Dissecting the polysaccharide-rich grape cell wall changes during winemaking using combined high-throughput and fractionation methods

ABSTRACT We have developed a novel assay specific to MraY, which catalyzes the first membrane step in the biosynthesis of bacterial cell wall peptidoglycan. This was accomplished by using UDP-MurNAc-Nε -dansylpentapeptide, a fluorescent derivative of the MraY nucleotide substrate, and a partially purified preparation of MraY solubilized from membranes of an Escherichia coli overproducing strain. Two versions of the assay were developed, one consisting of the high-pressure liquid chromatography separation of the substrate and product (dansylated lipid I) and the other, without separation and adapted to the high-throughput format, taking advantage of the different fluorescence properties of the nucleotide and lipid I in the reaction medium. The latter assay was validated with a set of natural and synthetic MraY inhibitors.

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Antagonism screen for inhibitors of bacterial cell wall biogenesis uncovers an inhibitor of undecaprenyl diphosphate synthase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1511751112 ◽

2015 ◽

Vol 112 (35) ◽

pp. 11048-11053 ◽

Cited By ~ 44

Author(s):

Maya A. Farha ◽

Tomasz L. Czarny ◽

Cullen L. Myers ◽

Liam J. Worrall ◽

Shawn French ◽

...

Keyword(s):

Cell Wall ◽

High Throughput ◽

High Throughput Screening ◽

Teichoic Acid ◽

Antibacterial Drug ◽

Wall Teichoic Acid ◽

Chemical Screening ◽

Cellular Processes ◽

Cell Wall Biogenesis ◽

Antagonistic Interactions

Drug combinations are valuable tools for studying biological systems. Although much attention has been given to synergistic interactions in revealing connections between cellular processes, antagonistic interactions can also have tremendous value in elucidating genetic networks and mechanisms of drug action. Here, we exploit the power of antagonism in a high-throughput screen for molecules that suppress the activity of targocil, an inhibitor of the wall teichoic acid (WTA) flippase in Staphylococcus aureus. Well-characterized antagonism within the WTA biosynthetic pathway indicated that early steps would be sensitive to this screen; however, broader interactions with cell wall biogenesis components suggested that it might capture additional targets. A chemical screening effort using this approach identified clomiphene, a widely used fertility drug, as one such compound. Mechanistic characterization revealed the target was the undecaprenyl diphosphate synthase, an enzyme that catalyzes the synthesis of a polyisoprenoid essential for both peptidoglycan and WTA synthesis. The work sheds light on mechanisms contributing to the observed suppressive interactions of clomiphene and in turn reveals aspects of the biology that underlie cell wall synthesis in S. aureus. Further, this effort highlights the utility of antagonistic interactions both in high-throughput screening and in compound mode of action studies. Importantly, clomiphene represents a lead for antibacterial drug discovery.

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High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchical clustering of their carbohydrate microarray binding profiles

Glycoconjugate Journal ◽

10.1007/s10719-007-9059-7 ◽

2007 ◽

Vol 25 (1) ◽

pp. 37-48 ◽

Cited By ~ 114

Author(s):

Isabel Moller ◽

Susan E. Marcus ◽

Ash Haeger ◽

Yves Verhertbruggen ◽

Rene Verhoef ◽

...

Keyword(s):

Cell Wall ◽

Monoclonal Antibodies ◽

Hierarchical Clustering ◽

Plant Cell ◽

High Throughput ◽

High Throughput Screening ◽

Plant Cell Wall

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A High Throughput Screen for Inhibitors of Fungal Cell Wall Synthesis

CrossRef Listing of Deleted DOIs ◽

10.1177/108705710200700408 ◽

2002 ◽

Vol 7 (4) ◽

pp. 359-366 ◽

Cited By ~ 8

Author(s):

Jonathan M. Evans ◽

Phillip G. Zaworski ◽

Christian N. Parker

Keyword(s):

Cell Wall ◽

Cell Growth ◽

High Throughput ◽

High Throughput Screening ◽

Osmotic Shock ◽

Fungal Cell Wall ◽

Cell Wall Synthesis ◽

Fungal Cell ◽

Unique Nature ◽

Pharmacologically Active

Fungal cell wall synthesis is essential for viability, requiring the activity of genes involved in environmental sensing, precursor synthesis, transport, secretion, and assembly. This multitude of potential targets, the availability of known agents targeting this pathway, and the unique nature of fungal cell wall synthesis make this pathway an appealing target for drug discovery. Here we describe the adaptation of an assay monitoring cell wall synthesis for high-throughput screening. The assay requires fungal cell growth, in the presence of the test compound, for 3 h before the cells are subjected to osmotic shock in the presence of a dye that stains DNA. Miniaturization of the assay to a 384-well plate format and removing a mechanical transfer led to subtle changes in the assay characteristics. Validation of the assay with a library of known pharmacologically active agents has identified a number of different classes of compounds that are active in this assay, causing aberrant cell wall morphology and in many cases the inhibition of fungal cell growth.

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