Publications by authors named "Clifton E Barry"

212 Publications

Targeting CoaBC through Chemical Inhibition of 4'-Phosphopantothenoyl-l-cysteine Synthetase (CoaB) Activity.

ACS Infect Dis 2021 06 3;7(6):1666-1679. Epub 2021 May 3.

MRC/NHLS/UCT Molecular Mycobacteriology Research Unit & DST/NRF Centre of Excellence for Biomedical TB Research & Wellcome Centre for Infectious Diseases Research in Africa, Institute of Infectious Disease and Molecular Medicine and Department of Pathology, Faculty of Health Sciences, University of Cape Town, Anzio Road, Observatory 7925, South Africa.

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. (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 and . Here, we describe the identification of compound , 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 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 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.
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http://dx.doi.org/10.1021/acsinfecdis.0c00904DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8205227PMC
June 2021

Tuberculosis Drug Discovery: A Decade of Hit Assessment for Defined Targets.

Front Cell Infect Microbiol 2021 15;11:611304. Epub 2021 Mar 15.

Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, United States.

More than two decades have elapsed since the publication of the first genome sequence of () 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 or defined targets where mechanism of action investigation has defined target-hit couples and structure-activity relationship studies have described the pharmacophore.
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http://dx.doi.org/10.3389/fcimb.2021.611304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005628PMC
June 2021

Resistance of Mycobacterium tuberculosis to indole 4-carboxamides occurs through alterations in drug metabolism and tryptophan biosynthesis.

Cell Chem Biol 2021 Mar 13. Epub 2021 Mar 13.

Tuberculosis Research Section, Laboratory of Clinical Immunology & Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA; Institute for Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town 7935, South Africa. Electronic address:

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.
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http://dx.doi.org/10.1016/j.chembiol.2021.02.023DOI Listing
March 2021

Fourteen-day PET/CT imaging to monitor drug combination activity in treated individuals with tuberculosis.

Sci Transl Med 2021 Feb;13(579)

Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, Division of Intramural Research, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD 20892, USA.

Early bactericidal activity studies monitor daily sputum bacterial counts in individuals with tuberculosis (TB) for 14 days during experimental drug treatment. The rate of change in sputum bacterial load over time provides an informative, but imperfect, estimate of drug activity and is considered a critical step in development of new TB drugs. In this clinical study, 160 participants with TB received isoniazid, pyrazinamide, or rifampicin, components of first-line chemotherapy, and moxifloxacin individually and in combination. In addition to standard bacterial enumeration in sputum, participants underwent 2-deoxy-2-[F]fluoro-d-glucose positron emission tomography and computerized tomography ([F]FDG-PET/CT) at the beginning and end of the 14-day drug treatment. Quantitating radiological responses to drug treatment provided comparative single and combination drug activity measures across lung lesion types that correlated more closely with established clinical outcomes when combined with sputum enumeration compared to sputum enumeration alone. Rifampicin and rifampicin-containing drug combinations were most effective in reducing both lung lesion volume measured by CT imaging and lesion-associated inflammation measured by PET imaging. Moxifloxacin was not superior to rifampicin in any measure by PET/CT imaging, consistent with its performance in recent phase 3 clinical trials. PET/CT imaging revealed synergy between isoniazid and pyrazinamide and demonstrated that the activity of pyrazinamide was limited to lung lesion, showing the highest FDG uptake during the first 2 weeks of drug treatment. [F]FDG-PET/CT imaging may be useful for measuring the activity of single drugs and drug combinations during evaluation of potential new TB drug regimens before phase 3 trials.
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http://dx.doi.org/10.1126/scitranslmed.abd7618DOI Listing
February 2021

Inhibiting Mycobacterium tuberculosis CoaBC by targeting an allosteric site.

Nat Commun 2021 01 8;12(1):143. Epub 2021 Jan 8.

Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK.

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.
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http://dx.doi.org/10.1038/s41467-020-20224-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7794376PMC
January 2021

Structure-Activity Relationships of Pyrazolo[1,5-]pyrimidin-7(4)-ones as Antitubercular Agents.

ACS Infect Dis 2021 02 6;7(2):479-492. Epub 2021 Jan 6.

Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, United States.

Pyrazolo[1,5-]pyrimidin-7(4)-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 (). 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 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.
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http://dx.doi.org/10.1021/acsinfecdis.0c00851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7887755PMC
February 2021

Antitubercular 2-Pyrazolylpyrimidinones: Structure-Activity Relationship and Mode-of-Action Studies.

