Competitive Fluorescence Polarization Assay Research Articles

Discovery of Small Molecule Inhibitors of the Interaction of the Thyroid Hormone Receptor with Transcriptional Coregulators

Thyroid hormone (3,5,3'-triiodo-L-thyronine, T3) is an endocrine hormone that exerts homeostatic regulation of basal metabolic rate, heart rate and contractility, fat deposition, and other phenomena (1, 2). T3 binds to the thyroid hormone receptors (TRs) and controls their regulation of transcription of target genes. The binding of TRs to thyroid hormone induces a conformational change in TRs that regulates the composition of the transcriptional regulatory complex. Recruitment of the correct coregulators (CoR) is important for successful gene regulation. In principle, inhibition of the TR-CoR interaction can have a direct influence on gene transcription in the presence of thyroid hormones. Herein we report a high throughput screen for small molecules capable of inhibiting TR coactivator interactions. One class of inhibitors identified in this screen was aromatic beta-aminoketones, which exhibited IC50 values of approximately 2 microm. These compounds can undergo a deamination, generating unsaturated ketones capable of reacting with nucleophilic amino acids. Several experiments confirm the hypothesis that these inhibitors are covalently bound to TR. Optimization of these compounds produced leads that inhibited the TR-CoR interaction in vitro with potency of approximately 0.6 microm and thyroid signaling in cellular systems. These are the first small molecules irreversibly inhibiting the coactivator binding of a nuclear receptor and suppressing its transcriptional activity.

Synthesis of O-galactosyl aldoximes as potent LacNAc-mimetic galectin-3 inhibitors

A panel of anomeric oxime ether derivatives of β-galactose were synthesized via the reaction of O-β- d-galactopyranosylhydroxylamine with aldehydes. The oxime ethers were evaluated as inhibitors against galectin-3 in a competitive fluorescence polarization assay. The best inhibitor, [ E]- O-(β- d-galactopyranosyl)-indole-3-carbaldoxime ( E- 52), had a K d value of 180 μM, which is 24 times better than methyl β- d-galactopyranoside ( K d = 4400 μM) and in the same range as methyl lactoside ( K d = 220 μM).

Detecting the C282Y and H63D Mutations of the HFE Gene by Holliday Junction-Based Allele-Specific Genotyping Methods

Hereditary hemochromatosis, one of the most common genetic diseases in Caucasians, is characterized by excessive iron deposition secondary to hyperabsorption of dietary iron and can potentially can lead to multiorgan failure if untreated. The C282Y and H63D mutations of the HFE gene are the most common mutations associated with symptomatic hemochromatosis. Recently, several genotyping methods have been used to identify hemochromatosis mutations and other single-nucleotide polymorphisms (SNPs) (1). There are advantages and limitations for each methodology in terms of cost and efficiency (2). We report here the application of a new SNP/point mutation genotyping platform developed by our group, the Holliday junction-based allele-specific genotyping (HAS) platform (3), to identify the C282Y and H63D mutations associated with hemochromatosis. For the HAS technology, we developed two detection modalities based on differences in the physical and biochemical properties between Holliday junctions (HJs) and duplex DNA or single-stranded DNA. The junctions that form in an allele-specific manner can be detected heterogeneously through gel electrophoresis (acrylamide or agarose; Fig. 1A⇓ ) or homogeneously through a fluorescence polarization (FP) competition assay (3). Using the HAS genotyping platform, we developed an assay for genotyping the C282Y and H63D mutations with both gel electrophoresis and FP for detection. Five primers (one forward, two reference, and two reverse primers) were designed for each of the two mutations (see Table 1 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol51/issue1/). PCR amplification was performed with a PTC-200 DNA Engine thermocycler (MJ Research, Inc.). For each point mutation locus, two PCR reactions in separate tubes were carried out in parallel for each DNA sample to amplify the target DNA with each of its two DNA references. Each PCR reaction contained four primers [one forward, one reference, reverse tail I, and reverse tail II (Table …

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 μg/ml. An X-ray costructure of a promising small molecule in this library complexed with ZipA 185-328 ( K I =12 μM) revealed that the compound binds to the same hydrophobic pocket as the FtsZ 367-383 peptide.

Allele-specific Holliday junction formation: a new mechanism of allelic discrimination for SNP scoring.

We report here a new mechanism for allelic discrimination--allele-specific Holliday Junction formation. The Holliday Junction (HJ) is a unique DNA structure that can be formed in a sequence-nonspecific manner by routine PCR. To cause the PCR-based HJ formation to occur in an allele-specific manner, the PCR primers are manipulated such that an extra mismatch next to a SNP of interest is introduced between a target and a reference amplicon and a GC-clamp is added. Based on this new mechanism, novel SNP genotyping methods were developed, including a homogeneous fluorescence polarization (FP) competition assay that requires neither labeled primers/probes nor expensive enzymes/substrates. Using this novel genotyping technology, we were able to convert >95% of SNP sequences into genotyping assays that work well under a universal set of assay conditions and achieved 100% accuracy in clinical samples.

