollo Drug Discovery Consulting, LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hembre, Early, Odingo, Shelton, Anoshchenko, Guzman, Flint, Dennison, McNeil, Korkegian, Ovechkina, Ornstein, Masquelin, Hipskind and Parish.)

The phenoxyalkylimidazoles (PAI) are an attractive chemical series with potent anti-tubercular activity targeting Mycobacterium tuberculosis respiration. Our aim was to determine if the PAI compounds are subject to efflux. Two analogs containing an oxadiazole had improved potency in the presence of the efflux inhibitors reserpine and carbonyl cyanide m-chlorophenylhydrazine, whereas the potency of analogs with a diazole was not affected., Competing Interests: The authors have declared that no competing interests exist.

Phenotypic screening of a Medicines for Malaria Venture compound library against Mycobacterium tuberculosis ( Mtb ) identified a cluster of pan-active 2-pyrazolylpyrimidinones. The biology triage of these actives using various tool strains of Mtb suggested a novel mechanism of action. The compounds were bactericidal against replicating Mtb and retained potency against clinical isolates of Mtb . Although selected MmpL3 mutant strains of Mtb showed resistance to these compounds, there was no shift in the minimum inhibitory concentration (MIC) against a mmpL3 hypomorph, suggesting mutations in MmpL3 as a possible resistance mechanism for the compounds but not necessarily as the target. RNA transcriptional profiling and the checkerboard board 2D-MIC assay in the presence of varying concentrations of ferrous salt indicated perturbation of the Fe-homeostasis by the compounds. Structure-activity relationship studies identified potent compounds with good physicochemical properties and in vitro microsomal metabolic stability with moderate selectivity over cytotoxicity against mammalian cell lines.

With the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis, there is a pressing need for new oral drugs with novel mechanisms of action. A number of scaffolds with potent anti-tubercular in vitro activity have been identified from phenotypic screening that appear to target MmpL3. However, the scaffolds are typically lipophilic, which facilitates partitioning into hydrophobic membranes, and several contain basic amine groups. Highly lipophilic basic amines are typically cytotoxic against mammalian cell lines and have associated off-target risks, such as inhibition of human ether-à-go-go related gene (hERG) and IKr potassium current modulation. The spirocycle compound 3 was reported to target MmpL3 and displayed promising efficacy in a murine model of acute tuberculosis (TB) infection. However, this highly lipophilic monobasic amine was cytotoxic and inhibited the hERG ion channel. Herein, the related spirocycles ( 1-2 ) are described, which were identified following phenotypic screening of the Eli Lilly corporate library against M. tuberculosis . The novel N-alkylated pyrazole portion offered improved physicochemical properties, and optimization led to identification of a zwitterion series, exemplified by lead 29 , with decreased HepG2 cytotoxicity as well as limited hERG ion channel inhibition. Strains with mutations in MmpL3 were resistant to 29 , and under replicating conditions, 29 demonstrated bactericidal activity against M. tuberculosis . Unfortunately, compound 29 had no efficacy in an acute model of TB infection; this was most likely due to the in vivo exposure remaining above the minimal inhibitory concentration for only a limited time., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

The introduction of DNA into bacterial cells is one of the foundational methods of bacterial genetics. Transformation of mycobacterial species is complicated due to the structure of the cell wall, which has a complex outer layer with low permeability. Electroporation has become a routine procedure in genetic studies. In this process, cells are subjected to a brief high-voltage electrical impulse which allows the entry of DNA. It can be used to introduce plasmid DNA, phage DNA, or oligonucleotides. This chapter presents methods for introducing DNA into a representative slow-growing species, M. tuberculosis, and a representative fast-growing species, M. smegmatis. Other mycobacteria can be transformed using variations of these methods, although the efficiency of transformation will vary.

We previously identified a diazaborine series with potential for development as a new tuberculosis drug. This series has activity in vitro and in vivo and targets cell wall biosynthesis via inhibition of InhA. The overall aim of this study was to determine whether InhA inhibitors have activity against non-replicating Mycobacterium tuberculosis. We tested the ability of two molecules of the diazaborine series to kill non-replicating M. tuberculosis in the nutrient starvation model; both molecules were bactericidal, reducing viability by >3 logs in 21 days. Activity showed similar kill rates to other InhA inhibitors (isoniazid and NITD-916). We conclude that inhibition of InhA is bactericidal against nutrient-starved non-replicating M. tuberculosis., Competing Interests: The authors have declared that no competing interests exist.