J Med Chem 2021 01 4;64(1):719-740. Epub 2021 Jan 4.

Drug Discovery and Development Centre (H3D), Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa.

Phenotypic screening of a Medicines for Malaria Venture compound library against () identified a cluster of pan-active 2-pyrazolylpyrimidinones. The biology triage of these actives using various tool strains of suggested a novel mechanism of action. The compounds were bactericidal against replicating and retained potency against clinical isolates of . Although selected MmpL3 mutant strains of 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 microsomal metabolic stability with moderate selectivity over cytotoxicity against mammalian cell lines.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01727DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7816196PMC
January 2021

Correction for Singh et al., "The Three RelE Homologs of Mycobacterium tuberculosis Have Individual, Drug-Specific Effects on Bacterial Antibiotic Tolerance".

J Bacteriol 2020 Nov 19;202(24). Epub 2020 Nov 19.

Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland 20892

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http://dx.doi.org/10.1128/JB.00554-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685548PMC
November 2020

MAIT cell-directed therapy of Mycobacterium tuberculosis infection.

Mucosal Immunol 2021 01 18;14(1):199-208. Epub 2020 Aug 18.

T Lymphocyte Biology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA.

Mucosal-associated invariant T (MAIT) cells are potential targets of vaccination and host-directed therapeutics for tuberculosis, but the role of MAIT cells during Mycobacterium tuberculosis (Mtb) infection in vivo is not well understood. Here we find that following Mtb infection MAIT cells mount minimal responses, and MAIT cell-deficient MR1 mice display normal survival. Preinfection expansion of MAIT cells through 5-OP-RU vaccination fails to protect against subsequent Mtb challenge. In fact, 5-OP-RU vaccination delays Mtb-specific CD4 T cell priming in lung-draining lymph nodes, and conversely MR1 deficiency or blockade accelerates T cell priming. The MAIT cell-mediated delay in T cell priming is partly dependent on TGF-β. Surprisingly, 5-OP-RU treatment during chronic infection drives MAIT cell expansion and an IL-17A-dependent reduction in bacterial loads. Thus, during early infection MAIT cells directly contribute to the notoriously slow priming of CD4 T cells, but later during infection MAIT cell stimulation may be an effective host-directed therapy for tuberculosis.
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http://dx.doi.org/10.1038/s41385-020-0332-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790750PMC
January 2021

PE/PPE proteins mediate nutrient transport across the outer membrane of .

Science 2020 03;367(6482):1147-1151

Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

has an unusual outer membrane that lacks canonical porin proteins for the transport of small solutes to the periplasm. We discovered that 3,3--di(methylsulfonyl)propionamide (3bMP1) inhibits the growth of , and resistance to this compound is conferred by mutation within a member of the proline-proline-glutamate (PPE) family, PPE51. Deletion of PPE51 rendered cells unable to replicate on propionamide, glucose, or glycerol. Growth was restored upon loss of the mycobacterial cell wall component phthiocerol dimycocerosate. Mutants in other proline-glutamate (PE)/PPE clusters, responsive to magnesium and phosphate, also showed a phthiocerol dimycocerosate-dependent growth compromise upon limitation of the corresponding substrate. Phthiocerol dimycocerosate determined the low permeability of the mycobacterial outer membrane, and the PE/PPE proteins apparently act as solute-specific channels.
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http://dx.doi.org/10.1126/science.aav5912DOI Listing
March 2020

Current and future treatments for tuberculosis.

BMJ 2020 03 2;368:m216. Epub 2020 Mar 2.

Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, Division of Intramural Medicine, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.

Guidelines on the treatment of tuberculosis (TB) have essentially remained the same for the past 35 years, but are now starting to change. Ongoing clinical trials will hopefully transform the landscape for treatment of drug sensitive TB, drug resistant TB, and latent TB infection. Multiple trials are evaluating novel agents, repurposed agents, adjunctive host directed therapies, and novel treatment strategies that will increase the probability of success of future clinical trials. Guidelines for HIV-TB co-infection treatment continue to be updated and drug resistance testing has been revolutionized in recent years with the shift from phenotypic to genotypic testing and the concomitant increased speed of results. These coming changes are long overdue and are sorely needed to address the vast disparities in global TB incidence rates. TB is currently the leading cause of death globally from a single infectious agent, but the work of many researchers and the contributions of many patients in clinical trials will reduce the substantial global morbidity and mortality of the disease.
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http://dx.doi.org/10.1136/bmj.m216DOI Listing
March 2020

Quantitative 18F-FDG PET-CT scan characteristics correlate with tuberculosis treatment response.

