Development of a fluorescence polarization assay to screen for inhibitors of the FtsZ/ZipA interaction.
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.
Kenny
Cynthia Hess
CH
Screening Sciences, Biophysics/Enzymology, Wyeth Research, Pearl River, NY 10965, USA. kennycl@wyeth.com
Ding
Weidong
W
Kelleher
Kerry
K
Benard
Susan
S
Dushin
Elizabeth Glasfeld
EG
Sutherland
Alan G
AG
Mosyak
Lidia
L
Kriz
Ronald
R
Ellestad
George
G
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005454:Fluorescence Polarization / Q000379:methods*; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D011743:Pyrimidines / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013501:Surface-Active Agents / Q000494:pharmacology
D001426:Bacterial Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D029968:Escherichia coli Proteins; C071525:FtsZ protein, Bacteria; D010446:Peptide Fragments; D011743:Pyrimidines; D013501:Surface-Active Agents; C103795:ZipA protein, E coli; D004121:Dimethyl Sulfoxide