The Mycobacterium tuberculosis protein MmpL3 performs an essential role in cell wall synthesis, since it effects the transport of trehalose monomycolates across the inner membrane. Numerous structurally diverse pharmacophores have been identified as inhibitors of MmpL3 largely based on the identification of resistant isolates with mutations in MmpL3. For some compounds, it is possible there are different primary or secondary targets. Here, we have investigated resistance to the spiral amine class of compounds. Isolation and sequencing of resistant mutants demonstrated that all had mutations in MmpL3. We hypothesized that if additional targets of this pharmacophore existed, then successive rounds to generate resistant isolates might reveal mutations in other loci. Since compounds were still active against resistant isolates, albeit with reduced potency, we isolated resistant mutants in this background at higher concentrations. After a second round of isolation with the spiral amine, we found additional mutations in MmpL3. To increase our chance of finding alternative targets, we ran a third round of isolation using a different molecule scaffold (AU1235, an adamantyl urea). Surprisingly, we obtained further mutations in MmpL3. Multiple mutations in MmpL3 increased the level and spectrum of resistance to different pharmacophores but did not incur a fitness cost in vitro These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores. IMPORTANCE Mycobacterium tuberculosis is a major global human pathogen, and new drugs and new drug targets are urgently required. Cell wall biosynthesis is a major target of current tuberculosis drugs and of new agents under development. Several new classes of molecules appear to have the same target, MmpL3, which is involved in the export and synthesis of the mycobacterial cell wall. However, there is still debate over whether MmpL3 is the primary or only target for these classes. We wanted to confirm the mechanism of resistance for one series. We identified mutations in MmpL3 which led to resistance to the spiral amine series. High-level resistance to these compounds and two other series was conferred by multiple mutations in the same protein (MmpL3). These mutations did not reduce growth rate in culture. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores., (Copyright © 2020 McNeil et al.)

Crude extracts of the marine sponge Chelonaplysilla sp. collected in Samoa, that were obtained from the NCI Open Repository (NCS 21903), inhibited Mycobacterium tuberculosis growth. Assay-guided fractionation of the extract led to the isolation and structural elucidation of the known diterpenoid macfarlandin D (1) and three new diterpenoids macfarlandins F (2), G (3), and H (4). Macfarlandin D (1) exhibited potent antimicrobial activity against M. tuberculosis with an MIC of 1.2 ± 0.4 µg mL -1 . Macfarlandins F (2), G (3), and H (4) exhibited significantly weaker antitubercular activities, revealing SAR for the macfarlandin antitubercular pharmacophore. The structures of compounds 2, 3, and 4 were elucidated via detailed analysis of NMR and MS data.

The enhanced intracellular survival (Eis) protein of Mycobacterium tuberculosis ( Mtb ) is a versatile acetyltransferase that multiacetylates aminoglycoside antibiotics abolishing their binding to the bacterial ribosome. When overexpressed as a result of promoter mutations, Eis causes drug resistance. In an attempt to overcome the Eis-mediated kanamycin resistance of Mtb , we designed and optimized structurally unique thieno[2,3- d ]pyrimidine Eis inhibitors toward effective kanamycin adjuvant combination therapy. We obtained 12 crystal structures of enzyme-inhibitor complexes, which guided our rational structure-based design of 72 thieno[2,3- d ]pyrimidine analogues divided into three families. We evaluated the potency of these inhibitors in vitro as well as their ability to restore the activity of kanamycin in a resistant strain of Mtb , in which Eis was upregulated. Furthermore, we evaluated the metabolic stability of 11 compounds in vitro . This study showcases how structural information can guide Eis inhibitor design.

Introduction : Tuberculosis is the leading cause of death from infectious disease. Current drug therapy requires a combination of antibiotics taken over >6 months. An urgent need for new agents that can shorten therapy is required. In order to develop new drugs, simple in vitro assays are required that can identify efficacious compounds rapidly and predict in vivo activity in the human. Areas covered : This review focusses on the most relevant in vitro assays that can be utilized in a drug discovery program and which mimic different aspects of infection or disease. The focus is largely on assays used to test >1000s of compounds reliably and robustly. However, some assays used for 10s to 100s of compounds are included where the utility outweighs the low capacity. Literature searches for high throughput screening, models and in vitro assays were undertaken. Expert opinion : Drug discovery and development in tuberculosis is extremely challenging due to the requirement for predicting drug efficacy in a disease with complex pathology in which bacteria exist in heterogeneous states in inaccesible locations. A combination of assays can be used to determine profiles against replicating, non-replicating, intracellular and tolerant bacteria.

The fatty acid synthase type II (FAS-II) multienzyme system builds the main chain of mycolic acids (MAs), important lipid pathogenicity factors of Mycobacterium tuberculosis (Mtb). Due to their original structure, the identification of the (3 R)-hydroxyacyl-ACP dehydratases, HadAB and HadBC, of Mtb FAS-II complex required in-depth work. Here, we report the discovery of a third dehydratase protein, HadD Mtb (Rv0504c), whose gene is non-essential and sits upstream of cmaA2 encoding a cyclopropane synthase dedicated to keto- and methoxy-MAs. HadD Mtb deletion triggered a marked change in Mtb keto-MA content and size distribution, deeply impacting the production of full-size molecules. Furthermore, abnormal MAs, likely generated from 3-hydroxylated intermediates, accumulated. These data strongly suggest that HadD Mtb catalyzes the 3-hydroxyacyl dehydratation step of late FAS-II elongation cycles during keto-MA biosynthesis. Phenotyping of Mtb hadD deletion mutant revealed the influence of HadD Mtb on the planktonic growth, colony morphology and biofilm structuration, as well as on low temperature tolerance. Importantly, HadD Mtb has a strong impact on Mtb virulence in the mouse model of infection. The effects of the lack of HadD Mtb observed both in vitro and in vivo designate this protein as a bona fide target for the development of novel anti-TB intervention strategies.

