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Rising antimicrobial resistance challenges our ability to combat bacterial infections. The problem is acute for tuberculosis (TB), the leading cause of death from infection before COVID-19. Here, we developed a framework for multiple pharmaceutical companies to share proprietary information and compounds with multiple laboratories in the academic and government sectors for a broad examination of the ability of β-lactams to kill Mycobacterium tuberculosis (Mtb). In the TB Drug Accelerator (TBDA), a consortium organized by the Bill & Melinda Gates Foundation, individual pharmaceutical companies collaborate with academic screening laboratories. We developed a higher order consortium within the TBDA in which four pharmaceutical companies (GlaxoSmithKline, Sanofi, MSD, and Lilly) collectively collaborated with screeners at Weill Cornell Medicine, the Infectious Disease Research Institute (IDRI), and the National Institute of Allergy and Infectious Diseases (NIAID), pharmacologists at Rutgers University, and medicinal chemists at the University of North Carolina to screen ∼8900 β-lactams, predominantly cephalosporins, and characterize active compounds. In a striking contrast to historical expectation, 18% of β-lactams screened were active against Mtb, many without a β-lactamase inhibitor. One potent cephaloporin was active in Mtb-infected mice. The steps outlined here can serve as a blueprint for multiparty, intra- and intersector collaboration in the development of anti-infective agents.

Phenotypic whole cell high-throughput screening of a ∼150,000 diverse set of compounds against Mycobacterium tuberculosis (Mtb) in cholesterol-containing media identified 1,3-diarylpyrazolyl-acylsulfonamide 1 as a moderately active hit. Structure-activity relationship (SAR) studies demonstrated a clear scope to improve whole cell potency to MIC values of

Resistance of bacterial pathogens against antibiotics is declared by WHO as a major global health threat. As novel antibacterial agents are urgently needed, we re-assessed the broad-spectrum myxobacterial antibiotic myxovalargin and found it to be extremely potent against Mycobacterium tuberculosis. To ensure compound supply for further development we studied myxovalargin biosynthesis in detail enabling production via fermentation of a native producer. Feeding experiments as well as functional genomics analysis suggested a structural revision, which was eventually corroborated by development of a concise total synthesis. The ribosome was identified as the molecular target based on resistant mutant sequencing and a cryo-EM structure revealed that myxovalargin binds within and completely occludes the exit tunnel, consistent with a mode of action to arrest translation during a late stage of translation initiation. Pharmacokinetic and initial in vivo efficacy studies indicated that myxovalargin and analogues show potential for development as an antibacterial agent.

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 [IC 50 ] = 0.07 μM), is active against extracellular bacteria in cholesterol-containing culture medium, and exhibits no cross-resistance with known antitubercular drugs.

Pyrazolo[1,5-a]pyrimidin-7(4H)-one was identified through high-throughput whole-cell screening as a potential antituberculosis lead. The core of this scaffold has been identified several times previously and has been associated with various modes of action against Mycobacterium tuberculosis (Mtb). We explored this scaffold through the synthesis of a focused library of analogues and identified key features of the pharmacophore while achieving substantial improvements in antitubercular activity. Our best hits had low cytotoxicity and showed promising activity against Mtb within macrophages. The mechanism of action of these compounds was not related to cell-wall biosynthesis, isoprene biosynthesis, or iron uptake as has been found for other compounds sharing this core structure. Resistance to these compounds was conferred by mutation of a flavin adenine dinucleotide (FAD)-dependent hydroxylase (Rv1751) that promoted compound catabolism by hydroxylation from molecular oxygen. Our results highlight the risks of chemical clustering without establishing mechanistic similarity of chemically related growth inhibitors.

Swapping the substituents in positions 2 and 4 of the previously synthesized but yet undisclosed 5-cyano-4-(methylthio)-2-arylpyrimidin-6-ones 4, ring closure, and further optimization led to the identification of the potent antitubercular 2-thio-substituted quinazolinone 26. Structure-activity relationship (SAR) studies indicated a crucial role for both meta-nitro substituents for antitubercular activity, while the introduction of polar substituents on the quinazolinone core allowed reduction of bovine serum albumin (BSA) binding (63c, 63d). While most of the tested quinazolinones exhibited no cytotoxicity against MRC-5, the most potent compound 26 was found to be mutagenic via the Ames test. This analogue exhibited moderate inhibitory potency against Mycobacterium tuberculosis thymidylate kinase, the target of the 3-cyanopyridones that lies at the basis of the current analogues, indicating that the whole-cell antimycobacterial activity of the present S-substituted thioquinazolinones is likely due to modulation of alternative or additional targets. Diminished antimycobacterial activity was observed against mutants affected in cofactor F-420 biosynthesis (fbiC), cofactor reduction (fgd), or deazaflavin-dependent nitroreductase activity rv3547 , indicating that reductive activation of the 3,5-dinitrobenzyl analogues is key to antimycobacterial activity.