EJNMMI Res 2020 Feb 10;10(1). Epub 2020 Feb 10.

Department of Science and Technology/National Research Foundation, Centre of Excellence for Biomedical Tuberculosis Research and South African Medical Research Council Centre for Tuberculosis Research, Cape Town, South Africa.

Background: There is a growing interest in the use of F-18 FDG PET-CT to monitor tuberculosis (TB) treatment response. Tuberculosis lung lesions are often complex and diffuse, with dynamic changes during treatment and persisting metabolic activity after apparent clinical cure. This poses a challenge in quantifying scan-based markers of burden of disease and disease activity. We used semi-automated, whole lung quantification of lung lesions to analyse serial FDG PET-CT scans from the Catalysis TB Treatment Response Cohort to identify characteristics that best correlated with clinical and microbiological outcomes.

Results: Quantified scan metrics were already associated with clinical outcomes at diagnosis and 1 month after treatment, with further improved accuracy to differentiate clinical outcomes after standard treatment duration (month 6). A high cavity volume showed the strongest association with a risk of treatment failure (AUC 0.81 to predict failure at diagnosis), while a suboptimal reduction of the total glycolytic activity in lung lesions during treatment had the strongest association with recurrent disease (AUC 0.8 to predict pooled unfavourable outcomes). During the first year after TB treatment lesion burden reduced; but for many patients, there were continued dynamic changes of individual lesions.

Conclusions: Quantification of FDG PET-CT images better characterised TB treatment outcomes than qualitative scan patterns and robustly measured the burden of disease. In future, validated metrics may be used to stratify patients and help evaluate the effectiveness of TB treatment modalities.
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http://dx.doi.org/10.1186/s13550-020-0591-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7010890PMC
February 2020

Setting Our Sights on Infectious Diseases.

ACS Infect Dis 2020 01 6;6(1):3-13. Epub 2019 Dec 6.

Global Health Institute of Merck, Ares Trading S.A. , a subsidiary of Merck KGaA Darmstadt Germany , Route de Crassier 1 , 1262 Eysins , Switzerland.

In May 2019, the Wellcome Centre for Anti-Infectives Research (CAIR) at the University of Dundee, UK, held an international conference with the aim of discussing some key questions around discovering new medicines for infectious diseases and a particular focus on diseases affecting Low and Middle Income Countries. There is an urgent need for new drugs to treat most infectious diseases. We were keen to see if there were lessons that we could learn across different disease areas and between the preclinical and clinical phases with the aim of exploring how we can improve and speed up the drug discovery, translational, and clinical development processes. We started with an introductory session on the current situation and then worked backward from clinical development to combination therapy, pharmacokinetic/pharmacodynamic (PK/PD) studies, drug discovery pathways, and new starting points and targets. This Viewpoint aims to capture some of the learnings.
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http://dx.doi.org/10.1021/acsinfecdis.9b00371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6958537PMC
January 2020

Plasticity of the Mycobacterium tuberculosis respiratory chain and its impact on tuberculosis drug development.

Nat Commun 2019 10 31;10(1):4970. Epub 2019 Oct 31.

Department of Microbiology and Immunology, Weill Cornell Medical College, New York, NY, 10065, USA.

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.
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http://dx.doi.org/10.1038/s41467-019-12956-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6823465PMC
October 2019

Targeting of Fumarate Hydratase from Using Allosteric Inhibitors with a Dimeric-Binding Mode.

J Med Chem 2019 12 27;62(23):10586-10604. Epub 2019 Sep 27.

Department of Chemistry , University of Cambridge , Lensfield Road , Cambridge CB2 1EW , U.K.

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 (), is a metabolic target that could satisfy this unmet demand. A key challenge in the targeting of 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 in vitro identified compounds with potent minimum inhibitory concentrations.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01203DOI Listing
December 2019

Mode-of-action profiling reveals glutamine synthetase as a collateral metabolic vulnerability of to bedaquiline.

Proc Natl Acad Sci U S A 2019 09 9;116(39):19646-19651. Epub 2019 Sep 9.

Division of Infectious Diseases, Weill Department of Medicine, Weill Cornell Medical College, New York, NY 10065;

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 (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.
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http://dx.doi.org/10.1073/pnas.1907946116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6765305PMC
September 2019

Inhibition of CorA-Dependent Magnesium Homeostasis Is Cidal in Mycobacterium tuberculosis.