Azole drugs such as econazole, are active on Mycobacterium tuberculosis and Mycobacterium smegmatis ; however, the identification of their target(s) is still pending. It has been reported that mutations in the non-essential system mmp L5- mmp S5 conferred resistance to econazole in M. tuberculosis . We herein report that an azole-resistant mutant screen in M. smegmatis rendered mutations in rsh A, encoding a non-essential anti-sigma H protein., Competing Interests: The authors declare that there are no conflicts of interest., (© 2019 The Authors.)

There is an urgent need for new anti-tubercular agents which can lead to a shortened treatment time by targeting persistent or non-replicating bacilli. In order to assess compound activity against non-replicating Mycobacterium tuberculosis, we developed a method to detect the bactericidal activity of novel compounds within 7 days. Our method uses incubation at low pH in order to induce a non-replicating state. We used a strain of M. tuberculosis expressing luciferase; we first confirmed the linear relationship between luminescence and viable bacteria (determined by colony forming units) under our assay conditions. We optimized the assay parameters in 96-well plates in order to achieve a reproducible assay. Our final assay used M. tuberculosis in phosphate-citrate buffer, pH 4.5 exposed to compounds for 7 days; viable bacteria were determined by luminescence. We recorded the minimum bactericidal concentration at pH 4.5 (MBC4.5) representing >2 logs of kill. We confirmed the utility of the assay with control compounds. The ionophores monensin, niclosamide, and carbonyl cyanide 3-chlorophenylhydrazone and the anti-tubercular drugs pretomanid and rifampicin were active, while several other drugs such as isoniazid, ethambutol, and linezolid were not., Competing Interests: Tanya Parish serves on the Editorial Board of PLOS ONE. This work was funded in part by Eli Lilly and Company in support of the mission of the Lilly TB Drug Discovery Initiative awarded to TP (https://www.lilly.com/). This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.

Iron is essential for the pathogenicity and virulence of Mycobacterium tuberculosis, which synthesises salicyl-capped siderophores (mycobactins) to acquire this element from the host. MbtA is the adenylating enzyme that catalyses the initial reaction of mycobactin biosynthesis and is solely expressed by mycobacteria. A 3200-member library comprised of lead-like, structurally diverse compounds was screened against M. tuberculosis for whole-cell inhibitory activity. A set of 846 compounds that inhibited the tubercle bacilli growth were then tested for their ability to bind to MbtA using a fluorescence-based thermal shift assay and NMR-based Water-LOGSY and saturation transfer difference (STD) experiments. We identified an attractive hit molecule, 5-hydroxyindol-3-ethylamino-(2-nitro-4-trifluoromethyl)benzene (5), that bound with high affinity to MbtA and produced a MIC 90 value of 13 μm. The ligand was docked into the MbtA crystal structure and displayed an excellent fit within the MbtA active pocket, adopting a binding mode different from that of the established MbtA inhibitor Sal-AMS., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

There is an urgent need for new treatments effective against Mycobacterium tuberculosis, the causative agent of tuberculosis. The 8-hydroxyquinoline series is a privileged scaffold with anticancer, antifungal, and antibacterial activities. We conducted a structure-activity relationship study of the series regarding its antitubercular activity using 26 analogs. The 8-hydroxyquinolines showed good activity against M. tuberculosis, with minimum inhibitory concentrations (MIC90) of <5 μM for some analogs. Small substitutions at C5 resulted in the most potent activity. Substitutions at C2 generally decreased potency, although a sub-family of 2-styryl-substituted analogs retained activity. Representative compounds demonstrated bactericidal activity against replicating M. tuberculosis with >4 log kill at 10× MIC over 14 days. The majority of the compounds demonstrated cytotoxicity (IC 50 of <100 μM). Further development of this series as antitubercular agents should address the cytotoxicity liability. However, the 8-hydroxyquinoline series represents a useful tool for chemical genomics to identify novel targets in M. tuberculosis., (© 2019 The Authors. Drug Development Research published by Wiley Periodicals, Inc.)

