Development of a fluorescence polarization assay to screen for inhibitors of the FtsZ/ZipA interaction

Dynamic Interaction of the Escherichia coli Cell Division ZipA and FtsZ Proteins Evidenced in Nanodiscs

“…However, only a 60% decrease in the fraction of bound Nd-ZipA was found upon the addition of CTZ-WT. These observations are in accordance with the different affinities of both peptides (15). As expected, the peptidemediated dissociation of the Nd*-ZipA⅐FtsZ polymer complex was similarly observed when the FtsZ polymer was formed in the presence of GTP and a GTP-regenerating system.…”

“…Fluorescence polarization competition assays using two different peptides (CTZ-WT and CTZ-MUT, as described under supplemental Methods) designed to mimic a segment of the C-terminal region of FtsZ demonstrated that they could bind with high affinity to the FtsZ-binding domain of ZipA (15). The binding affinity of CTZ-MUT to the C-terminal region of ZipA is an order of magnitude higher than the binding of CTZ-WT (15). We have used fluorescence correlation spectroscopy competition assays to measure the ability of these peptides to inhibit the binding of Nd-ZipA to FtsZ polymers.…”

“…Our knowledge on the association of the proto-ring proteins FtsZ and ZipA is up to now mainly based on genetic studies (2), with only a limited number of structural and biophysical studies on ZipA-FtsZ associations being available (13)(14)(15)(16). Previous results to measure the binding of a variant of ZipA, lacking the transmembrane region (sZipA) to FtsZ-GDP (16), indicated that independently of the oligomer size (up to hexamers), the binding affinity was moderate, and only one sZipA was bound to any of the different FtsZ species present in the solution.…”

Gelatinase-mediated migration and invasion of cancer cells

“…A library of 250 000 compounds has now been screened using this methodology to identify E. coli FtsZ/ZipA protein-protein interaction inhibitors. Among the screened compounds, 29 hits were found (Kenny et al, 2003). This approach offers a direct way to assay for small molecules in the absence of structural, or practically any other information about the target protein.…”

Assay Design for High‐Throughput Screening

“…The biochemical assays utilize purified recombinant or native proteins to recapitulate biological process in a well. Such assays assess enzymatic activities [24,25], receptor-ligand binding [26], or protein-protein interactions [27] in cell-free e nvironment. This approach has been a standard mainstream approach in the pharmaceutical industry for decades.…”