Antimicrob Agents Chemother 2019 10 23;63(10). Epub 2019 Sep 23.

Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland, USA

Mechanisms of magnesium homeostasis in 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/Co transporter. Mutations in domains of CorA that are predicted to regulate the pore opening in response to Mg ions conferred resistance to this scaffold. The pyrimidinetrione amides were cidal against the pathogen under both actively replicating and nonreplicating conditions 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 homeostasis by CorA is an attractive target for tuberculosis drug discovery and encourage identification of improved CorA inhibitors.
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http://dx.doi.org/10.1128/AAC.01006-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6761525PMC
October 2019

Changes in inflammatory protein and lipid mediator profiles persist after antitubercular treatment of pulmonary and extrapulmonary tuberculosis: A prospective cohort study.

Cytokine 2019 11 18;123:154759. Epub 2019 Jun 18.

Inflammation and Innate Immunity Unit, Laboratory of Clinical Immunology and Microbiology, NIAID, NIH, Bethesda 20892, MD, USA. Electronic address:

Background: The identification of meaningful biomarkers of tuberculosis (TB) has potential to improve diagnosis, disease staging and prediction of treatment outcomes. It has been shown that active pulmonary TB (PTB) is associated with qualitative and quantitative changes in systemic immune profile, suggesting a chronic inflammatory imbalance. Here we characterized the profile of PTB and extrapulmonary TB (EPTB) in a prospective cohort study.

Methods: We measured a panel of 27 inflammatory cytokines, soluble receptors, and lipid mediators in peripheral blood from patients with PTB or EPTB from a prospective clinical study in China. Multidimensional analyses were performed to describe associations between plasma levels of biomarkers and different TB disease presentation profiles.

Results: Mycobacterium tuberculosis infection induced changes in both the expression and correlation profiles of plasma mediators of inflammation in patients with PTB compared to those with EPTB. Increases in mycobacterial loads in sputum smears were associated with rises in concentrations of several molecules involved in TB pathogenesis, such as IL-1β, IFN-α, IL-10 and PGF2α. Moreover, PTB patients presenting with severe disease exhibited a distinct inflammatory profile hallmarked by heightened levels of TNF-α, IL-1β, IL17, IL-18 and IL-27. Interestingly, while antitubercular treatment (ATT) resulted in early changes of plasma concentrations of markers in PTB, changes were delayed in EPTB patients. Exploratory analyses of the molecular degree of perturbation (MDP) of the inflammatory mediators before and during ATT suggested the occurrence of infection and/or treatment-induced long lasting "inflammatory imprinting" of biomarker profiles in TB. At 24 weeks post ATT commencement, markers underlying the observed increases in MDP scores were IL-27 in PTB and IL-1β in EPTB patients.

Conclusion: Our findings describe systemic and durable changes in the concentrations of inflammatory cytokines and lipid mediators in both PTB and EPTB and emphasize the role of M. tuberculosis bacterial burden and site of disease in modulating patient immune biomarkers.
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http://dx.doi.org/10.1016/j.cyto.2019.154759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6739167PMC
November 2019

Molecular degree of perturbation of plasma inflammatory markers associated with tuberculosis reveals distinct disease profiles between Indian and Chinese populations.

Sci Rep 2019 05 29;9(1):8002. Epub 2019 May 29.

Instituto Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, 40296-710, Brazil.

Tuberculosis (TB) is a chronic inflammatory disease caused by Mycobacterium tuberculosis infection which causes tremendous morbidity and mortality worldwide. Clinical presentation of TB patients is very diverse and disease heterogeneity is associated with changes in biomarker signatures. Here, we compared at the molecular level the extent of individual inflammatory perturbation of plasma protein and lipid mediators associated with TB in patients in China versus India. We performed a cross-sectional study analyzing the overall degree of inflammatory perturbation in treatment-naïve pulmonary TB patients and uninfected individuals from India (TB: n = 97, healthy: n = 20) and China (TB: n = 100, healthy: n = 11). We employed the molecular degree of perturbation (MDP) adapted to plasma biomarkers to examine the overall changes in inflammation between these countries. M. tuberculosis infection caused a significant degree of molecular perturbation in patients from both countries, with higher perturbation detected in India. Interestingly, there were differences in biomarker perturbation patterns and the overall degree of inflammation. Patients with severe TB exhibited increased MDP values and Indian patients with this condition exhibited even higher degree of perturbation compared to Chinese patients. Network analyses identified IFN-α, IFN-β, IL-1RI and TNF-α as combined biomarkers that account for the overall molecular perturbation in the entire study population. Our results delineate the magnitude of the systemic inflammatory perturbation in pulmonary TB and reveal qualitative changes in inflammatory profiles between two countries with high disease prevalence.
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http://dx.doi.org/10.1038/s41598-019-44513-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6541651PMC
May 2019

Tuberculosis drugs' distribution and emergence of resistance in patient's lung lesions: A mechanistic model and tool for regimen and dose optimization.