Summary: We characterized Mycobacterium bovis BCG isolates found in lung and brain samples from a previously vaccinated patient with IFNγR1 deficiency. The isolates collected displayed distinct genomic and phenotypic features consistent with host adaptation and associated changes in antibiotic susceptibility and virulence traits. Background: We report a case of a patient with partial recessive IFNγR1 deficiency who developed disseminated BCG infection after neonatal vaccination (BCG-vaccine). Distinct M. bovis BCG-vaccine derived clinical strains were recovered from the patient’s lungs and brain. Methods: BCG strains were phenotypically (growth, antibiotic susceptibility, lipid) and genetically (whole genome sequencing) characterized. Mycobacteria cell infection models were used to assess apoptosis, necrosis, cytokine release, autophagy, and JAK-STAT signaling. Results: Clinical isolates BCG-brain and BCG-lung showed distinct Rv0667 rpoB mutations conferring high- and low-level rifampin resistance; the latter displayed clofazimine resistance through Rv0678 gene (MarR-like transcriptional regulator) mutations. BCG-brain and BCG-lung showed mutations in fadA2, fadE5, and mymA operon genes, respectively. Lipid profiles revealed reduced levels of PDIM in BCG-brain and BCG-lung and increased TAGs and Mycolic acid components in BCG-lung, compared to parent BCG-vaccine. In vitro infected cells showed that the BCG-lung induced a higher cytokine release, necrosis, and cell-associated bacterial load effect when compared to BCG-brain; conversely, both strains inhibited apoptosis and altered JAK-STAT signaling. Conclusions: During a chronic-disseminated BCG infection, BCG strains can evolve independently at different sites likely due to particular microenvironment features leading to differential antibiotic resistance, virulence traits resulting in dissimilar responses in different host tissues.

Translational frameshift errors are often deleterious to the synthesis of functional proteins and could therefore be promoted therapeutically to kill bacteria. TrmD (tRNA-(N(1)G37) methyltransferase) is an essential tRNA modification enzyme in bacteria that prevents +1 errors in the reading frame during protein translation and represents an attractive potential target for the development of new antibiotics. Here, we describe the application of a structure-guided fragment-based drug discovery approach to the design of a new class of inhibitors against TrmD in Mycobacterium abscessus. Fragment library screening, followed by structure-guided chemical elaboration of hits, led to the rapid development of drug-like molecules with potent in vitro TrmD inhibitory activity. Several of these compounds exhibit activity against planktonic M. abscessus and M. tuberculosis as well as against intracellular M. abscessus and M. leprae, indicating their potential as the basis for a novel class of broad-spectrum mycobacterial drugs.

Tuberculosis (TB) is an air-borne infectious disease and is the leading cause of death among all infectious diseases globally. The current treatment regimen for TB is overtly long and patient non-compliance often leads to drug resistant TB resulting in a need to develop new drugs that will act via novel mechanisms. In this research work, we selected Mycobacterium membrane protein large (MmpL3) as the drug target and indole-2-carboximide as our molecule of interest for further designing new molecules. A homology model was prepared for the Mycobacterium tuberculosis MmpL3 from the crystal structure of Mycobacterium smegmatis MmpL3. A series of indoles which are known to be MmpL3 inhibitors were docked in the prepared protein and the binding site properties were identified. Based on that, 10 molecules were designed and synthesized and their antitubercular activities evaluated. We identified four hits among which the highest potency candidate possessed a minimum inhibitory concentration (MIC) of 1.56 μM at 2-weeks. Finally, molecular dynamics simulation studies were done with 3b and a previously reported MmpL3 inhibitor to understand the intricacies of their binding in real time and to correlate the experimental findings with the simulation data.

SUMMARYNatural products provide a rich source of potential antimicrobials for use in treating infectious diseases for which drug resistance has emerged. Foremost among these is tuberculosis. Assessment of the antimycobacterial activity of nargenicin, a natural product that targets the replicative DNA polymerase of Staphylococcus aureus, revealed that it is a bactericidal genotoxin that induces a DNA damage response in Mycobacterium tuberculosis (Mtb) and inhibits growth by blocking the replicative DNA polymerase, DnaE1. Cryo-electron microscopy revealed that binding of nargenicin to Mtb DnaE1 requires the DNA substrate such that nargenicin is wedged between the terminal base pair and the polymerase and occupies the position of both the incoming nucleotide and templating base. Comparative analysis across three bacterial species suggests that the activity of nargenicin is partly attributable to the DNA binding affinity of the replicative polymerase. This work has laid the foundation for target-led drug discovery efforts focused on Mtb DnaE1.

A new series of novel diphenyl ether based 2-oxo-dihydropyridine derivatives were synthesized and screened for their in vitro antimycobacterial and antibacterial activities. Most of the synthesized compounds showed significant activity against Mycobacterium tuberculosis H37Rv strain in comparison to triclosan. Among them, compounds 3k and 3q were found to be most active against Mycobacterium tuberculosis H37Rv strain with MIC of 31 and 32 mM respectively. All the compounds were found to be more active against Bacillus subtilis and Staphylococcus aureus than against Pseudomonas aeruginosa and Escherichia coli. Several compounds were found to safe against Vero and HepG2 cell lines. Molecular docking study was utilized to explore the binding mode of the synthesized compounds to the target enzyme InhA. The results showed reasonable binding interactions of synthesized molecules and good dock score. Molecular dynamics studies were performed in order to support the docking results. The compound 3k-InhA complex was found to be more stable and exhibited more interaction when compared to Triclosan. The compounds followed Lipinski rule of five and displayed acceptable pharmacokinetic properties depicted via in silico studies.

