Pathway-Selective Sensitization of Mycobacterium tuberculosis for Target-Based Whole-Cell Screening

Abrahams, Garth L.; Kumar, Anuradha; Savvi, Suzana; Hung, Alvin W.; Wen, Shulin; Abell, Chris; Barry, Clifton E.; Sherman, David R.; Boshoff, Helena I.; Mizrahi, Valerie

doi:10.1016/j.chembiol.2012.05.020

Cited by 123 publications

(173 citation statements)

References 39 publications

(51 reference statements)

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“…The identification of novel small molecules that penetrate the bacterial cell envelope and efficiently and selectively inactivate a bacterial target remains a major bottleneck. Target-based whole-cell screens, which use bacteria underexpressing a selected protein to bias hit compounds toward inhibitors of that protein (37)(38)(39)(40)(41), promise to widen this bottleneck and are best applied to proteins that represent well-validated targets. The strategy we used to identify NadE as such a validated target is scalable and can be applied, in principle, to any gene in the Mtb genome.…”

Section: Discussionmentioning

confidence: 99%

A genetic strategy to identify targets for the development of drugs that prevent bacterial persistence

Kim

O’Brien

Sharma

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

135

143

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Antibacterial drug development suffers from a paucity of targets whose inhibition kills replicating and nonreplicating bacteria. The latter include phenotypically dormant cells, known as persisters, which are tolerant to many antibiotics and often contribute to failure in the treatment of chronic infections. This is nowhere more apparent than in tuberculosis caused by Mycobacterium tuberculosis, a pathogen that tolerates many antibiotics once it ceases to replicate. We developed a strategy to identify proteins that Mycobacterium tuberculosis requires to both grow and persist and whose inhibition has the potential to prevent drug tolerance and persister formation. This strategy is based on a tunable dualcontrol genetic switch that provides a regulatory range spanning three orders of magnitude, quickly depletes proteins in both replicating and nonreplicating mycobacteria, and exhibits increased robustness to phenotypic reversion. Using this switch, we demonstrated that depletion of the nicotinamide adenine dinucleotide synthetase (NadE) rapidly killed Mycobacterium tuberculosis under conditions of standard growth and nonreplicative persistence induced by oxygen and nutrient limitation as well as during the acute and chronic phases of infection in mice. These findings establish the dual-control switch as a robust tool with which to probe the essentiality of Mycobacterium tuberculosis proteins under different conditions, including those that induce antibiotic tolerance, and NadE as a target with the potential to shorten current tuberculosis chemotherapies.

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Section: Discussionmentioning

confidence: 99%

A genetic strategy to identify targets for the development of drugs that prevent bacterial persistence

Kim

O’Brien

Sharma

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

135

143

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“…M. tuberculosis H37RvMA pMSP12:GFP in GAST-Fe media [32], and M. tuberculosis H37Rv:pCHERRY3 [31] in 7H9 + OADC. The MIC values in the GAST-Fe assay were consistently lower (2-3 fold lower for CM8, CM12, and CM14 and 14 fold lower for CM15).…”

Section: Discussionmentioning

confidence: 99%

“…CM8, CM12, CM14 and CM15 were screened using two independent methods as described below. Here, the standard broth microdilution method was performed using M. tuberculosis H37RvMA pMSP12:GFP [32]. A 10 ml culture was grown in glycerol-alanine-salts with 0.05% Tween 80 and iron (0.05%; GAST-Fe) pH 6.6, to an OD600 of 0.6 -0.7.…”

Section: Minimum Inhibitory Concentration Determinationmentioning

confidence: 99%

“…Briefly, the US assay (similar to the assay used for the MTS), utilized a H37Rv strain transformed with a plasmid encoding a red fluorescent reporter (pCHERRY3 [31]), a gift from Tanya Parish (Addgene plasmid # 24659)), in albumin-containing media [Middlebrook 7H9 broth, (Becton, Dickinson and C., USA) enriched with 10% OADC, 0.2% glycerol and 0.05% Tween 80; 7H9+OADC]. The UCT method utilized a standard broth microdilution method with M. tuberculosis H37RvMA pMSP12:GFP in glycerol-alanine-salts with 0.05% Tween 80 and iron (GAST-Fe) [32]. Consistently lower MICs were observed in the GFP GAST-Fe assay.…”