PLoS Med 2019 04 2;16(4):e1002773. Epub 2019 Apr 2.

Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, California, United States of America.

Background: The sites of mycobacterial infection in the lungs of tuberculosis (TB) patients have complex structures and poor vascularization, which obstructs drug distribution to these hard-to-reach and hard-to-treat disease sites, further leading to suboptimal drug concentrations, resulting in compromised TB treatment response and resistance development. Quantifying lesion-specific drug uptake and pharmacokinetics (PKs) in TB patients is necessary to optimize treatment regimens at all infection sites, to identify patients at risk, to improve existing regimens, and to advance development of novel regimens. Using drug-level data in plasma and from 9 distinct pulmonary lesion types (vascular, avascular, and mixed) obtained from 15 hard-to-treat TB patients who failed TB treatments and therefore underwent lung resection surgery, we quantified the distribution and the penetration of 7 major TB drugs at these sites, and we provide novel tools for treatment optimization.

Methods And Findings: A total of 329 plasma- and 1,362 tissue-specific drug concentrations from 9 distinct lung lesion types were obtained according to optimal PK sampling schema from 15 patients (10 men, 5 women, aged 23 to 58) undergoing lung resection surgery (clinical study NCT00816426 performed in South Korea between 9 June 2010 and 24 June 2014). Seven major TB drugs (rifampin [RIF], isoniazid [INH], linezolid [LZD], moxifloxacin [MFX], clofazimine [CFZ], pyrazinamide [PZA], and kanamycin [KAN]) were quantified. We developed and evaluated a site-of-action mechanistic PK model using nonlinear mixed effects methodology. We quantified population- and patient-specific lesion/plasma ratios (RPLs), dynamics, and variability of drug uptake into each lesion for each drug. CFZ and MFX had higher drug exposures in lesions compared to plasma (median RPL 2.37, range across lesions 1.26-22.03); RIF, PZA, and LZD showed moderate yet suboptimal lesion penetration (median RPL 0.61, range 0.21-2.4), while INH and KAN showed poor tissue penetration (median RPL 0.4, range 0.03-0.73). Stochastic PK/pharmacodynamic (PD) simulations were carried out to evaluate current regimen combinations and dosing guidelines in distinct patient strata. Patients receiving standard doses of RIF and INH, who are of the lower range of exposure distribution, spent substantial periods (>12 h/d) below effective concentrations in hard-to-treat lesions, such as caseous lesions and cavities. Standard doses of INH (300 mg) and KAN (1,000 mg) did not reach therapeutic thresholds in most lesions for a majority of the population. Drugs and doses that did reach target exposure in most subjects include 400 mg MFX and 100 mg CFZ. Patients with cavitary lesions, irrespective of drug choice, have an increased likelihood of subtherapeutic concentrations, leading to a higher risk of resistance acquisition while on treatment. A limitation of this study was the small sample size of 15 patients, performed in a unique study population of TB patients who failed treatment and underwent lung resection surgery. These results still need further exploration and validation in larger and more diverse cohorts.

Conclusions: Our results suggest that the ability to reach and maintain therapeutic concentrations is both lesion and drug specific, indicating that stratifying patients based on disease extent, lesion types, and individual drug-susceptibility profiles may eventually be useful for guiding the selection of patient-tailored drug regimens and may lead to improved TB treatment outcomes. We provide a web-based tool to further explore this model and results at http://saviclab.org/tb-lesion/.
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http://dx.doi.org/10.1371/journal.pmed.1002773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6445413PMC
April 2019

Discovery and Structure-Activity-Relationship Study of N-Alkyl-5-hydroxypyrimidinone Carboxamides as Novel Antitubercular Agents Targeting Decaprenylphosphoryl-β-d-ribose 2'-Oxidase.

J Med Chem 2018 11 5;61(22):9952-9965. Epub 2018 Nov 5.

Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases , National Institutes of Health , Bethesda , Maryland 20892 , United States.