A new series of novel triclosan (2,4,40-trichloro-20-hydroxydiphenylether) analogues were designed, synthesized, and screened for their in vitro antimycobacterial and antibacterial activities. Most of the compounds showed significant activity against Mycobacterium tuberculosis H37Rv strain with minimum inhibitory concentration (MIC) values in 20–40 μM range in GAST/Fe medium when compared with triclosan (43 μM) in the first week of assay, and after additional incubation, seven compounds, that is, 2a, 2c, 2g, 2h, 2i, 2j, and 2m, exhibited MIC values at the concentration of 20–40 μM. The compounds also showed more significant activity against Bacillus subtilis and Staphylococcus aureus. The synthesized compounds showed druggable properties, and the predicted ADME (absorption, distribution, metabolism, and excretion) properties were within the acceptable limits. The in silico studies predicted better interactions of compounds with target protein residues and a higher dock score in comparison with triclosan. Molecular dynamics simulation study of the most active compound 2i was performed in order to further explore the stability of the protein– ligand complex and the protein–ligand interaction in detail.

Combination chemotherapy can increase treatment efficacy and suppress drug resistance. Knowledge of how to engineer rational, mechanism-based drug combinations, however, remains lacking. Although studies of drug activity have historically focused on the primary drug-target interaction, growing evidence has emphasized the importance of the subsequent consequences of this interaction. Bedaquiline (BDQ) is the first new drug for tuberculosis (TB) approved in more than 40 y, and a species-selective inhibitor of the Mycobacterium tuberculosis (Mtb) ATP synthase. Curiously, BDQ-mediated killing of Mtb lags significantly behind its inhibition of ATP synthase, indicating a mode of action more complex than the isolated reduction of ATP pools. Here, we report that BDQ-mediated inhibition of Mtb's ATP synthase triggers a complex metabolic response indicative of a specific hierarchy of ATP-dependent reactions. We identify glutamine synthetase (GS) as an enzyme whose activity is most responsive to changes in ATP levels. Chemical supplementation with exogenous glutamine failed to affect BDQ's antimycobacterial activity. However, further inhibition of Mtb's GS synergized with and accelerated the onset of BDQ-mediated killing, identifying Mtb's glutamine synthetase as a collateral, rather than directly antimycobacterial, metabolic vulnerability of BDQ. These findings reveal a previously unappreciated physiologic specificity of ATP and a facet of mode-of-action biology we term collateral vulnerability, knowledge of which has the potential to inform the development of rational, mechanism-based drug combinations.

The Tuberculosis Drug Accelerator, an experiment designed to facilitate collaboration in tuberculosis drug discovery by breaking down barriers among competing labs and institutions, has reached a 10-year landmark. We review the consortium’s achievements, advantages and limitations and advocate for the application of similar models to other diseases.

Coenzyme A (CoA) is a ubiquitous cofactor present in all living cells and estimated to be required for up to 9% of intracellular enzymatic reactions. Mycobacterium tuberculosis (Mtb) relies on its own ability to biosynthesize CoA to meet the needs of the myriad enzymatic reactions that depend on this cofactor for activity. As such, the pathway to CoA biosynthesis is recognized as a potential source of novel tuberculosis drug targets. In prior work, we genetically validated CoaBC as a bactericidal drug target in Mtb in vitro and in vivo. Here, we describe the identification of compound 1f, a small molecule inhibitor of the 4′-phosphopantothenoyl-l-cysteine synthetase (PPCS; CoaB) domain of the bifunctional Mtb CoaBC, and show that this compound displays on-target activity in Mtb. Compound 1f was found to inhibit CoaBC uncompetitively with respect to 4′-phosphopantothenate, the substrate for the CoaB-catalyzed reaction. Furthermore, metabolomic profiling of wild-type Mtb H37Rv following exposure to compound 1f produced a signature consistent with perturbations in pantothenate and CoA biosynthesis. As the first report of a direct small molecule inhibitor of Mtb CoaBC displaying target-selective whole-cell activity, this study confirms the druggability of CoaBC and chemically validates this target.

More than two decades have elapsed since the publication of the first genome sequence of Mycobacterium tuberculosis (Mtb) which, shortly thereafter, enabled methods to determine gene essentiality in the pathogen. Despite this, target-based approaches have not yielded drugs that have progressed to clinical testing. Whole-cell screening followed by elucidation of mechanism of action has to date been the most fruitful approach to progressing inhibitors into the tuberculosis drug discovery pipeline although target-based approaches are gaining momentum. This review discusses scaffolds that have been identified over the last decade from screens of small molecule libraries against Mtb or defined targets where mechanism of action investigation has defined target-hit couples and structure-activity relationship studies have described the pharmacophore.

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.

Coenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent and selective inhibitors of M. tuberculosis CoaB, which we show to bind to a cryptic allosteric site within CoaB., The bifunctional enzyme CoaBC catalyses the second and third step in the Coenzyme A (CoA) biosynthesis pathway and is of interest as a M. tuberculosis drug target. Here, the authors present the full-length crystal structure of Mycobacterium smegmatis CoaBC, which is regulated by CoA and CoA thioesters and forms a dodecamer and by performing a high-throughput screen they identify selective inhibitors of M. tuberculosis CoaB that bind to an allosteric site within CoaB.

Antimicrobial susceptibility testing is the mainstay of tuberculosis drug development programs. In this chapter, we describe methods for determination of the minimum inhibitory concentration of compounds against Mycobacterium tuberculosis growing in liquid media as a function of carbon source, detergent, and environmental stress imposed by acidic pH as well as reactive nitrogen intermediates. Methods for determining the effect of bovine serum albumin in the growth medium on antimicrobial susceptibility are also described. Finally, we provide a method for antimicrobial susceptibility testing on agar medium.

Mycobacterium tuberculosis resides in the lungs in various lesion types with unique microenvironmental conditions. This diversity is in line with heterogeneous disease progression and divergent drug efficiency. Fluorescent reporter strains can be used to decipher the micromilieu and to guide future treatment regimens. Current reporters using replicating plasmids, however, are not suitable for long-term mouse infections or studies in non-human primates. Using a combination of recombinant DNA and protein optimization techniques, we have developed reporter strains based on integrative plasmids, which exhibit stimulus-response characteristics and fluorescence intensities comparable to those based on replicating plasmids. We successfully applied the concepts by constructing a multi-color reporter strain able to detect simultaneous changes in environmental pH, Mg2+ concentrations, and protein expression levels.

In Escherichia coli, expression of the RelE and HipA toxins in the absence of their cognate antitoxins has been associated with generating multidrug-tolerant “persisters.” Here we show that unlike persisters of E. coli, persisters of Mycobacterium tuberculosis selected with one drug do not acquire cross-resistance to other classes of drugs. M. tuberculosis has three homologs of RelE arranged in operons with their apparent antitoxins. Each toxin individually arrests growth of both M. tuberculosis and E. coli, an effect that is neutralized by coexpression of the cognate antitoxin. Overexpression or deletion of each of the RelE toxins had a toxin- and drug-specific effect on the proportion of bacilli surviving antibiotic killing. All three toxins were upregulated in vivo, but none of the deletions affected survival during murine infection. RelE2 overexpression increased bacterial survival rates in the presence of rifampin in vitro, while deletion significantly decreased survival rates. Strikingly, deletion of this toxin had no discernible effect on the level of persisters seen in rifampin-treated mice. Our results suggest that, in vivo, RelE-generated persisters are unlikely to play a significant role in the generation of bacilli that survive in the face of multidrug therapy or in the generation of multidrug-resistant M. tuberculosis.

Candida auris is an emerging fatal fungal infection that has resulted in several outbreaks in hospitals and care facilities. Current treatment options are limited by the development of drug resistance. Identification of new pharmaceuticals to combat these drug-resistant infections will thus be required to overcome this unmet medical need. We have established a bioluminescent ATP-based assay to identify new compounds and potential drug combinations showing effective growth inhibition against multiple strains of multidrug-resistant Candida auris. The assay is robust and suitable for assessing large compound collections by high-throughput screening (HTS). Utilizing this assay, we conducted a screen of 4,314 approved drugs and pharmacologically active compounds that yielded 25 compounds, including 6 novel anti-Candida auris compounds and 13 sets of potential two-drug combinations. Among the drug combinations, the serine palmitoyltransferase inhibitor myriocin demonstrated a combinational effect with flucytosine against all tested isolates during screening. This combinational effect was confirmed in 13 clinical isolates of Candida auris.

The synthesis, absolute stereochemical configuration, complete biological characterization, mechanism of action and resistance, and pharmacokinetic properties of (S)-(−)-acidomycin are described. Acidomycin possesses promising antitubercular activity against a series of contemporary drug susceptible and drug-resistant M. tuberculosis strains (MICs = 0.096–6.2 μM), but is inactive against non-tuberculosis mycobacteria, gram-positive and gram-negative pathogens (MICs > 1000 μM). Complementation studies with biotin biosynthetic pathway intermediates and subsequent biochemical studies confirmed acidomycin inhibits biotin synthesis with a K(i) of approximately 1 μM through the competitive inhibition of biotin synthase (BioB) and also stimulates unproductive cleavage of S-adenosylmethionine (SAM) to generate the toxic metabolite 5′-deoxyadenosine. Cell studies demonstrate acidomycin selectively accumulates in M. tuberculosis providing a mechanistic basis for the observed antibacterial activity. The development of spontaneous resistance by M. tuberculosis to acidomycin was difficult and only low-level resistance to acidomycin was observed by overexpression of BioB. Collectively, the results provide a foundation to advance acidomycin and highlight BioB as a promising target.

