Bioluminescent Reporters for Rapid Mechanism of Action Assessment in Tuberculosis Drug Discovery

Naran, Krupa; Moosa, A.; Barry, Clifton E.; Boshoff, Helena I.; Mizrahi, Valerie; Warner, Digby F.

doi:10.1128/aac.01178-16

Cited by 42 publications

(73 citation statements)

References 53 publications

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“…For mechanism of action of these compounds against Mtb, the results obtained from triage assays 53,54 , the lack of hypersensitivity observed in a cydKO mutant, and measurement of OCR eliminate mycobacterial respiration as target. In contrast, the immediate and sustained luminescence signal in the PiniB-LUX reporter assay strongly implicates cell wall homoeostasis as a likely primary target 57 . Moreover, the detection of late-stage (after day 4) triggering of recA promoter activity suggests some effect on DNA metabolism as well.…”

Section: Discussionmentioning

confidence: 92%

“…Notably, no significant change in the OCR was observed following treatment with 16, also reinforcing the notion that QcrB is not the primary target (Supplementary Methods and Supplementary Figure 1). Bioluminescence reporter assays 57 were used to establish if the compounds 15-17 disrupt cell wall homoeostasis or are genotoxic in Mtb. All three compounds elicited a strong signal using the PiniB-LUX reporter (Supplementary Figure 2), triggering an immediate luminescence signal on day 1 which was sustained throughout the 10-day assay, as was observed for the isoniazid control.…”

Section: Resultsmentioning

confidence: 99%

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Accessible and distinct decoquinate derivatives active against Mycobacterium tuberculosis and apicomplexan parasites

et al. 2018

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The quinolone decoquinate is coadministered with feed for treatment of parasites which cause coccidiosis in poultry. However, from a drug-development perspective, the biological activity is often not adequately exploited due to poor physicochemical properties. Here we convert decoquinate into N-alkyl quinolone amides that, in contrast to decoquinate, are active against the tuberculosis bacterium with MIC 90 values ranging from 1.4 to 3.64 µM, and quinoline O-carbamates active against apicomplexan parasites that cause malaria, toxoplasmosis, and neosporosis with IC 50 values of 0.32-1.5 nM for the best derivative. Uniquely for the TB-active amides, disruption of cell wall homoeostasis is identified as one target. With IC 50 values against fetal lung fibroblast cells of 40 to >100 μM, the derivatives are selective for the pathogens. Structures of the most active derivatives are determined by NMR spectroscopy and X-ray crystallography. Analogues lacking the decyl side chain of decoquinate are inactive.

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Section: Discussionmentioning

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Accessible and distinct decoquinate derivatives active against Mycobacterium tuberculosis and apicomplexan parasites

et al. 2018

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“…Fusion of the iniBAC promoter to β‐galactosidase or luciferase results in a strong inducible reporter signal . A bacterial luciferase reporter, generated using the iniBAC promoter, generated a reporter strain that can be used to quickly assess mechanism of action of novel molecules, by identifying compounds that have isoniazid‐like activity and cell wall stress triggering activities . An iniBAC reporter was also used in a genetic screen in Mycobacterium marinum to discover new genes controlling cell wall stress responses.…”

Section: Pathway Specific Transcriptional Reportersmentioning

confidence: 99%

Mycobacterium tuberculosis Reporter Strains as Tools for Drug Discovery and Development

Abramovitch

2018

IUBMB Life

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Reporter strains have proven to be powerful tools to study Mycobacterium tuberculosis (Mtb) physiology. Transcriptional and translational reporter strains are engineered by fusing a readout gene, encoding a fluorescent, luminescent or enzymatic protein, downstream of a promoter or in-frame with a gene of interest. When the reporter is expressed, it generates a signal that acts as a synthetic phenotype, enabling the study of physiologies that might have otherwise been hidden. This review will discuss approaches for generating reporter strains in Mtb and how they can be used as tools for high-throughput genetic and small molecule screening and as biomarkers for examining Mtb responses to drug or immune stresses during animal infections. Fluorescent reporter strains have an added benefit in that they can be used for single-cell studies both in vitro and in vivo, thus enabling the study of mechanisms underlying phenotypic heterogeneity. Recent examples of the use of Mtb reporter strains will be presented with a focus on how they can be used as tools for drug discovery and development. © 2018 IUBMB Life, 70(9):818-825, 2018.

