PMID:14656529
| Citation |
Kenny, CH, Ding, W, Kelleher, K, Benard, S, Dushin, EG, Sutherland, AG, Mosyak, L, Kriz, R and Ellestad, G (2003) Development of a fluorescence polarization assay to screen for inhibitors of the FtsZ/ZipA interaction. Anal. Biochem. 323:224-33 |
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| Abstract |
A fluorescence polarization competition assay has been developed to screen for inhibitors of the Escherichia coli FtsZ/ZipA protein-protein interaction. A previously published X-ray costructure demonstrated that a 17-amino-acid peptide, corresponding to FtsZ C-terminal residues 367-383 (FtsZ(367-383)), interacts with the C-terminal FtsZ binding domain of ZipA (ZipA(185-328)). Phage display was employed to identify a unique but related peptide which when further modified and labeled was shown to have a higher affinity to ZipA(185-328) than the FtsZ(367-383) peptide and binds to the same site. This peptide had a six fold increase in fluorescence polarization upon binding to ZipA(185-328) compared to a two fold increase for the FtsZ(367-383) fluorophore. As a result, assay parameters using the phage display peptide were further optimized and adapted for the high-throughput screen. A high-throughput screen of 250,000 compounds identified 29 hits with inhibition equal to or greater than 30% at 50 microg/ml. An X-ray costructure of a promising small molecule in this library complexed with ZipA(185-328) (KI=12 microM) revealed that the compound binds to the same hydrophobic pocket as the FtsZ(367-383) peptide. |
| Links | |
| Keywords |
Amino Acid Sequence; Bacterial Proteins/antagonists & inhibitors; Bacterial Proteins/metabolism; Binding, Competitive; Carrier Proteins/antagonists & inhibitors; Carrier Proteins/metabolism; Cell Cycle Proteins/antagonists & inhibitors; Cell Cycle Proteins/metabolism; Cytoskeletal Proteins/antagonists & inhibitors; Cytoskeletal Proteins/metabolism; Dimethyl Sulfoxide/pharmacology; Escherichia coli/drug effects; Escherichia coli/metabolism; Escherichia coli Proteins/antagonists & inhibitors; Escherichia coli Proteins/metabolism; Fluorescence Polarization/methods; Models, Biological; Models, Molecular; Molecular Sequence Data; Peptide Fragments/metabolism; Protein Binding/drug effects; Protein Binding/physiology; Pyrimidines/pharmacology; Structure-Activity Relationship; Surface-Active Agents/pharmacology |
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