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Development of a high‐throughput screening approach targeting inhibitors of bacterial cell wall synthesis
Author(s) -
White Courtney L,
Gober James W
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.lb203
Subject(s) - mreb , caulobacter crescentus , high throughput screening , peptidoglycan , computational biology , bacterial cell structure , biology , microbiology and biotechnology , lipid ii , penicillin binding proteins , protein–protein interaction , cell wall , bacteria , bacterial protein , chemistry , cell , biochemistry , cytoskeleton , genetics
Many current antimicrobials act upon the synthesis of the peptidoglycan (PG) cell wall. The proteins that synthesize, assemble, and degrade PG are critical for cell viability. However, evolving antibiotic resistance places a new urgency in prokaryotic biology for new targets. In rod‐shaped bacteria, a large complex of protein interactions coordinate PG synthesis. At the heart of this complex exists an association recently found between two well‐conserved proteins ‐ an actin homolog (MreB) and a bitopic membrane protein (RodZ), both vital for cell wall maintenance. The interaction is confirmed in several species by various methods, one including a bacterial two‐hybrid system. We have adapted this system to high‐throughput screening (HTS) to search for inhibitors of the Caulobacter crescentus MreB‐RodZ interaction. This 384‐ well adaptation involves several original protocol modifications for final assay validation. With this assay, we have screened more than 70,000 unique compounds, producing 485 potential hits. A secondary counter‐screen and confirmatory experiments determine hits specific to RodZ‐MreB interaction. This work demonstrates two‐hybrid HTS assay feasibility and may potentially provide novel inhibitors of these proteins. It also may prove useful for testing of other essential protein interactions. This work is funded by a NIH Exploratory Research Grant.