Fluorescence polarization (FP) has been used to develop high throughput screening (HTS) assays for nuclear receptor-ligand displacement and kinase inhibition. FP is a solution-based, homogeneous technique requiring no immobilization or separation of reaction components. The FP-based estrogen receptor (ER) assay is based on the competition of fluorescein-labeled estradiol and estrogen-like compounds for binding to ER. These studies determined the Kd for this interaction to be 3 nM for ERα and 2 nM for ERβ; IC50 values for 17β-estradiol, tamoxifen, 4-OH-tamoxifen, and diethylstibestrol were determined to be 5.6, 189, 26, and 3.5 nM, respectively. In a screen of 50 lead compounds from a transcriptional activation screen, 21 compounds had IC50 values below 10 μM, with one having an almost 100-fold higher affinity for ERβ over ERα. These data show that an FP-based competitive binding assay can be used to screen diverse compounds with a broad range of binding affinities for ERs. The FP-based protein-tyrosine kinase (PTK) assay uses fluorescein-labeled phosphopeptides bound to anti-phosphotyrosine antibodies. Phosphopeptides generated by a kinase compete for this binding. In c-Src kinase reactions, polarization decreased with time as reaction products displaced the fluorescein-labeled phosphopeptide from the anti-phosphotyrosine antibodies. The experimentally determined IC50 of AG 1478 was 400 pM, while Genistein did not inhibit the epidermal growth factor receptor at similar concentrations. Like the FP-based PTK assay, the protein kinase C (PKC) assay utilizes competition. PKC isoforms had different turnover rates for the peptide substrate. The IC50 for staurosporine was less than 10 nM for all PKC isoforms. Tyrosine phosphatase assays use direct binding rather than competition. Increasing concentrations of T-cell protein-tyrosine phosphatase (TC PTP) increased the rate of dephosphorylation. This change in polarization was dependent on TC PTP and was inhibited by 50,μM Na3VO4. The IC50 of Na3VO4 was 4 nM for TC PTP. These data demonstrate that a FP-based assay can detect kinase and phosphatase activity. Homogeneous, fluorescent techniques such as FP are now methods of choice for screening many types of drug targets. New HTS instrumentation and assay methods like these make FP a technology easily incorporated into HTS.
Over the last few years, an increased awarenes of endocrine disrupting chemicals (EDCs)
Fluorescence polarization (FP) equilibrium binding assays differ from other types of binding studies in one important regard: they require no steps to separate free from bound tracer and are therefore fast, simple and accurate.
Over the last few years, an increased awareness of endocrine disrupting chemicals (EDCs) and their potential to affect wildlife and humans has produced a demand for practical screening methods to identify endocrine activity in a wide range of environmental and industrial chemicals. While it is clear that in vivo methods will be required to identify adverse effects produced by these chemicals, in vitro assays can define particular mechanisms of action and have the potential to be employed as rapid and low-cost screens for use in large scale EDC screening programs. Traditional estrogen receptor (ER) binding assays are useful for characterizing a chemical's potential to be an estrogen-acting EDC, but they involve displacement of a radioactive ligand from crude receptor preparations at low temperatures. The usefulness of these assays for realistically determining the ER binding interactions of weakly estrogenic environmental and industrial compounds that have low aqueous solubility is unclear. In this report, we present a novel fluorescence polarization (FP) method that measures the capacity of a competitor chemical to displace a high affinity fluorescent ligand from purified, recombinant human ER-[alpha] at room temperature. The ER-[alpha] binding interactions generated for 15 natural and synthetic compounds were found to be similar to those determined with traditional receptor binding assays. We also discuss the potential to employ this FP technology to binding studies involving ER-ss and other receptors. Thus, the assay introduced in this study is a nonradioactive receptor binding method that shows promise as a high throughput screening method for large-scale testing of environmental and industrial chemicals for ER binding interactions.ImagesFigure 2Figure 3Figure 4
Phosphorylation of the tumor suppressor p53 is generally thought to modify the properties of the protein in four of its five independent domains. We used synthetic peptides to directly study the effects of phosphorylation on the non-sequence-specific DNA binding and conformation of the C-terminal, basic domain. The peptides corresponded to amino acids 361-393 and were either nonphosphorylated or phosphorylated at the protein kinase C (PKC) site, Ser378, or the casein kinase II (CKII) site, Ser392, or bis-phosphorylated on both the PKC and the CKII sites. A fluorescence polarization analysis revealed that either the recombinant p53 protein or the synthetic peptides bound to two unrelated target DNA fragments. Phosphorylation of the peptide at the PKC or the CKII sites clearly decreased DNA binding, and addition of a second phosphate group almost completely abolished binding. Circular dichroism spectroscopy showed that the peptides assumed identical unordered structures in aqueous solutions. The unmodified peptide, unlike the Ser378 phosphorylated peptide, changed conformation in the presence of DNA. The inherent ability of the peptides to form an alpha-helix could be detected when circular dichroism and nuclear magnetic resonance spectra were taken in trifluoroethanol-water mixtures. A single or double phosphorylation destabilized the helix around the phosphorylated Ser378 residue but stabilized the helix downstream in the sequence.
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