Current treatments for Mycobacterium tuberculosis infections require long and complicated regimens that can lead to patient non-compliance, increasing incidences of antibiotic-resistant strains, and lack of efficacy against latent stages of disease. Thus, new therapeutics are needed to improve tuberculosis standard of care. One strategy is to target protein homeostasis pathways by inhibiting molecular chaperones such as GroEL/ES (HSP60/10) chaperonin systems. M. tuberculosis has two GroEL homologs: GroEL1 is not essential but is important for cytokine-dependent granuloma formation, while GroEL2 is essential for survival and likely functions as the canonical housekeeping chaperonin for folding proteins. Another strategy is to target the protein tyrosine phosphatase B (PtpB) virulence factor that M. tuberculosis secretes into host cells to help evade immune responses. In the present study, we have identified a series of GroEL/ES inhibitors that inhibit M. tuberculosis growth in liquid culture and biochemical function of PtpB in vitro. With further optimization, such dual-targeting GroEL/ES and PtpB inhibitors could be effective against all stages of tuberculosis - actively replicating bacteria, bacteria evading host cell immune responses, and granuloma formation in latent disease - which would be a significant advance to augment current therapeutics that primarily target actively replicating bacteria., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Mycobacterium tuberculosis is the leading cause of morbidity and death resulting from infectious disease worldwide. The incredible disease burden, combined with the long course of drug treatment and an increasing incidence of antimicrobial resistance among M. tuberculosis isolates, necessitates novel drugs and drug targets for treatment of this deadly pathogen. Recent work has produced several promising clinical candidates targeting components of the electron transport chain (ETC) of M. tuberculosis , highlighting this pathway's potential as a drug target. Menaquinone is an essential component of the M. tuberculosis ETC, as it functions to shuttle electrons through the ETC to produce the electrochemical gradient required for ATP production for the cell. We show that inhibitors of MenA, a component of the menaquinone biosynthetic pathway, are highly active against M. tuberculosis MenA inhibitors are bactericidal against M. tuberculosis under both replicating and nonreplicating conditions, with 10-fold higher bactericidal activity against nutrient-starved bacteria than against replicating cultures. MenA inhibitors have enhanced activity in combination with bedaquiline, clofazimine, and inhibitors of QcrB, a component of the cytochrome bc 1 oxidase. Together, these data support MenA as a viable target for drug treatment against M. tuberculosis MenA inhibitors not only kill M. tuberculosis in a variety of physiological states but also show enhanced activity in combination with ETC inhibitors in various stages of clinical trial testing., (Copyright © 2019 Berube et al.)

Mycobacterium tuberculosis adenosine kinase (MtbAdoK) is an essential enzyme of Mtb and forms part of the purine salvage pathway within mycobacteria. Evidence suggests that the purine salvage pathway might play a crucial role in Mtb survival and persistence during its latent phase of infection. In these studies, we adopted a structural approach to the discovery, structure-guided design, and synthesis of a series of adenosine analogues that displayed inhibition constants ranging from 5 to 120 nM against the enzyme. Two of these compounds exhibited low micromolar activity against Mtb with half maximal effective inhibitory concentrations of 1.7 and 4.0 μM. Our selectivity and preliminary pharmacokinetic studies showed that the compounds possess a higher degree of specificity against MtbAdoK when compared with the human counterpart and are well tolerated in rodents, respectively. Finally, crystallographic studies showed the molecular basis of inhibition, potency, and selectivity and revealed the presence of a potentially therapeutically relevant cavity unique to the MtbAdoK homodimer.

A family of compounds typified by an anthranilic amide 1 was identified from a whole-cell screening effort targeted at identifying compounds that disrupt pH homeostasis in Mycobacterium tuberculosis . 1 demonstrated bactericidal activity against non-replicating M. tuberculosis in pH 4.5 buffer (MBC 4.5 = 6.3 μM). Exploration of the structure-activity relations failed to simplify the scaffold. The antitubercular activity proved dependent on the lipophilicity and planarity of the molecule and directly correlated with mammalian cytotoxicity. Further studies revealed a pH-dependent correlation between the family's disruption of M. tuberculosis membrane potential and antitubercular activity, with active compounds causing a drop in membrane potential at concentrations below their MBC 4.5 . A second compound family, identified in the same screening effort and typified by imidazo(4,5- e )(2,1,3)benzothiadiazole 2 , provided a contrasting profile. As with 1 , structure-activity profiling of 2 (MBC 4.5 = 25 μM) failed to minimize the initial scaffold, mammalian cytotoxicity was observed for a majority of the active compounds, and many of the active compounds disrupted M. tuberculosis membrane potential. However, unlike the anthranilic amide compounds, the benzothiadiazole compounds disrupted M. tuberculosis membrane potential primarily at concentrations above the MBC 4.5 in a pH-independent fashion. These differences suggest an alternative mechanism of action for the benzothiadiazole compounds. As a result, while the cytotoxicity of the anthranilic amides limits their utility to tool compounds, benzothiadiazole 2 presents an attractive target for more focused SAR exploration.