Section: Minimum Inhibitory Concentration Determinationmentioning

confidence: 99%

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Versatility of 7-Substituted Coumarin Molecules as Antimycobacterial Agents, Neuronal Enzyme Inhibitors and Neuroprotective Agents

Kapp

Visser

Sampson

et al. 2017

Preprint

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An in vitro medium-throughput screen using M. tuberculosis H37Rv was employed to screen an in-house library of structurally diverse compounds for antimycobacterial activity. From this initial screen, eleven 7-substituted coumarin derivatives with confirmed monoamine oxidase-B and cholinesterase inhibitory activities, demonstrated growth inhibition of more than 50% at a 50 µM concentration. This prompted further exploration of all the 7-substituted coumarins in our library, nineteen in total, as potential antimycobacterial agents. Four derivatives showed promising antimycobacterial activity with MIC99 values of 8.31 -29.70 µM and 44.15 -57.17 µM on M. tuberculosis H37Rv in independent assays using Gaste-Fe and 7H9 + OADC media, respectively. These compounds were found to bind to albumin which may explain the variations in MIC between the two assays. Preliminary antimycobacterial evaluation of moxifloxacin resistant M. tuberculosis show that these compounds are able to maintain their activity in fluoroquinolone resistant mycobacteria. Analysis of structure activity relationships for antimycobacterial versus neuronal enzyme inhibitory activity indicate that structural modification on position 4 and/or 7 of the coumarin scaffold may be utilized to improve selectivity towards either inhibition of neuronal enzymes or antimycobacterial effect. Cytotoxicity evaluations of the compounds indicate moderate cytotoxicity with slight selectivity towards mycobacteria. Further neuroprotective assays on SH-SY5Y human neuroblastoma cells indicate significant neuroprotection for selected compounds irrespective of their neuronal enzyme inhibitory properties. These coumarin molecules are thus interesting lead compounds that may provide insight into the design of new antimicrobacterial and/or neuroprotective agents.

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“…In these settings researchers were able to screen entire libraries of under-expression strains against whole chemical libraries and successfully identify inhibitors of growth and their MOA in a single screen, greatly reducing both the time and resources necessary for selecting interesting hits. The first steps providing proof-of-concept studies toward applying this approach to mycobacteria have been made already: Abrahams and coworkers used promoter replacement to decrease the expression of icl1, lysA, and panC, and they were able to validate these genes as important for M. tuberculosis survival in vitro (129). They then used the panC knockdown strain in a target-based whole-cell screen (TB-WCS) of a small compound library (600 compounds) to identify 37 hits.…”

Section: Leveraging Biologymentioning

confidence: 99%

Genetic Strategies for Identifying New Drug Targets

2014

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Genetic strategies have yet to come into their own as tools for antibiotic development. While holding a lot of initial promise, they have only recently started to bear fruit in the quest for new drug targets. An ever-increasing body of knowledge is showing that genetics can lead to significant improvements in the success and efficiency of drug discovery. Techniques such as high-frequency transposon mutagenesis and expression modulation have matured and have been applied successfully not only to the identification and characterization of new targets, but also to their validation as tractable weaknesses of bacteria. Past experience shows that choosing targets must not rely on gene essentiality alone, but rather needs to incorporate knowledge of the system as a whole. The ability to manipulate genes and their expression is key to ensuring that we understand the entire set of processes that are affected by drug treatment. Focusing on exacerbating these perturbations, together with the identification of new targets to which resistance has not yet occurred-both enabled by genetic approaches-may point us toward the successful development of new combination therapies engineered based on underlying biology.

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Pathway-Selective Sensitization of Mycobacterium tuberculosis for Target-Based Whole-Cell Screening

Cited by 123 publications

References 39 publications

A genetic strategy to identify targets for the development of drugs that prevent bacterial persistence

A genetic strategy to identify targets for the development of drugs that prevent bacterial persistence

Versatility of 7-Substituted Coumarin Molecules as Antimycobacterial Agents, Neuronal Enzyme Inhibitors and Neuroprotective Agents

Genetic Strategies for Identifying New Drug Targets

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