Magnesium plays an important role in infection with Mycobacterium tuberculosis ( Mtb) as a signal of the extracellular environment, as a cofactor for many enzymes, and as a structural element in important macromolecules. Raltegravir, an antiretroviral drug that inhibits HIV-1 integrase is known to derive its potency from selective sequestration of active-site magnesium ions in addition to binding to a hydrophobic pocket. In order to determine if essential Mtb-related phosphoryl transfers could be disrupted in a similar manner, a directed screen of known molecules with integrase inhibitor-like pharmacophores ( N-alkyl-5-hydroxypyrimidinone carboxamides) was performed. Initial hits afforded compounds with low-micromolar potency against Mtb, acceptable cytotoxicity and PK characteristics, and robust SAR. Elucidation of the target of these compounds revealed that they lacked magnesium dependence and instead disappointingly inhibited a known promiscuous target in Mtb, decaprenylphosphoryl-β-d-ribose 2'-oxidase (DprE1, Rv3790).
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http://dx.doi.org/10.1021/acs.jmedchem.8b00883DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6257622PMC
November 2018

Storage lipid studies in tuberculosis reveal that foam cell biogenesis is disease-specific.

PLoS Pathog 2018 08 30;14(8):e1007223. Epub 2018 Aug 30.

Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ, United States of America.

Foam cells are lipid-laden macrophages that contribute to the inflammation and tissue damage associated with many chronic inflammatory disorders. Although foam cell biogenesis has been extensively studied in atherosclerosis, how these cells form during a chronic infectious disease such as tuberculosis is unknown. Here we report that, unlike the cholesterol-laden cells of atherosclerosis, foam cells in tuberculous lung lesions accumulate triglycerides. Consequently, the biogenesis of foam cells varies with the underlying disease. In vitro mechanistic studies showed that triglyceride accumulation in human macrophages infected with Mycobacterium tuberculosis is mediated by TNF receptor signaling through downstream activation of the caspase cascade and the mammalian target of rapamycin complex 1 (mTORC1). These features are distinct from the known biogenesis of atherogenic foam cells and establish a new paradigm for non-atherogenic foam cell formation. Moreover, they reveal novel targets for disease-specific pharmacological interventions against maladaptive macrophage responses.
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http://dx.doi.org/10.1371/journal.ppat.1007223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6117085PMC
August 2018

The present state of the tuberculosis drug development pipeline.

Curr Opin Pharmacol 2018 10 23;42:81-94. Epub 2018 Aug 23.

Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, United States. Electronic address:

Tuberculosis now ranks as the leading cause of death in the world due to a single infectious agent. Current standard of care treatment can achieve very high cure rates for drug-sensitive disease but requires a 6-month duration of chemotherapy. Drug-resistant disease requires significantly longer treatment durations with drugs associated with a higher risk of adverse events. Thus, there is a pressing need for a drug regimen that is safer, shorter in duration and superior to current front-line chemotherapy in terms of efficacy. The TB drug pipeline contains several candidates that address one or more of the required attributes of chemotherapeutic regimens that may redefine the standard of care of this disease. Several new drugs have been reported and novel targets have been identified allowing regimens containing new compounds to trickle into clinical studies. Furthermore, a recent paradigm-shift in understanding the pharmacokinetics of anti-tubercular drugs is revolutionizing the way we select compounds for clinical progression.
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http://dx.doi.org/10.1016/j.coph.2018.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6204086PMC
October 2018

A semi-automatic technique to quantify complex tuberculous lung lesions on F-fluorodeoxyglucose positron emission tomography/computerised tomography images.

EJNMMI Res 2018 Jun 25;8(1):55. Epub 2018 Jun 25.

Division of Nuclear Medicine, Department of Medical Imaging and Clinical Oncology, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa.

Background: There is a growing interest in the use of F-FDG PET-CT to monitor tuberculosis (TB) treatment response. However, TB causes complex and widespread pathology, which is challenging to segment and quantify in a reproducible manner. To address this, we developed a technique to standardise uptake (Z-score), segment and quantify tuberculous lung lesions on PET and CT concurrently, in order to track changes over time. We used open source tools and created a MATLAB script. The technique was optimised on a training set of five pulmonary tuberculosis (PTB) cases after standard TB therapy and 15 control patients with lesion-free lungs.