Effective treatment of tuberculosis is challenged by the rapid development of Mycobacterium tuberculosis (Mtb) multidrug resistance that presumably could be overcome with novel multi-target drugs. Aminoacyl-tRNA synthetases (AARSs) are an essential part of protein biosynthesis machinery and attractive targets for drug discovery. Here, we experimentally verify a hypothesis of simultaneous targeting of structurally related AARSs by a single inhibitor. We previously identified a new class of mycobacterial leucyl-tRNA synthetase inhibitors, N-benzylidene-N'-thiazol-2-yl-hydrazines. Molecular docking of a library of novel N-benzylidene-N'-thiazol-2-yl-hydrazine derivatives into active sites of M. tuberculosis LeuRS (MtbLeuRS) and MetRS (MtbMetRS) resulted in a panel of the best ranking compounds, which were then evaluated for enzymatic potency. Screening data revealed 11 compounds active against MtbLeuRS and 28 compounds active against MtbMetRS. The hit compounds display dual inhibitory potency as demonstrated by IC50 values for both enzymes. Compound 3 is active against Mtb H37Rv cells in in vitro bioassays.

Severe malaria due to Plasmodium falciparum remains a significant global health threat. DXR, the second enzyme in the MEP pathway, plays an important role to synthesize building blocks for isoprenoids. This enzyme is a promising drug target for malaria due to its essentiality as well as its absence in humans. In this study, we designed and synthesized a series of α,β-unsaturated analogues of fosmidomycin, a natural product that inhibits DXR in P. falciparum. All compounds were evaluated as inhibitors of P. falciparum. The most promising compound, 18a, displays on-target, potent inhibition against the growth of P. falciparum (IC(50) = 13 nM) without significant inhibition of HepG2 cells (IC(50) > 50 μM). 18a was also tested in a luciferase-based Plasmodium berghei mouse model of malaria and showed exceptional in vivo efficacy. Together, the data support MEPicide 18a as a novel, potent, and promising drug candidate for the treatment of malaria.

New drugs and molecular targets are urgently needed to address the emergence and spread of drug-resistant tuberculosis. Mycobacterium tuberculosis ( Mtb) inosine 5'-monophosphate dehydrogenase 2 ( MtbIMPDH2) is a promising yet controversial potential target. The inhibition of MtbIMPDH2 blocks the biosynthesis of guanine nucleotides, but high concentrations of guanine can potentially rescue the bacteria. Herein we describe an expansion of the structure-activity relationship (SAR) for the benzoxazole series of MtbIMPDH2 inhibitors and demonstrate that minimum inhibitory concentrations (MIC) of ≤1 μM can be achieved. The antibacterial activity of the most promising compound, 17b (Q151), is derived from the inhibition of MtbIMPDH2 as demonstrated by conditional knockdown and resistant strains. Importantly, guanine does not change the MIC of 17b, alleviating the concern that guanine salvage can protect Mtb in vivo. These findings suggest that MtbIMPDH2 is a vulnerable target for tuberculosis.

Six known compounds; β-sitosterol, ursolic acid, methyl chlorogenate, ethyl chlorogenate, rutin and mannitol were isolated from the leaf extracts of Pavetta crassipes (Rubiaceae), a Nigerian medicinal plant used in the indigenous treatment of tuberculosis. Separation and isolation of the compounds were achieved by chromatographic techniques and the structures of isolated compounds were established by spectroscopic and chemical methods. The isolated compounds were screened for antimycobacterial activities against Mycobacterium tuberculosis H37Rv employing the green fluorescence protein reporter microplate assay and the broth microdilution method. Ursolic acid, methyl chlorogenate and ethyl chlorogenate were found moderately active in the broth microdilution assay with MICs of 200, 100 and 50 µg/ml, respectively while methyl chlorogenate and ethyl chlorogenate were active in the protein reporter microplate assay with MICs of 200 and 100 µg ml-1, respectively. The presence of antimycobacterial terpenoids and quinate esters in leaves of Pavetta crassipes provides scientific evidence for the ethnomedicinal use of the plant as a traditional anti-tuberculosis remedy.

[Image: see text] 5′-O-[N-(Salicyl)sulfamoyl]adenosine (Sal-AMS, 1) is a nucleoside antibiotic that inhibits incorporation of salicylate into siderophores required for bacterial iron acquisition and has potent activity against Mycobacterium tuberculosis (Mtb). Cinnolone analogues exemplified by 5 were designed to replace the acidic acyl-sulfamate functional group of 1 (pK(a) = 3) by a more stable sulfonamide linkage (pK(a) = 6.0) in an attempt to address potential metabolic liabilities and improve membrane permeability. We showed 5 potently inhibited the mycobacterial salicylate ligase MbtA (apparent K(i) = 12 nM), blocked production of the salicylate-capped siderophores in whole-cell Mtb, and exhibited excellent antimycobacterial activity under iron-deficient conditions (minimum inhibitor concentration, MIC = 2.3 μM). To provide additional confirmation of the mechanism of action, we demonstrated the whole-cell activity of 5 could be fully antagonized by the addition of exogenous salicylate to the growth medium. Although the total polar surface area (tPSA) of 5 still exceeds the nominal threshold value (140 Å) typically required for oral bioavailability, we were pleasantly surprised to observe introduction of the less acidic and conformationally constrained cinnolone moiety conferred improved drug disposition properties as evidenced by the 7-fold increase in volume of distribution in Sprague–Dawley rats.