Bacterial persisters are a subpopulation of cells that exhibit phenotypic resistance during exposure to a lethal dose of antibiotics. They are difficult to target and thought to contribute to the long treatment duration required for tuberculosis. Understanding the molecular and cellular biology of persisters is critical to finding new tuberculosis drugs that shorten treatment. This review focuses on mycobacterial persisters and describes the challenges they pose in tuberculosis therapy, their characteristics and formation, how persistence leads to resistance, and the current approaches being used to target persisters within mycobacterial drug discovery.

The menaquinone biosynthetic pathway presents a promising drug target against Mycobacterium tuberculosis and potentially other Gram-positive pathogens. In the present study, the essentiality, steady state kinetics of MenA from M. tuberculosis and the mechanism of MenA inhibition by Ro 48-8071 were characterized. MenA [isoprenyl diphosphate:1,4-dihydroxy-2-naphthoate (DHNA) isoprenyltransferase] catalyzes a critical reaction in menaquinone biosynthesis that involves the conversion of cytosolic DHNA, to membrane bound demethylmenaquinone by transferring a hydrophobic 45-carbon isoprenoid chain (in the case of mycobacteria) to the ring nucleus of DHNA. Rv0534c previously identified as the gene encoding MenA in M. tuberculosis complemented a menA deletion in E. coli and an E. coli host expressing Rv0534c exhibited an eight-fold increase in MenA specific activity over the control strain harboring empty vector under similar assay conditions. Expression of Rv0534c is essential for mycobacterial survival and the native enzyme from M. tuberculosis H37Rv was characterized using membrane preparations as it was not possible to solubilize and purify the recombinant enzyme. The enzyme is absolutely dependent on the presence of a divalent cation for optimal activity with Mg+2 being the most effective and is active over a wide pH range, with pH 8.5 being optimal. The apparent Km values for DHNA and farnesyl diphosphate were found to be 8.2 and 4.3 μM, respectively. Ro 48-8071, a compound previously reported to inhibit mycobacterial MenA activity, is non-competitive with regard to DHNA and competitive with regard to the isoprenyldiphosphate substrate., Competing Interests: The authors have declared that no competing interests exist.

AN12855 is a direct, cofactor-independent inhibitor of InhA in Mycobacterium tuberculosis In the C3HeB/FeJ mouse model with caseous necrotic lung lesions, AN12855 proved efficacious with a significantly lower resistance frequency than isoniazid. AN12855 drug levels were better retained in necrotic lesions and caseum where the majority of hard to treat, extracellular bacilli reside. Owing to these combined attributes, AN12855 represents a promising alternative to the frontline antituberculosis agent isoniazid., (Copyright © 2019 Robertson et al.)

There is a need for new combination regimens for tuberculosis. Identifying synergistic drug combinations can avoid toxic side effects and reduce treatment times. Using a fluorescent rifampicin conjugate, we demonstrated that synergy between cell wall inhibitors and rifampicin was associated with increased accumulation of rifampicin. Increased accumulation was also associated with increased cellular permeability., Competing Interests: The authors declare that there are no conflicts of interest., (© 2019 The Authors.)

To find new inhibitors of Mycobacterium tuberculosis that have novel mechanisms of action, we miniaturized a high throughput screen to identify compounds that disrupt pH homeostasis. We adapted and validated a 384-well format assay to determine intrabacterial pH using a ratiometric green fluorescent protein. We screened 89000 small molecules under nonreplicating conditions and confirmed 556 hits that reduced intrabacterial pH (below pH 6.5). We selected five compounds that disrupt intrabacterial pH homeostasis and also showed some activity against nonreplicating bacteria in a 4-stress model, but with no (or greatly reduced) activity against replicating bacteria. The compounds selected were two benzamide sulfonamides, a benzothiadiazole, a bissulfone, and a thiadiazole, none of which are known antibacterial agents. All of these five compounds demonstrated bactericidal activity against nonreplicating bacteria in buffer. Four of the five compounds demonstrated increased activity under low pH conditions. None of the five compounds acted as ionophores or as general disrupters of membrane potential. These compounds are useful starting points for work to elucidate their mechanism of action and their utility for drug discovery.

Tuberculosis is a disease of global importance for which novel drugs are urgently required. We developed a whole-cell phenotypic screen which can be used to identify inhibitors of Mycobacterium tuberculosis growth. We used recombinant strains of virulent M. tuberculosis which express far-red fluorescent reporters and used fluorescence to monitor growth in vitro. We optimized our high throughput assays using both 96-well and 384-well plates; both formats gave assays which met stringent reproducibility and robustness tests. We screened a compound set of 1105 chemically diverse compounds previously shown to be active against M. tuberculosis and identified primary hits which showed ≥ 90% growth inhibition. We ranked hits and identified three chemical classes of interest-the phenoxyalkylbenzamidazoles, the benzothiophene 1-1 dioxides, and the piperidinamines. These new compound classes may serve as starting points for the development of new series of inhibitors that prevent the growth of M. tuberculosis. This assay can be used for further screening, or could easily be adapted to other strains of M. tuberculosis., Competing Interests: Tanya Parish serves on the Editorial Board of PLOS ONE. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

The diaminoquinazoline series has good potency against Mycobacterium tuberculosis Resistant isolates have mutations in Rv3161c, a putative dioxygenase. We carried out metabolite analysis on a wild-type strain and an Rv3161c mutant strain after exposure to a diaminoquinazoline. The parental compound was found in intracellular extracts from the mutant but not the wild type. A metabolite consistent with a monohydroxylated form was identified in the wild type. These data support the hypothesis that Rv3161c metabolizes diaminoquinazolines in M. tuberculosis ., (Copyright © 2018 Melief et al.)

