Development of High Throughput Screening Assays Using Fluorescence Polarization: Nuclear Receptor-Ligand–Binding and Kinase /Phosphatase Assays

Abstract

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.

Controllable drug uptake and nongenomic response through estrogen-anchored cyclodextrin drug complex

“…The binding of the CDE 1 -Ada-DOX inclusion complex to recombinant human ERα fragments consisting of amino acid residues 1-116 at the C-terminus (His tag C-terminus, Molecular Mass =12,200 Da) (catalogue number: ab153776; Abcam Plc, Cambridge, UK) was investigated using the fluorescence polarization method as described previously. 31 Briefly, human ERα fragments were reconstituted in phosphate-buffered saline to the final concentration of 0.8 µM, and CDE 1 -Ada-DOX complex samples at concentrations from 0.04 µM to 1.26 µM were added to the protein solution. The samples were mixed well at room temperature and subject to analysis immediately.…”

“…31 The polarization (mP) data over the concentration of the CDE 1 -Ada-DOX inclusion complex in the absence or presence of human ERα fragments at a fixed concentration of 0.08 µM are shown in Figure 7B-D. The mP values were increased when the concentration of the CDE 1 -Ada-DOX inclusion complex was increased without adding the ERα fragments, with a K d of 0.018 µM.…”

Breath biomarkers in toxicology

“…Once an AOP is established from in vivo laboratory study, one small section of the chemical reaction chain is reproduced in vitro to represent the full pathway. To date, AOP tests are mostly implemented in multi-well plates and the analyses are based on liquid-phase immunochemistry, although some liquid chromatography work may also be performed (Kavlock et al 2012;Parker et al 2000;Inglese et al 2007). A complementary approach has been proposed termed the aggregate exposure pathway (AEP), which is meant to serve as the precursor analog to the AOP (Teeguarden et al 2016).…”

Identification of 2-oxohistidine Interacting Proteins Using E. coli Proteome Chips

“…On the other hand, fluorescence polarization assay is a homogeneous binding detection method to mimic the interaction between two compounds in the cellular solution environment (55)(56)(57)(58)(59). We used fluorescence polarization assays to investigate the binding between fluorescent peptide probes and the 10 identified interacting proteins.…”