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“…S6). We focused on induction of the iniBAC operon, which is rapidly upregulated due inhibition of cell-wall synthesis and is used to screen for cell-wall acting compounds (12, 13). We observed that cycloserine’s induction of iniBAC genes was mild compared to other cell-wall acting antibiotics (~1.5 fold and ~3.5 fold respectively), consistent with the weak association of cycloserine to other cell-wall antibacterials (Figure 3 Right and Fig S6 Top).…”

Section: Resultsmentioning

confidence: 99%

Morphological profiling of tubercule bacilli identifies drug pathways of action

Smith

Pullen

Olson

et al. 2020

Preprint

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34 35 36 Keywords 37 tuberculosis, drug discovery, cell morphology, high throughput 38 39 40 41 42 43 2 Abstract 44Morphological profiling is a method to classify target pathways of antibacterials based on how 45 bacteria respond to treatment through changes to cellular shape and spatial organization. Here, we utilized 46 the cell-to-cell variation in morphological features of Mycobacterium tuberculosis bacilli to develop a rapid 47 profiling platform called Morphological Evaluation and Understanding of Stress (MorphEUS). MorphEUS 48 classified 94% of tested drugs correctly into broad categories according to modes of action previously 49 identified in the literature. In the other 6%, MorphEUS pointed to key off-target or secondary bactericidal 50 activities. We observed cell-wall damaging activity induced by bedaquiline and moxifloxacin through 51 secondary effects downstream from their main target pathways. We implemented MorphEUS to correctly 52 classify three compounds in a blinded study and identified an off-target effect for one compound that was 53 not readily apparent in previous studies. We anticipate that the ability of MorphEUS to rapidly identify 54 pathways of drug action and the proximal cause of bactericidal activity in tubercule bacilli will make it 55 applicable to other pathogens and cell types where morphological responses are subtle and 56 heterogeneous. 58 Significance Statement 59Tuberculosis is a leading cause of death in the world and requires treatment with an arduous 60 multidrug regimen. Many new tuberculosis drugs are in development, and the drug development pipeline 61 would benefit from more rapid methods to learn drug mechanism of action and off-target effects. Here, we 62 describe a high throughput imaging method for rapidly classifying drugs into categories based on the 63 primary and secondary cellular damage called Morphological Evaluation and Understanding of drug-Stress 64 (MorphEUS). We anticipate that MorphEUS will assist in rapidly pinpointing causes of cellular death in 65 response to drug treatment in tuberculosis and other bacterial pathogens. 66 67 68 69 3 Main Text 70 Introduction 71Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), causes more deaths 72 annually than any other infectious agent (1). Tuberculosis treatment is lengthy, lasting between four months 73 to over a year (1). The difficult regimen, rate of relapse, and incidence of drug resistant Mtb has motivated 74 a significant effort to develop new antibacterial compounds that are effective in sterilizing Mtb infection (2). 75Many new drug classes and derivative compounds have been developed (2), but rapidly identifying the 76 primary and secondary pathways of action of each compound is often a protracted process due to the 77 difficulty in generating resistant mutants, and dissecting the broad-reaching metabolic effects of drug 78 treatment leading to death (3). Furthermore, drug action on bacterial cells can elicit dynamic responses in 79 multiple pathways both on-and off-target, s...

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Bioluminescent Reporters for Rapid Mechanism of Action Assessment in Tuberculosis Drug Discovery

Cited by 42 publications

References 53 publications

Accessible and distinct decoquinate derivatives active against Mycobacterium tuberculosis and apicomplexan parasites

Accessible and distinct decoquinate derivatives active against Mycobacterium tuberculosis and apicomplexan parasites

Mycobacterium tuberculosis Reporter Strains as Tools for Drug Discovery and Development

Morphological profiling of tubercule bacilli identifies drug pathways of action

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