In vitro drug discovery models for Mycobacterium tuberculosis relevant for host infection

Vitamin B 12 -dependent enzymes function in core biochemical pathways in Mycobacterium tuberculosis , an obligate pathogen whose metabolism in vivo is poorly understood. Although M. tuberculosis can access vitamin B 12 in vitro , it is uncertain whether the organism is able to scavenge B 12 during host infection. This question is crucial to predictions of metabolic function, but its resolution is complicated by the absence in the M. tuberculosis genome of a direct homologue of BtuFCD, the only bacterial B 12 transport system described to date. We applied genome-wide transposon mutagenesis to identify M. tuberculosis mutants defective in their ability to use exogenous B 12 . A small proportion of these mapped to Rv1314c , identifying the putative PduO-type ATP : co(I)rrinoid adenosyltransferase as essential for B 12 assimilation. Most notably, however, insertions in Rv1819c dominated the mutant pool, revealing an unexpected function in B 12 acquisition for an ATP-binding cassette (ABC)-type protein previously investigated as the mycobacterial BacA homologue. Moreover, targeted deletion of Rv1819c eliminated the ability of M. tuberculosis to transport B 12 and related corrinoids in vitro . Our results establish an alternative to the canonical BtuCD-type system for B 12 uptake in M. tuberculosis , and elucidate a role in B 12 metabolism for an ABC protein implicated in chronic mycobacterial infection.

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Mycobacterium tuberculosis Gyrase Inhibitors as a New Class of Antitubercular Drugs

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03913-14 ◽

2015 ◽

Vol 59 (4) ◽

pp. 1868-1875 ◽

Cited By ~ 23

Author(s):

Delia Blanco ◽

Esther Perez-Herran ◽

Mónica Cacho ◽

Lluís Ballell ◽

Julia Castro ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Discovery ◽

Oral Absorption ◽

Cell Activity ◽

Cross Resistance ◽

Drug Candidate ◽

Antibacterial Drug ◽

Gyrase Inhibitors

ABSTRACTOne way to speed up the TB drug discovery process is to search for antitubercular activity among compound series that already possess some of the key properties needed in anti-infective drug discovery, such as whole-cell activity and oral absorption. Here, we present MGIs, a new series ofMycobacterium tuberculosisgyrase inhibitors, which stem from the long-term efforts GSK has dedicated to the discovery and development of novel bacterial topoisomerase inhibitors (NBTIs). The compounds identified were found to be devoid of fluoroquinolone (FQ) cross-resistance and seem to operate through a mechanism similar to that of the previously described NBTI GSK antibacterial drug candidate. The remarkablein vitroandin vivoantitubercular profiles showed by the hits has prompted us to further advance the MGI project to full lead optimization.

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Bioluminescence screening in vitro (Bio-Siv) assays for high-volume antimycobacterial drug discovery.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.6.1536 ◽

1996 ◽

Vol 40 (6) ◽

pp. 1536-1541 ◽

Cited By ~ 40

Author(s):

T M Arain ◽

A E Resconi ◽

M J Hickey ◽

C K Stover

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Discovery ◽

High Volume ◽

Luciferase Gene ◽

Direct Identification ◽

Mycobacterium Intracellulare ◽

Large Numbers ◽

Antimycobacterial Agents ◽

Screening In Vitro

Bioluminescence-based assays to indicate antimicrobial susceptibility have been developed and validated for recombinant strains of Mycobacterium tuberculosis, Mycobacterium bovis BCG, Mycobacterium avium, and Mycobacterium intracellulare expressing an integrated eukaryotic luciferase gene. MICs determined with these bioluminescence assays for several antimycobacterial agents, including isoniazid, ethambutol, rifampin, amikacin, streptomycin, ciprofloxacin, and clarithromycin, compared favorably with traditional BACTEC methods and visual estimations of the inhibitory end point. Assay methodology has been optimized for the analysis of large numbers of novel compounds and is simple, inexpensive, and labor efficient. The availability of these four recombinant mycobacteria has permitted a strategy for drug discovery employing the nonpathogenic BCG strain for mass screening purposes with subsequent confirmation of activity against the pathogenic mycobacteria. Furthermore, evidence suggests that the BCG-based screen may allow the direct identification of bactericidal agents.