Tuberculosis is a massive global burden and Mycobacterium tuberculosis is increasingly resistant to first- and second-line drugs. There is an acute need for new anti-mycobacterial drugs with novel targets. We previously evaluated a series of 2-aminothiazoles with activity against Mycobacterium tuberculosis . In this study, we identify the glycolytic enzyme enolase as the target of these molecules using pull down studies. We demonstrate that modulation of the level of enolase expression affects sensitivity to 2-aminothiazoles; increased expression leads to resistance while decreased protein levels increase sensitivity. Exposure to 2-aminothiazoles results in increased levels of metabolites preceding the action of enolase in the glycolytic pathway and decreased ATP levels. We demonstrate that 2-aminothiazoles inhibit the activity of the human α-enolase, which could also account for the cytotoxicity of some of those molecules. If selectivity for the bacterial enzyme over the human enzyme could be achieved, enolase would represent an attractive target for M. tuberculosis drug discovery and development efforts.

There is an urgent need for the development of shorter, simpler and more tolerable drugs to treat antibiotic tolerant populations of Mycobacterium tuberculosis. We previously identified a series of hydrazones active against M. tuberculosis. We selected five representative compounds for further analysis. All compounds were active against non-replicating M. tuberculosis, with two compounds demonstrating greater activity under hypoxic conditions than aerobic culture. Compounds had bactericidal activity with MBC/MIC of < 4 and demonstrated an inoculum-dependent effect against aerobically replicating bacteria. Bacterial kill kinetics demonstrated a faster rate of kill against non-replicating bacilli generated by nutrient starvation. Compounds had limited activity against other bacterial species. In conclusion, we have demonstrated that hydrazones have some attractive properties in terms of their anti-tubercular activity., Competing Interests: Tanya Parish serves on the Editorial Board of PLOS ONE. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Mycobacterium abscessus infections are increasing worldwide. Current drug regimens are largely ineffective, yet the current development pipeline for M. abscessus is alarmingly sparse. Traditional discovery efforts for M. abscessus assess the capability of a new drug to inhibit bacterial growth under nutrient-rich growth conditions, but this does not predict the impact when used in the clinic. The disconnect between in vitro and in vivo activity is likely due to the genetic and physiological adaptation of the bacteria to the environmental conditions encountered during infection; these include low oxygen tension and nutrient starvation. We sought to fill a gap in the drug discovery pipeline by establishing an assay to identify novel compounds with bactericidal activity against M. abscessus under non-replicating conditions. We developed and validated a novel screen using nutrient starvation to generate a non-replicating state. We used alamarBlue ® to measure metabolic activity and demonstrated this correlates with bacterial viability under these conditions. We optimized key parameters and demonstrated reproducibility. Using this assay, we determined that niclosamide was bactericidal against non-replicating bacilli, highlighting its potential to be included in M. abscessus regimens. In contrast, most other drugs currently used in the clinic for M. abscessus infections, were completely inactive, potentially explaining their poor efficacy. Thus, our assay allows for rapid identification of bactericidal compounds in a model using conditions that are more relevant in vivo . This screen can be used in a high-throughput way to identify novel agents with properties that promise an increase in efficacy, while also shortening treatment times.

Objective: We have previously codon-optimized a number of red fluorescent proteins for use in Mycobacterium tuberculosis (mCherry, tdTomato, Turbo-635). We aimed to expand this repertoire to include DsRed, another widely used and flexible red fluorescent protein., Results: We generated expression constructs with a full length DsRed under the control of one of three strong, constitutive promoters (P hsp60 , P rpsA or P G13 ) for use in mycobacteria. We confirmed that full length DsRed (225 amino acids) was expressed and fluoresced brightly. In contrast to mCherry, truncated versions of DsRed lacking several amino acids at the N-terminus were not functional. Thus, we have expanded the repertoire of optimized fluorescent proteins for mycobacteria.

With the emergence of multidrug-resistant strains of Mycobacterium tuberculosis there is a pressing need for new oral drugs with novel mechanisms of action. Herein, we describe the identification of a novel morpholino-thiophenes (MOT) series following phenotypic screening of the Eli Lilly corporate library against M. tuberculosis strain H37Rv. The design, synthesis, and structure-activity relationships of a range of analogues around the confirmed actives are described. Optimized leads with potent whole cell activity against H37Rv, no cytotoxicity flags, and in vivo efficacy in an acute murine model of infection are described. Mode-of-action studies suggest that the novel scaffold targets QcrB, a subunit of the menaquinol cytochrome c oxidoreductase, part of the bc1-aa3-type cytochrome c oxidase complex that is responsible for driving oxygen-dependent respiration.