Fluorescence Polarization Competition Assay: The Range of Resolvable Inhibitor Potency Is Limited by the Affinity of the Fluorescent Ligand

For the development of fluorescence polarization (FP) competition assays, there is a widespread belief that tight-binding fluorescent ligands should be avoided to identify inhibitors of low or intermediate potency in the screening of small-molecule compound libraries. It is demonstrated herein that this statement is a misconception; in fact, the higher the affinity of the fluorescent ligand, the wider the range of inhibitor potency that can be resolved. An approximate estimate for the low end of inhibitor K(i) values that can be resolved is the K(d) value of the fluorescent ligand. Because FP competition assays are typically conducted under nonstoichiometric titration conditions, it is suggested that a fluorescent ligand of highest affinity that also has an adequate quantum yield to satisfy such conditions be selected.

Development and Validation of a Competitive AKT Serine/Threonine Kinase Fluorescence Polarization Assay Using a Product-Specific Anti-phospho-serine Antibody

A competitive fluorescence polarization (FP) assay has been developed for the serine/threonine kinase, AKT. The FP assay has been formatted in a 384-well microtiter plate and automated using a pipeting workstation with performance suitable for high-throughput screening. The assay design utilizes a fluorescent phosphorylated peptide complexed to a product-specific anti-phospho-serine antibody. When unlabeled substrate is phosphorylated, by the kinase, the product competes with the fluorescent phosphorylated peptide for the antibody. The fluorescent phosphorylated peptide is then released from the antibody into solution resulting in a loss in polarization signal. Seven fluorescent phosphorylated peptides and 19 antibodies were evaluated for this assay. RARTSpSFAEPGK-Fl peptide and anti-phospho-GSK-3α Ser21 antibody gave the best affinity and change in polarization signal. The apparent kinetic constants were calculated for the FP assay and were consistent with reported values. The FP assay was validated with known inhibitors and the results compared to a radioactive Flashplate transfer assay, utilizing [ 33P]ATP and a biotinylated substrate, also developed in our laboratory. The IC 50 values generated were comparable between the two methods suggesting the competitive FP assay and Flashplate assay have similar sensitivities and abilities to identify inhibitors during screening.

Identification of a High-Affinity Anti-Phosphoserine Antibody for the Development of a Homogeneous Fluorescence Polarization Assay of Protein Kinase C

In the last few years, fluorescence polarization (FP) has been applied to the development of robust, homogeneous, high throughput assays in molecular recognition research, such as ligand-protein interactions. Recently, this technology has been applied to the development of homogeneous tyrosine kinase assays, since there are high-affinity anti-phosphotyrosine antibodies available. Unlike tyrosine kinases, application of FP to assay development for serine/threonine kinases has been impeded because of lack of high-affinity anti-phosphoserine/threonine antibodies. In the present study, we report the discovery of a high-affinity, monoclonal anti-phosphoserine antibody, 2B9, with a Kd of 250 ± 34 pM for a phosphoserine-containing peptide tracer, fluorescein-RFARKGS(PO4)LRQKNV. Our data suggest that 2B9 is selective for fluorescein-RFARKGS(PO4)LRQKNV. The antibody and tracer have been used for the development of a competitive FP assay for protein kinase C (PKC) in 384-well plates. Phosphatidylserine, which enhances the kinase activity of PKC in a Ca2+-dependent manner and has a structure similar to that of phosphoserine, did not interfere with binding of the peptide tracer to the antibody in the FP assay. The data indicate that the FP assay is more sensitive and robust than the scintillation proximity assay for PKC. The FP assay developed here can be used for rapid screening of hundreds of thousands of compounds for discovery of therapeutic leads for PKC-related diseases.

On the Interaction between a Bactericidal Antibody and a PorA Epitope ofNeisseria meningitidisin Outer Membrane Vesicles: A Competitive Fluorescence Polarization Immunoassay

This paper describes a method for determining the affinity constant (Ka) of the binding between an antibody Fab fragment and a membrane-embedded protein epitope under equilibrium conditions. Monoclonal antibody MN12H2, directed against outer membrane protein PorA ofNeisseria meningitidis,is used in a competitive fluorescence polarization assay with a cyclic peptide–fluorescein conjugate as a tracer antigen. Displacement experiments with PorA-containing and PorA-deficient meningococcal outer membrane vesicles revealed highly specific binding of MN12H2 Fab to the membrane-embedded PorA P1.16 epitope withKaof 1.5 × 108m−1.

Identification of the binding site for plasma prekallikrein in human high molecular weight kininogen. A region from residues 185 to 224 of the kininogen light chain retains full binding activity.

We studied the ability of fragments of the light chain of human high molecular weight kininogen to bind to plasma prekallikrein. In a competitive fluorescence polarization assay, kallikrein-cleaved light chain (light chain-2; residues 49-255), a cyanogen bromide fragment (residues 185-242), and a tryptic peptide (T-7; residues 185-224) had binding affinities of approximately 20 nM, equivalent to the value for the intact light chain (residues 1-255) of high-molecular-weight kininogen. In contrast, fragments consisting of residues 49-184 and 243-255 showed no binding activity (Kd much greater than 1,000 nM). Direct titrations of fluorescein-labeled derivatives of light chain-2 and peptide T-7 with prekallikrein confirmed that T-7 retained full binding activity for prekallikrein (Kd = 12 +/- 2 nM for labeled light chain-2; Kd = 7 +/- 1 nM for labeled T-7). These results localize the binding site of high molecular weight kininogen for prekallikrein within a region of 40 amino acids (residues 185-224) that resides in the near carboxyl terminus of the light chain of kininogen.