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Rapid Microbiologic and Pharmacologic Evaluation of Experimental Compounds against Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.50.4.1245-1250.2006 ◽

2006 ◽

Vol 50 (4) ◽

pp. 1245-1250 ◽

Cited By ~ 26

Author(s):

Veronica Gruppo ◽

Christine M. Johnson ◽

Karen S. Marietta ◽

Hataichanok Scherman ◽

Erin E. Zink ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Discovery ◽

High Throughput Screening ◽

Mouse Serum ◽

Serum Samples ◽

Microtiter Plate Assay ◽

Plate Assay ◽

Drug Concentrations

ABSTRACT The assessment of physiochemical and pharmacological properties at early stages of drug discovery can accelerate the conversion of hits and leads into candidates for further development. A strategy for streamlined evaluation of compounds against Mycobacterium tuberculosis in the early preclinical stage is presented in this report. As a primary assay to rapidly select experimental compounds with sufficient in vitro activity, the growth inhibition microtiter plate assay was devised as an alternative to current methods. This microdilution plate assay is a liquid culture method based on spectrophotometric readings of the bacillary growth. The performance of this method was compared to the performance of two established susceptibility methods using clinical available tuberculosis (TB) drugs. Data generated from all three assays were similar for all of the tested compounds. A second simple bioassay was devised to assess the oral bioavailability of compounds prior to extensive in vivo efficacy testing. The bioassay estimates drug concentrations in collected serum samples by a microdilution MIC plate method using M. tuberculosis. In the same assay, the MIC of the compound is also determined in the presence of 10% mouse serum as an indication of protein binding. The method was validated using different clinically available TB drugs, and results are discussed in this report. With these methodological advances, screening of compounds against tuberculosis in the preclinical phase will be rapid, can be adapted to semi-high-throughput screening, and will add relevant physicochemical and basic pharmacological criteria to the decision process of drug discovery.

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The promises and limitations of N-acetylcysteine as a potentiator of first-line and second-line tuberculosis drugs

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01703-20 ◽

2021 ◽

Author(s):

Catherine Vilchèze ◽

William R. Jacobs

Keyword(s):

Mycobacterium Tuberculosis ◽

Liver Injury ◽

Multidrug Resistant ◽

Human Macrophage ◽

Second Line ◽

Macrophage Cell ◽

First Line ◽

Host Infection

N-acetylcysteine (NAC) is most commonly used for the treatment of acetaminophen overdose and acetaminophen-induced liver injury. In patients infected with Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), NAC is given to treat hepatotoxicity induced by TB drugs. We had previously shown that cysteine, a derivative of NAC, potentiated the activity of isoniazid, a first-line TB drug, by preventing the emergence of INH resistance and persistence in M. tuberculosis in vitro. Herein, we demonstrate that in vitro, NAC has the same boosting activity with various combinations of first- and second-line TB drugs against drug-susceptible and multidrug-resistant M. tuberculosis strains. Similar to cysteine, NAC increased M. tuberculosis respiration. However, in M. tuberculosis-infected mice, the addition of NAC did not augment the activity of first- or second-line TB drugs. A comparison of the activity of NAC combined with TB drugs in murine and human macrophage cell lines revealed that studies in mice might not be recapitulated during host infection in vivo.

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The EXIT Strategy: an Approach for Identifying Bacterial Proteins Exported during Host Infection

mBio ◽

10.1128/mbio.00333-17 ◽

2017 ◽

Vol 8 (2) ◽

Cited By ~ 9

Author(s):

E. F. Perkowski ◽

K. E. Zulauf ◽

D. Weerakoon ◽

J. D. Hayden ◽

T. R. Ioerger ◽

...

Keyword(s):