Objective: Our aim was to identify natural products with anti-tubercular activity., Results: A set of ~ 500 purified natural product compounds was screened for inhibition against the human pathogen Mycobacterium tuberculosis. A series of cyclic hexapeptides with anti-tubercular activity was identified. Five analogs from a set of sixteen closely related compounds were active, with minimum inhibitory concentrations ranging from 2.3 to 8.9 μM. Eleven structural analogs had no significant activity (MIC > 20 μM) demonstrating structure activity relationship. Sequencing of resistant mutant isolates failed to identify changes accounting for the resistance phenotype.

NF-κB is an important mediator of immune activity and its activation is essential in mounting immune response to pathogens. Here, we describe the optimization and implementation of a high-throughput screening platform that utilizes high content imaging and analysis to monitor NF-κB nuclear translocation. We screened 38,991 compounds from three different small molecule libraries and identified 103 compound as hits; 31% of these were active in a dose response assay. Several of the molecules lacked cytotoxicity or had a selectivity index of more than 2-fold. Our image-based approach provides an important first step towards identifying small molecules with immunomodulatory activity., Competing Interests: The authors have read the journal's policy and the authors of this manuscript have the following competing interests: TP is on the Editorial board for PLOS One. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

New antitubercular agents are needed to combat the spread of multidrug- and extensively drug-resistant strains of Mycobacterium tuberculosis . The frontline antitubercular drug isoniazid (INH) targets the mycobacterial enoyl-ACP reductase, InhA. Resistance to INH is predominantly through mutations affecting the prodrug-activating enzyme KatG. Here, we report the identification of the diazaborines as a new class of direct InhA inhibitors. The lead compound, AN12855, exhibited in vitro bactericidal activity against replicating bacteria and was active against several drug-resistant clinical isolates. Biophysical and structural investigations revealed that AN12855 binds to and inhibits the substrate-binding site of InhA in a cofactor-independent manner. AN12855 showed good drug exposure after i.v. and oral delivery, with 53% oral bioavailability. Delivered orally, AN12855 exhibited dose-dependent efficacy in both an acute and chronic murine model of tuberculosis infection that was comparable with INH. Combined, AN12855 is a promising candidate for the development of new antitubercular agents., Competing Interests: The authors declare that they have no conflict of interest.

Despite increased research efforts to find new treatments for tuberculosis in recent decades, compounds with novel mechanisms of action are still required. We previously identified a series of novel aryl-oxadiazoles with anti-tubercular activity specific for bacteria using butyrate as a carbon source. We explored the structure activity relationship of this series. Structural modifications were performed in all domains to improve potency and physico-chemical properties. A number of compounds displayed sub-micromolar activity against M. tuberculosis utilizing butyrate, but not glucose as the carbon source. Compounds showed no or low cytotoxicity against eukaryotic cells. Three compounds were profiled in mouse pharmacokinetic studies. Plasma clearance was low to moderate but oral exposure suggested solubility-limited drug absorption in addition to first pass metabolism. The presence of a basic nitrogen in the linker slightly increased solubility, and salt formation optimized aqueous solubility. Our findings suggest that the 1,3,4-oxadiazoles are useful tools and warrant further investigation., (Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The imidazopyridines are a promising new class of antitubercular agents with potent activity in vitro and in vivo We isolated mutants of Mycobacterium tuberculosis resistant to a representative imidazopyridine; the mutants had large shifts (>20-fold) in MIC. Whole-genome sequencing revealed mutations in Rv1339, a hypothetical protein of unknown function. We isolated mutants resistant to three further compounds from the series; resistant mutants isolated from two of the compounds had single nucleotide polymorphisms in Rv1339 and resistant mutants isolated from the third compound had single nucleotide polymorphisms in QcrB, the proposed target for the series. All the strains were resistant to two compounds, regardless of the mutation, and a strain carrying the QcrB T313I mutation was resistant to all of the imidazopyridine derivatives tested, confirming cross-resistance. By monitoring pH homeostasis and ATP generation, we confirmed that compounds from the series were targeting QcrB; imidazopyridines disrupted pH homeostasis and depleted ATP, providing further evidence of an effect on the electron transport chain. A representative compound was bacteriostatic against replicating bacteria, consistent with a mode of action against QcrB. The series had a narrow inhibitory spectrum, with no activity against other bacterial species. No synergy or antagonism was seen with other antituberculosis drugs under development. In conclusion, our data support the hypothesis that the imidazopyridine series functions by reducing ATP generation via inhibition of QcrB., (Copyright © 2018 O'Malley et al.)

