scholarly journals Whole-cell screening of oxidative enzymes using genetically encoded sensors

2021 ◽  
Author(s):  
Tsvetan Kardashliev ◽  
Alexandra Weingartner ◽  
Elvira Romero ◽  
Ulrich Schwaneberg ◽  
Marco Fraaije ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Oxidative Enzyme ◽  
Oxidative Enzymes ◽  
Whole Cell ◽  
Genetically Encoded Sensors

Genetically encoded biosensors enable efficient high-throughput screening of oxidative enzyme libraries.

scholarly journals High-throughput Screening and Sensitized Bacteria Identify an M. tuberculosis Dihydrofolate Reductase Inhibitor with Whole Cell Activity

PLoS ONE ◽  
2012 ◽  
Vol 7 (6) ◽  
pp. e39961 ◽  
Author(s):  
Anuradha Kumar ◽  
Meng Zhang ◽  
Linyun Zhu ◽  
Reiling P. Liao ◽  
Charles Mutai ◽  
...  
Keyword(s):  
High Throughput ◽  
Dihydrofolate Reductase ◽  
High Throughput Screening ◽  
Reductase Inhibitor ◽  
Cell Activity ◽  
Whole Cell

scholarly journals Rapid Methods for High-Throughput Detection of Sulfoxides

2009 ◽  
Vol 75 (14) ◽  
pp. 4711-4719 ◽  
Author(s):  
Janna Shainsky ◽  
Netta-Lee Derry ◽  
Yael Leichtmann-Bardoogo ◽  
Thomas K. Wood ◽  
Ayelet Fishman
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Colorimetric Detection ◽  
Selective Inhibition ◽  
Saturation Mutagenesis ◽  
Screening Methods ◽  
Acid Activation ◽  
Special Equipment ◽  
Whole Cell ◽  
Microtiter Plates

ABSTRACT Enantiopure sulfoxides are prevalent in drugs and are useful chiral auxiliaries in organic synthesis. The biocatalytic enantioselective oxidation of prochiral sulfides is a direct and economical approach for the synthesis of optically pure sulfoxides. The selection of suitable biocatalysts requires rapid and reliable high-throughput screening methods. Here we present four different methods for detecting sulfoxides produced via whole-cell biocatalysis, three of which were exploited for high-throughput screening. Fluorescence detection based on the acid activation of omeprazole was utilized for high-throughput screening of mutant libraries of toluene monooxygenases, but no active variants have been discovered yet. The second method is based on the reduction of sulfoxides to sulfides, with the coupled release and measurement of iodine. The availability of solvent-resistant microtiter plates enabled us to modify the method to a high-throughput format. The third method, selective inhibition of horse liver alcohol dehydrogenase, was used to rapidly screen highly active and/or enantioselective variants at position V106 of toluene ortho-monooxygenase in a saturation mutagenesis library, using methyl-p-tolyl sulfide as the substrate. A success rate of 89% (i.e., 11% false positives) was obtained, and two new mutants were selected. The fourth method is based on the colorimetric detection of adrenochrome, a back-titration procedure which measures the concentration of the periodate-sensitive sulfide. Due to low sensitivity during whole-cell screening, this method was found to be useful only for determining the presence or absence of sulfoxide in the reaction. The methods described in the present work are simple and inexpensive and do not require special equipment.

scholarly journals Selective and Brain Penetrant Neuropeptide Y Y2 Receptor Antagonists Discovered by Whole-Cell High-Throughput Screening

2009 ◽  
Vol 77 (1) ◽  
pp. 46-57 ◽  
Author(s):  
Shaun P. Brothers ◽  
S. Adrian Saldanha ◽  
Timothy P. Spicer ◽  
Michael Cameron ◽  
Becky A. Mercer ◽  
...  
Keyword(s):  
Neuropeptide Y ◽  
High Throughput ◽  
High Throughput Screening ◽  
Receptor Antagonists ◽  
Whole Cell ◽  
Y2 Receptor

scholarly journals Identification of Novel Inhibitors ofM. tuberculosisGrowth Using Whole Cell Based High-Throughput Screening

2012 ◽  
Vol 7 (8) ◽  
pp. 1377-1384 ◽  
Author(s):  
Sarah A. Stanley ◽  
Sarah Schmidt Grant ◽  
Tomohiko Kawate ◽  
Noriaki Iwase ◽  
Motohisa Shimizu ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Whole Cell

Union is strength: target-based and whole-cell high throughput screens in antibacterial discovery

2021 ◽  
Author(s):  
Cristina Landeta ◽  
Adrian Mejia-Santana
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Enzymatic Reactions ◽  
Whole Cell ◽  
Bacterial Physiology ◽  
Whole Cells ◽  
Discovery Research ◽  
High Throughput Screens

Antimicrobial resistance is one of the greatest global health challenges today. For over three decades antibacterial discovery research and development has been focused on cell-based and target-based high throughput assays. Target-based screens use diagnostic enzymatic reactions to look for molecules that can bind directly and inhibit the target. Target-based screens are only applied to proteins that can be successfully expressed, purified and the activity of which can be effectively measured using a biochemical assay. Often times the molecules found in these in vitro screens are not active in cells due to poor permeability or efflux. On the other hand, cell-based screens use whole cells and look for growth inhibition. These screens give higher number of hits than target-based assays and can simultaneously test many targets of one process or pathway in their physiological context. Both strategies have pros and cons when used separately. In the past decade and a half our increasing knowledge of bacterial physiology has led to the development of innovative and sophisticated technologies to perform high throughput screening combining these two strategies and thus minimizing their disadvantages. In this review we discuss recent examples of high throughput approaches that used both target-based and whole-cell screening to find new antibacterials, the new insights they have provided and how this knowledge can be applied to other in vivo validated targets to develop new antimicrobials.

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