A major constraint in reducing tuberculosis epidemic is the emergence of strains resistant to one or more of clinically approved antibiotics, which emphasizes the need of novel drugs with novel targets. Genetic knockout strains of Mycobacterium tuberculosis (Mtb) have established that tryptophan (Trp) biosynthesis is essential for the bacterium to survive in vivo and cause disease in animal models. An anthranilate-like compound, 6-FABA, was previously shown to synergize with the host immune response to Mtb infection in vivo. Herein, we present a class of anthranilate-like compounds endowed with good antimycobacterial activity and low cytotoxicity. We show how replacing the carboxylic moiety with a hydrazide led to a significant improvement in both activity and cytotoxicity relative to the parent compound 6-FABA. Several new benzohydrazides (compounds 20–31, 33, 34, 36, 38 and 39) showed good activities against Mtb (0.625 ≤ MIC≤6.25 μM) and demonstrated no detectable cytotoxicity against Vero cell assay (CC50 ≥ 1360 μM). The target preliminary studies confirmed the hypothesis that this new class of compounds inhibits Trp biosynthesis. Taken together, these findings indicate that fluorophenylbenzohydrazides represent good candidates to be assessed for drug discovery.

Mycobacterium tuberculosis thymidylate kinase (MtTMPK) has emerged as an attractive target for rational drug design. We recently investigated new families of non-nucleoside MtTMPK inhibitors in an effort to diversify MtTMPK inhibitor chemical space. We here report a new series of MtTMPK inhibitors by combining the Topliss scheme with rational drug design approaches, fueled by two co-crystal structures of MtTMPK in complex with developed inhibitors. These efforts furnished the most potent MtTMPK inhibitors in our assay, with two analogues displaying low micromolar MIC values against H37Rv Mtb. Prepared inhibitors address new sub-sites in the MtTMPK nucleotide binding pocket, thereby offering new insights into its druggability. We studied the role of efflux pumps as well as the impact of cell wall permeabilizers for selected compounds to potentially provide an explanation for the lack of correlation between potent enzyme inhibition and whole-cell activity.

Nicotinamide adenine dinucleotide (NAD+) synthetase catalyzes the last step in NAD+ biosynthesis. Depletion of NAD+ is bactericidal for both active and dormant Mycobacterium tuberculosis (Mtb). By inhibiting NAD+ synthetase (NadE) from Mtb, we expect to eliminate NAD+ production which will result in cell death in both growing and nonreplicating Mtb. NadE inhibitors have been investigated against various pathogens, but few have been tested against Mtb. Here, we report on the expansion of a series of urea-sulfonamides, previously reported by Brouillette et al. Guided by docking studies, substituents on a terminal phenyl ring were varied to understand the structure-activity-relationships of substituents on this position. Compounds were tested as inhibitors of both recombinant Mtb NadE and Mtb whole cells. While the parent compound displayed very weak inhibition against Mtb NadE (IC50 = 1000 μM), we observed up to a 10-fold enhancement in potency after optimization. Replacement of the 3,4-dichloro group on the phenyl ring of the parent compound with 4-nitro yielded 4f, the most potent compound of the series with an IC50 value of 90 μM against Mtb NadE. Our modeling results show that these urea-sulfonamides potentially bind to the intramolecular ammonia tunnel, which transports ammonia from the glutaminase domain to the active site of the enzyme. This hypothesis is supported by data showing that, even when treated with potent inhibitors, NadE catalysis is restored when treated with exogenous ammonia. Most of these compounds also inhibited Mtb cell growth with MIC values of 19-100 μg/mL. These results improve our understanding of the SAR of the urea-sulfonamides, their mechanism of binding to the enzyme, and of Mtb NadE as a potential antitubercular drug target.

Though phenotypic and target-based high-throughput screening approaches have been employed to discover new antibiotics, the identification of promising therapeutic candidates remains challenging. Each approach provides different information, and understanding their results can provide hypotheses for a mechanism of action (MoA) and reveal actionable chemical matter. Here, we describe a framework for identifying efficacy targets of bioactive compounds. High throughput biophysical profiling against a broad range of targets coupled with machine learning was employed to identify chemical features with predicted efficacy targets for a given phenotypic screen. We validate the approach on data from a set of 55 000 compounds in 24 historical internal antibacterial phenotypic screens and 636 bacterial targets screened in high-throughput biophysical binding assays. Models were built to reveal the relationships between phenotype, target, and chemotype, which recapitulated mechanisms for known antibacterials. We also prospectively identified novel inhibitors of dihydrofolate reductase with nanomolar antibacterial efficacy against Mycobacterium tuberculosis. Molecular modeling provided structural insight into target-ligand interactions underlying selective killing activity toward mycobacteria over human cells.

Tryptophan biosynthesis represents an important potential drug target for new anti-TB drugs. We identified a series of indole-4-carboxamides with potent antitubercular activity. In vitro, Mycobacterium tuberculosis (Mtb) acquired resistance to these compounds through three discrete mechanisms: (1) a decrease in drug metabolism via loss-of-function mutations in the amidase that hydrolyses these carboxamides, (2) an increased biosynthetic rate of tryptophan precursors via loss of allosteric feedback inhibition of anthranilate synthase (TrpE), and (3) mutation of tryptophan synthase (TrpAB) that decreased incorporation of 4-aminoindole into 4-aminotryptophan. Thus, these indole-4-carboxamides act as prodrugs of a tryptophan antimetabolite, 4-aminoindole.