Mycobacterium tuberculosis is an expert and deadly pathogen, causing the disease tuberculosis (TB) in humans. It has several notable features: the ability to enter non-replicating states for long periods and cause latent infection; metabolic remodelling during chronic infection; a thick, waxy cell wall; slow growth rate in culture; and intrinsic drug resistance and antibiotic tolerance. As a pathogen, M. tuberculosis has a complex relationship with its host, is able to replicate inside macrophages, and expresses diverse immunomodulatory molecules. M. tuberculosis currently causes over 1.8 million deaths a year, making it the world's most deadly human pathogen.

There is a pressing need to develop novel anti-tubercular drugs. High-throughput phenotypic screening yields chemical series that inhibit bacterial growth. Target identification for such series is challenging, but necessary for optimization of target engagement and the development of series into clinical drugs. We constructed a library of recombinant Mycobacterium tuberculosis strains each expressing a single protein from an inducible promoter as a tool for target identification. The library of 1733 clones was arrayed in 96-well plates for rapid screening and monitoring growth. The library contains the majority of the annotated essential genes as well as genes involved in cell wall and fatty acid biosynthesis, virulence factors, regulatory proteins, efflux, and respiration pathways. We evaluated the growth kinetics and plasmid stability over three passages for each clone in the library. We determined expression levels (mRNA and/or protein) in 396 selected clones. We screened the entire library and identified the Alr-expressing clone as the only recombinant strain, which grew in the presence of d-cycloserine (DCS). We confirmed that the Alr-expressing clone was resistant to DCS (7-fold shift in minimum inhibitory concentration). The library represents a new tool that can be used to screen for compound resistance and other phenotypes.

We identified a series of novel 7-phenyl benzoxaborole compounds with activity against Mycobacterium tuberculosis. Compounds had a range of activity with inhibitory concentrations (IC 90 ) as low as 5.1 μM and no cytotoxicity against eukaryotic cells (IC 50  > 50 μM). Compounds were active against intracellular mycobacteria cultured in THP-1 macrophages. We isolated and characterized resistant mutants with mutations in NADH dehydrogenase (Ndh) or the regulatory protein Mce3R. Mutations suggest that Ndh may be the target of this series., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

As an obligate aerobe, Mycobacterium tuberculosis uses its electron transport chain (ETC) to produce energy via oxidative phosphorylation. This pathway has recently garnered a lot of attention and is a target for several new antimycobacterials. We tested the respiratory adaptation of M. tuberculosis to phenoxyalkylbenzimidazoles (PABs), compounds proposed to target QcrB, a component of the cytochrome bc 1 complex. We show that M. tuberculosis is able to reroute its ETC to provide temporary resistance to PABs. However, combination treatment of PAB with agents targeting other components of the electron transport chain overcomes this respiratory flexibility. PAB in combination with clofazimine resulted in synergistic killing of M. tuberculosis under both replicating and nonreplicating conditions. PABs in combination with bedaquiline demonstrated antagonism at early time points, particularly under nonreplicating conditions. However, this antagonistic effect disappeared within 3 weeks, when PAB-BDQ combinations became highly bactericidal; in some cases, they were better than either drug alone. This study highlights the potential for combination treatment targeting the ETC and supports the development of PABs as part of a novel drug regimen against M. tuberculosis ., (Copyright © 2017 Berube and Parish.)

The phenoxy alkyl benzimidazoles (PABs) have good antitubercular activity. We expanded our structure-activity relationship studies to determine the core components of PABs required for activity. The most potent compounds had minimum inhibitory concentrations against Mycobacterium tuberculosis in the low nanomolar range with very little cytotoxicity against eukaryotic cells as well as activity against intracellular bacteria. We isolated resistant mutants against PAB compounds, which had mutations in either Rv1339, of unknown function, or qcrB, a component of the cytochrome bc 1 oxidase of the electron transport chain. QcrB mutant strains were resistant to all PAB compounds, whereas Rv1339 mutant strains were only resistant to a subset, suggesting that QcrB is the target. The discovery of the target for PAB compounds will allow for the improved design of novel compounds to target intracellular M. tuberculosis.

Isoniazid inhibits Mycobacterium tuberculosis InhA and is a key component of drug regimens that treat tuberculosis. However, the high rate of resistance against isoniazid is a contributing factor to the emergence of multi-drug resistance strains of M. tuberculosis. The 4-hydroxy-2-pyridine NITD-916 is a direct inhibitor of M. tuberculosis InhA that has comparable efficacy to isoniazid in mouse models of TB infection but a lower frequency of resistance. To characterize resistance mechanisms against NITD-916 we isolated resistant mutants in H37Rv (Euro-American lineage) and HN878 (East-Asian lineage) strains of M. tuberculosis. The resistance frequency was similar in both strains. Mutations were identified in residues within or near to the active of InhA or in the fabG1inhA promoter region. All mutants were resistant to NITD-916 but were not cross resistant to isoniazid, despite homology to SNPs identified in isoniazid resistant clinical isolates., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)