Results: We compared the proposed method to a fixed threshold (SUV > 1) and manual segmentation by two readers and piloted the technique successfully on scans of five control patients and five PTB cases (four cured and one failed treatment case), at diagnosis and after 1 and 6 months of treatment. There was a better correlation between the Z-score-based segmentation and manual segmentation than SUV > 1 and manual segmentation in terms of overall spatial overlap (measured in Dice similarity coefficient) and specificity (1 minus false positive volume fraction). However, SUV > 1 segmentation appeared more sensitive. Both the Z-score and SUV > 1 showed very low variability when measuring change over time. In addition, total glycolytic activity, calculated using segmentation by Z-score and lesion-to-background ratio, correlated well with traditional total glycolytic activity calculations. The technique quantified various PET and CT parameters, including the total glycolytic activity index, metabolic lesion volume, lesion volumes at different CT densities and combined PET and CT parameters. The quantified metrics showed a marked decrease in the cured cases, with changes already apparent at month one, but remained largely unchanged in the failed treatment case.

Conclusions: Our technique is promising to segment and quantify the lung scans of pulmonary tuberculosis patients in a semi-automatic manner, appropriate for measuring treatment response. Further validation is required in larger cohorts.
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http://dx.doi.org/10.1186/s13550-018-0411-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6020088PMC
June 2018

Construction of Fluorescent Analogs to Follow the Uptake and Distribution of Cobalamin (Vitamin B) in Bacteria, Worms, and Plants.

Cell Chem Biol 2018 08 17;25(8):941-951.e6. Epub 2018 May 17.

School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK. Electronic address:

Vitamin B is made by only certain prokaryotes yet is required by a number of eukaryotes such as mammals, fish, birds, worms, and Protista, including algae. There is still much to learn about how this nutrient is trafficked across the domains of life. Herein, we describe ways to make a number of different corrin analogs with fluorescent groups attached to the main tetrapyrrole-derived ring. A further range of analogs were also constructed by attaching similar fluorescent groups to the ribose ring of cobalamin, thereby generating a range of complete and incomplete corrinoids to follow uptake in bacteria, worms, and plants. By using these fluorescent derivatives we were able to demonstrate that Mycobacterium tuberculosis is able to acquire both cobyric acid and cobalamin analogs, that Caenorhabditis elegans takes up only the complete corrinoid, and that seedlings of higher plants such as Lepidium sativum are also able to transport B.
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http://dx.doi.org/10.1016/j.chembiol.2018.04.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6125784PMC
August 2018

Transmission of Mycobacterium tuberculosis From Patients Who Are Nucleic Acid Amplification Test Negative.

Clin Infect Dis 2018 11;67(11):1653-1659

Johns Hopkins University School of Medicine, Baltimore, Maryland.

Background: Among adults with signs and symptoms of pulmonary tuberculosis (TB), recognition of transmissible TB has implications for airborne infection isolation and public health activities. Sputum smear-negative TB patients account for around one-fifth of tuberculosis transmission. The tuberculosis transmission risk of TB patients with negative results on nucleic acid amplification test (NAAT) of respiratory specimens has not been established. We sought to estimate the tuberculosis transmission risk of NAAT-negative TB patients.

Methods: We retrospectively reviewed Maryland TB program data collected from 2004 to 2009, during which time NAAT using the Mycobacterium Tuberculosis Direct Test (MTD) was performed routinely. Patients with sputum Mycobacterium tuberculosis (M.tb) isolates having matching genotypes were assigned to clusters. Transmission sequence was approximated by collection order of individuals' first culture-positive specimens. Minimum transmission risks of NAAT (MTD)-negative TB patients and of smear-negative TB patients were estimated based on individuals' positions within clusters.

Results: Among 809 patients with culture-confirmed TB, M.tb genotypes were available for 782 (96.7%). For NAA-negative TB patients, the minimum transmission risk estimate was 5.1% (95% CI 0-11.4). For smear-negative TB patients, the minimum transmission risk estimate was 11.2% (95% CI 7.2-15.3).

Conclusions: Minimum transmission risk of NAAT-negative TB patients was lower than that of smear-negative TB patients. However, transmission risk of NAA-negative TB patients appears to not be negligible.
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http://dx.doi.org/10.1093/cid/ciy365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233677PMC
November 2018

2-Mercapto-Quinazolinones as Inhibitors of Type II NADH Dehydrogenase and Mycobacterium tuberculosis: Structure-Activity Relationships, Mechanism of Action and Absorption, Distribution, Metabolism, and Excretion Characterization.

ACS Infect Dis 2018 06 26;4(6):954-969. Epub 2018 Mar 26.

Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences , University of Dundee , Sir James Black Centre , Dundee , DD1 5EH , United Kingdom.

Mycobacterium tuberculosis ( MTb) possesses two nonproton pumping type II NADH dehydrogenase (NDH-2) enzymes which are predicted to be jointly essential for respiratory metabolism. Furthermore, the structure of a closely related bacterial NDH-2 has been reported recently, allowing for the structure-based design of small-molecule inhibitors. Herein, we disclose MTb whole-cell structure-activity relationships (SARs) for a series of 2-mercapto-quinazolinones which target the ndh encoded NDH-2 with nanomolar potencies. The compounds were inactivated by glutathione-dependent adduct formation as well as quinazolinone oxidation in microsomes. Pharmacokinetic studies demonstrated modest bioavailability and compound exposures. Resistance to the compounds in MTb was conferred by promoter mutations in the alternative nonessential NDH-2 encoded by ndhA in MTb. Bioenergetic analyses revealed a decrease in oxygen consumption rates in response to inhibitor in cells in which membrane potential was uncoupled from ATP production, while inverted membrane vesicles showed mercapto-quinazolinone-dependent inhibition of ATP production when NADH was the electron donor to the respiratory chain. Enzyme kinetic studies further demonstrated noncompetitive inhibition, suggesting binding of this scaffold to an allosteric site. In summary, while the initial MTb SAR showed limited improvement in potency, these results, combined with structural information on the bacterial protein, will aid in the future discovery of new and improved NDH-2 inhibitors.
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http://dx.doi.org/10.1021/acsinfecdis.7b00275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5996347PMC
June 2018

Role of Chemical Biology in Tuberculosis Drug Discovery and Diagnosis.

ACS Infect Dis 2018 04 7;4(4):458-466. Epub 2018 Feb 7.

Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology , National Institute of Allergy and Infectious Disease, NIH , Bethesda , Maryland 20892 , United States.

The use of chemical techniques to study biological systems (often referred to currently as chemical biology) has become a powerful tool for both drug discovery and the development of novel diagnostic strategies. In tuberculosis, such tools have been applied to identifying drug targets from hit compounds, matching high-throughput screening hits against large numbers of isolated protein targets and identifying classes of enzymes with important functions. Metabolites unique to mycobacteria have provided important starting points for the development of innovative tools. For example, the unique biology of trehalose has provided both novel diagnostic strategies as well as probes of in vivo biological processes that are difficult to study any other way. Other mycobacterial metabolites are potentially valuable starting points and have the potential to illuminate new aspects of mycobacterial pathogenesis.
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http://dx.doi.org/10.1021/acsinfecdis.7b00242DOI Listing
April 2018

Complement pathway gene activation and rising circulating immune complexes characterize early disease in HIV-associated tuberculosis.

Proc Natl Acad Sci U S A 2018 01 16;115(5):E964-E973. Epub 2018 Jan 16.

Department of Medicine, Imperial College London, London W2 1PG, United Kingdom;

The transition between latent and active tuberculosis (TB) occurs before symptom onset. Better understanding of the early events in subclinical disease will facilitate the development of diagnostics and interventions that improve TB control. This is particularly relevant in the context of HIV-1 coinfection where progression of TB is more likely. In a recent study using [F]-fluoro-2-deoxy-d-glucose positron emission/computed tomography (FDG-PET/CT) on 35 asymptomatic, HIV-1-infected adults, we identified 10 participants with radiographic evidence of subclinical disease, significantly more likely to progress than the 25 participants without. To gain insight into the biological events in early disease, we performed blood-based whole genome transcriptomic analysis on these participants and 15 active patients with TB. We found transcripts representing the classical complement pathway and Fcγ receptor 1 overabundant from subclinical stages of disease. Levels of circulating immune (antibody/antigen) complexes also increased in subclinical disease and were highly correlated with C1q transcript abundance. To validate our findings, we analyzed transcriptomic data from a publicly available dataset where samples were available in the 2 y before TB disease presentation. Transcripts representing the classical complement pathway and Fcγ receptor 1 were also differentially expressed in the 12 mo before disease presentation. Our results indicate that levels of antibody/antigen complexes increase early in disease, associated with increased gene expression of C1q and Fcγ receptors that bind them. Understanding the role this plays in disease progression may facilitate development of interventions that prevent this, leading to a more favorable outcome and may also be important to diagnostic development.
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http://dx.doi.org/10.1073/pnas.1711853115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5798330PMC
January 2018