Cell Wall ◽

Mycobacterium Tuberculosis ◽

Host Environment ◽

Bacterial Proteins ◽

Novel Method ◽

Host Infection ◽

Membrane Cell ◽

Significant Effort

ABSTRACT Exported proteins of bacterial pathogens function both in essential physiological processes and in virulence. Past efforts to identify exported proteins were limited by the use of bacteria growing under laboratory (in vitro) conditions. Thus, exported proteins that are exported only or preferentially in the context of infection may be overlooked. To solve this problem, we developed a genome-wide method, named EXIT (exported in vivo technology), to identify proteins that are exported by bacteria during infection and applied it to Mycobacterium tuberculosis during murine infection. Our studies validate the power of EXIT to identify proteins exported during infection on an unprecedented scale (593 proteins) and to reveal in vivo induced exported proteins (i.e., proteins exported significantly more during in vivo infection than in vitro). Our EXIT data also provide an unmatched resource for mapping the topology of M. tuberculosis membrane proteins. As a new approach for identifying exported proteins, EXIT has potential applicability to other pathogens and experimental conditions. IMPORTANCE There is long-standing interest in identifying exported proteins of bacteria as they play critical roles in physiology and virulence and are commonly immunogenic antigens and targets of antibiotics. While significant effort has been made to identify the bacterial proteins that are exported beyond the cytoplasm to the membrane, cell wall, or host environment, current methods to identify exported proteins are limited by their use of bacteria growing under laboratory (in vitro) conditions. Because in vitro conditions do not mimic the complexity of the host environment, critical exported proteins that are preferentially exported in the context of infection may be overlooked. We developed a novel method to identify proteins that are exported by bacteria during host infection and applied it to identify Mycobacterium tuberculosis proteins exported in a mouse model of tuberculosis. IMPORTANCE There is long-standing interest in identifying exported proteins of bacteria as they play critical roles in physiology and virulence and are commonly immunogenic antigens and targets of antibiotics. While significant effort has been made to identify the bacterial proteins that are exported beyond the cytoplasm to the membrane, cell wall, or host environment, current methods to identify exported proteins are limited by their use of bacteria growing under laboratory (in vitro) conditions. Because in vitro conditions do not mimic the complexity of the host environment, critical exported proteins that are preferentially exported in the context of infection may be overlooked. We developed a novel method to identify proteins that are exported by bacteria during host infection and applied it to identify Mycobacterium tuberculosis proteins exported in a mouse model of tuberculosis.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01006-19 ◽

2019 ◽

Vol 63 (10) ◽

Cited By ~ 2

Author(s):

Yumi Park ◽

Yong-Mo Ahn ◽

Surendranadha Jonnala ◽

Sangmi Oh ◽

Julia M. Fisher ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Discovery ◽

Macrophage Infection ◽

Content Type ◽

Magnesium Homeostasis ◽

Direct Binding ◽

Pore Opening

ABSTRACT 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 Mg2+/Co2+ transporter. Mutations in domains of CorA that are predicted to regulate the pore opening in response to Mg2+ 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 Mg2+ homeostasis by CorA is an attractive target for tuberculosis drug discovery and encourage identification of improved CorA inhibitors.

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Tuberkulose - Screening

Therapeutische Umschau ◽

10.1024/0040-5930/a000181 ◽

2011 ◽

Vol 68 (7) ◽

pp. 381-387

Author(s):

Otto Schoch

Keyword(s):

Mycobacterium Tuberculosis ◽

Interferon Gamma ◽

Wird Eine ◽

Interferon Gamma Release Assays

Das primäre Ziel der Aktivitäten zur bevölkerungsbezogenen Tuberkulosekontrolle ist die Identifizierung von Patienten mit sputummikroskopisch positiver Lungentuberkulose. Wenn diese Patienten umgehend therapiert werden, haben sie nicht nur eine optimale Heilungschance, sondern übertragen auch den Krankheitserreger nicht weiter auf andere Personen. Das Screening, die systematische Suche nach Tuberkulose, erfolgt in der Regel radiologisch bei der Suche nach Erkrankten, während immunologische Teste bei der Suche nach einer Infektion mit Mycobacterium tuberculosis zur Anwendung kommen. Diese Infektion, die ein erhöhtes Risiko für die Entwicklung einer Tuberkulose-Erkrankung mit sich bringt, wird im Rahmen der Umgebungsuntersuchungen oder bei Hochrisikogruppen gesucht. Neben dem traditionellen in vivo Mantoux Hauttest stehen heute die neueren in vitro Blutteste, die sogenannten Interferon Gamma Release Assays (IGRA) zur Verfügung, die unter anderem den Vorteil einer höheren Spezifität mit sich bringen, weil die verwendeten Antigene der Mykobakterien-Wand beim Impfstamm Bacille Calmitte Guerin (BCG) und bei den meisten atypischen Mykobakterien nicht vorhanden sind. Zudem kann bei Immunsupprimierten dank einer mitgeführten Positivkontrolle eine Aussage über die Wahrscheinlichkeit eines falsch negativen Testresultates gemacht werden. Bei neu diagnostizierter Infektion mit Mycobacterium tuberculosis wird eine präventive Chemotherapie mit Isoniazid während 9 Monaten durchgeführt.

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