A series of Mycobacterium tuberculosis TMPK (MtbTMPK) inhibitors based on a reported compound 3 were synthesized and evaluated for their capacity to inhibit MtbTMPK catalytic activity and the growth of a virulent M. tuberculosis strain (H37Rv). Modifications of the scaffold of 3 failed to afford substantial improvements in MtbTMPK inhibitory activity and antimycobacterial activity. Optimization of the substitution pattern of the D ring of 3 resulted in compound 21j with improved MtbTMPK inhibitory potency (three-fold) and H37Rv growth inhibitory activity (two-fold). Moving the 3-chloro substituent of 21j to the para-position afforded isomer 21h, which, despite a 10-fold increase in IC50-value, displayed promising whole cell activity (minimum inhibitory concentration (MIC) = 12.5 &mu, M).

The viability of Mycobacterium tuberculosis (Mtb) depends on energy generated by its respiratory chain. Cytochrome bc1-aa3 oxidase and type-2 NADH dehydrogenase (NDH-2) are respiratory chain components predicted to be essential, and are currently targeted for drug development. Here we demonstrate that an Mtb cytochrome bc1-aa3 oxidase deletion mutant is viable and only partially attenuated in mice. Moreover, treatment of Mtb-infected marmosets with a cytochrome bc1-aa3 oxidase inhibitor controls disease progression and reduces lesion-associated inflammation, but most lesions become cavitary. Deletion of both NDH-2 encoding genes (Δndh-2 mutant) reveals that the essentiality of NDH-2 as shown in standard growth media is due to the presence of fatty acids. The Δndh-2 mutant is only mildly attenuated in mice and not differently susceptible to clofazimine, a drug in clinical use proposed to engage NDH-2. These results demonstrate the intrinsic plasticity of Mtb’s respiratory chain, and highlight the challenges associated with targeting the pathogen’s respiratory enzymes for tuberculosis drug development., New tuberculosis therapies, targeting respiratory chain components of Mycobacterium tuberculosis, are under development. Here the authors show that, contrary to common belief, some of these components are not essential for pathogen viability and/or virulence in animal models of infection.

With the growing worldwide prevalence of antibiotic-resistant strains of tuberculosis (TB), new targets are urgently required for the development of treatments with novel modes of action. Fumarate hydratase (fumarase), a vulnerable component of the citric acid cycle in Mycobacterium tuberculosis (Mtb), is a metabolic target that could satisfy this unmet demand. A key challenge in the targeting of Mtb fumarase is its similarity to the human homolog, which shares an identical active site. A potential solution to this selectivity problem was previously found in a high-throughput screening hit that binds in a nonconserved allosteric site. In this work, a structure-activity relationship study was carried out with the determination of further structural biology on the lead series, affording derivatives with sub-micromolar inhibition. Further, the screening of this series against Mtb in vitro identified compounds with potent minimum inhibitory concentrations.

Effective treatment of tuberculosis is challenged by the rapid development of Mycobacterium tuberculosis (Mtb) multidrug resistance that presumably could be overcome with novel multi-target drugs. Aminoacyl-tRNA synthetases (AARSs) are an essential part of protein biosynthesis machinery and attractive targets for drug discovery. Here, we experimentally verify a hypothesis of simultaneous targeting of structurally related AARSs by a single inhibitor. We previously identified a new class of mycobacterial leucyl-tRNA synthetase inhibitors, N-benzylidene-N′-thiazol-2-yl-hydrazines. Molecular docking of a library of novel N-benzylidene-N′-thiazol-2-yl-hydrazine derivatives into active sites of M. tuberculosis LeuRS (MtbLeuRS) and MetRS (MtbMetRS) resulted in a panel of the best ranking compounds, which were then evaluated for enzymatic potency. Screening data revealed 11 compounds active against MtbLeuRS and 28 compounds active against MtbMetRS. The hit compounds display dual inhibitory potency as demonstrated by IC(50) values for both enzymes. Compound 3 is active against Mtb H37Rv cells in in vitro bioassays.

Diphenyl ether derivatives inhibit mycobacterial cell wall synthesis by inhibiting an enzyme, enoyl-acyl carrier protein reductase (InhA), which catalyses the last step in the fatty acid synthesis cycle of genus Mycobacterium. To select and validate a protein crystal structure of enoyl-acyl carrier protein reductase of Mycobacterium tuberculosis for designing inhibitors using molecular modelling, a cross-docking and correlation study was performed. A series of novel 1-(3-(3-hydroxy-4-phenoxyphenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl) ethan-1-ones were synthesized from this model and screened for their antitubercular activity against M. tuberculosis H37Rv. Compound PYN-8 showed good antitubercular activity on M. tuberculosis H37Rv (MIC = 4–7 µM) and Mycobacterium bovis (% inhibition at 10 µM = 95.91%). Cytotoxicity of all the synthesized derivatives was assessed using various cell lines, and they were found to be safe. Structure of PYN-8 was also confirmed by single-crystal X-ray diffraction. The molecular modelling studies also corroborated the biological activity of the compounds. Further, in silico findings revealed that all these tested compounds exhibited good ADME properties and drug likeness and thus may be considered as potential candidates for further drug development.

Mycobacterium abscessus (Mab) is a rapidly growing species of multidrug-resistant nontuberculous mycobacteria (NTM) that has emerged as a growing threat to individuals with cystic fibrosis, and other pre-existing chronic lung diseases. Mab pulmonary infections are difficult, or sometimes impossible, to treat and result in accelerated lung function decline and premature death. There is therefore an urgent need to develop novel antibiotics with improved efficacy. tRNA (m1G37) methyltransferase (TrmD) is a promising target for novel antibiotics. It is essential in Mab and other mycobacteria, improving reading frame maintenance on the ribosome to prevent frameshift errors. In this work a fragment-based approach was employed with the merging of two fragments bound to the active site, followed by structure-guided elaboration to design potent nanomolar inhibitors against Mab TrmD. Several of these compounds exhibit promising activity against mycobacterial species, including Mycobacterium tuberculosis and Mycobacterium leprae in addition to Mab, supporting the use of TrmD as a target for the development of antimycobacterial compounds.

Mechanisms of magnesium homeostasis in Mycobacterium tuberculosis are poorly understood. Here, we describe the characterization of a pyrimidinetrione amide scaffold that disrupts magnesium homeostasis in the pathogen by direct binding to the CorA Mg(2+)/Co(2+) transporter. Mutations in domains of CorA that are predicted to regulate the pore opening in response to Mg(2+) ions conferred resistance to this scaffold. The pyrimidinetrione amides were cidal against the pathogen under both actively replicating and nonreplicating conditions in vitro and were efficacious against the organism during macrophage infection. However, the compound lacked efficacy in infected mice, possibly due to limited exposure. Our results indicate that inhibition of Mg(2+) homeostasis by CorA is an attractive target for tuberculosis drug discovery and encourage identification of improved CorA inhibitors.

Translational frameshift errors are often deleterious to the synthesis of functional proteins as they lead to the production of truncated or inactive proteins. TrmD (tRNA-(N(1)G37) methyltransferase) is an essential tRNA modification enzyme in bacteria that prevents +1 errors in the reading frame during protein translation and has been identified as a therapeutic target for several bacterial infections. Here we validate TrmD as a target inMycobacterium abscessusand describe the application of a structure-guided fragment-based drug discovery approach for the design of a new class of inhibitors against this enzyme. A fragment library screening followed by structure-guided chemical elaboration of hits led to the development of compounds with potentin vitroTrmD inhibitory activity. Several of these compounds exhibit activity against planktonicM. abscessus and Mycobacterium tuberculosis.The compounds were further active in macrophage infection models againstMycobacterium lepraeandM. abscessussuggesting the potential for novel broad-spectrum mycobacterial drugs.

Isoniazid (INH) remains a cornerstone for treatment of drug susceptible tuberculosis (TB), yet the quantitative structure-activity relationships for INH are not well documented in the literature. In this paper, we have evaluated a systematic series of INH analogs against contemporary Mycobacterium tuberculosis strains from different lineages and a few non-tuberculous mycobacteria (NTM). Deletion of the pyridyl nitrogen atom, isomerization of the pyridine nitrogen to other positions, replacement of the pyridine ring with isosteric heterocycles, and modification of the hydrazide moiety of INH abolishes antitubercular activity. Similarly, substitution of the pyridine ring at the 3-position is not tolerated while substitution at the 2-position is permitted with 2-methyl-INH 9 displaying antimycobacterial activity comparable to INH. To assess the specific activity of this series of INH analogs against mycobacteria, we assayed them against a panel of gram-positive and gram-negative bacteria, as well as a few fungi. As expected INH and its analogs display a narrow spectrum of activity and are inactive against all non-mycobacterial strains evaluated, except for 4, which has modest inhibitory activity against Cryptococcus neoformans. Our findings provide an updated analysis of the structure-activity relationship of INH that we hope will serve as useful resource for the community.

Elucidating the target or mechanism of action of potential drugs in the discovery pipeline is an integral component of most programs. For antibacterial compounds, generation of resistant mutants followed by whole genome sequencing has often been successful in uncovering the proteins involved in regulating compound activation, uptake, efflux and importantly, target processes. When this process succeeds, we are quick to declare a target. In a study reported by Sing and Dhar et al. (in press), the combination of resistant mutant generation, whole genome sequencing and recombineering to identify the target of a Mycobacterium tuberculosis growth inhibitor, pointed to a mechanism involving a scaffolding protein, Wag31, involved in polar elongation of mycobacterial cells. Time-lapse microscopy and electron microscopy confirmed the view that this inhibitor resulted in interruption of nascent cell wall biosynthesis. However, co-expression as well as regulated titration of the putative Wag31 target demonstrated that the wild-type allele was dominant and showed no synergy with the inhibitor. The most plausible explanation from their results was that this inhibitor interfered with the interaction of Wag31 with one of its interacting partners in the elongation complex.