High-throughput scintillation proximity assay for stearoyl-CoA desaturase-1

Stearoyl-CoA desaturase (SCD) catalyzes the synthesis of monounsaturated fatty acids and has been implicated in a number of disease states, including obesity and diabetes. To find small-molecule inhibitor leads, a high-throughput scintillation proximity assay (SPA) was developed using the hydrophobic binding characteristics of a glass microsphere scintillant bead to capture SCD1 from a crude lysate of recombinant SCD1 in Sf9 lysate coupled with the strong binding characteristics of an azetidine compound ([(3)H]AZE). The SPA assay was stable over 24 h and could detect compounds with micromolar to nanomolar potencies. A robust 1536-well high-throughput screening assay was developed with good signal-to-noise ratio (10:1) and excellent Z' factor (0.8). A screening collection of 1.6 million compounds was screened at 11 μM, and approximately 7700 compounds were identified as initial hits, exhibiting at least 35% inhibition of [(3)H]AZE binding. Further screening and confirmation with an SCD enzyme activity assay led to a number of new structural leads for inhibition of the enzyme. The SPA assay complements the enzyme activity assay for SCD1 as a tool for the discovery of novel leads in drug discovery.     

Development of a microplate-based scintillation proximity assay for MraY using a modified substrate

MraY is an established target for the discovery of antibacterial agents. The conventional assay for MraY uses radioactive substrate and analysis of products after paper chromatography or butanol extraction. Synthesis of radiolabeled substrate has been done in vitro using purified enzymes or by growing cells on radiolabeled precursors. The authors report a simple and rapid method to chemically radiolabel MraY substrate, UDP-MurNAc-pentapeptide. Specific activity obtained by this method was more than 100 times higher than the conventionally labeled substrate, and yields are high enough to support the requirements of high-throughput screening (HTS). The authors have developed a microplate-based homogeneous assay for MraY in which the product is captured on wheat germ agglutinin (WGA) scintillation proximity assay (SPA) beads. The assay was validated by showing inhibition by specific inhibitors of MraY but not by inhibitors of other enzymes of peptidoglycan synthesis. The assay uses wild-type membranes of Escherichia coli, giving it an advantage over recently described assays that need the protein to be overexpressed. In addition, it has an advantage over the high-throughput MraY-MurG coupled assay reported in the literature because it is MraY specific, and therefore hits obtained in this assay do not need further deconvolution. It has potential for use in HTS approaches to find novel inhibitors of MraY.     

A scintillation proximity assay for poly(ADP-ribose) polymerase

Poly(ADP-ribose) polymerase (PARP) is an abundant nuclear protein in most of the eukaryotic tissues. When activated by DNA damage, PARP synthesizes poly(ADP-ribose) from NAD. Conventional radioactive PARP enzyme assay requires the separation of the polymer product from the NAD substrate, a rate-limiting step that hampers large-scale chemical library screening to identify novel small-molecule PARP inhibitors. By using biotinylated NAD, we have developed a scintillation proximity assay (SPA) for PARP. We demonstrated that PARP can incorporate the biotinylated ADP-ribose units into the radioactive poly(ADP-ribose) polymer, which can directly bind and excite the streptavidin-conjugated scintillation beads. PARP-SPA can be readily adapted to a 96-well format for automatic high-throughput screening for PARP inhibitors.     

High-throughput cell-based screening using scintillation proximity assay for the discovery of inositol phosphatase inhibitors

Inositol monophosphatase is a potential drug target for developing lithium-mimetic agents for the treatment of bipolar disorder. Enzyme-based assays have been traditionally used in compound screening to identify inositol monophosphatase inhibitors. A cell-based screening assay in which the compound needs to cross the cell membrane before reaching the target enzyme offers a new approach for discovering novel structure leads of the inositol monophosphatase inhibitor. The authors have recently reported a high-throughput measurement of G-protein-coupled receptor activation by determining inositol phosphates in cell extracts using scintillation proximity assay. This cell-based assay has been modified to allow the determination of inositol monophosphatase activity instead of G-protein-coupled receptors. The enzyme is also assayed in its native form and physiological environment. The authors have applied this cell-based assay to the high-throughput screening of a large compound collection and identified several novel inositol monophosphatase inhibitors.     

Development of a robust scintillation proximity assay for protein tyrosine phosphatase 1B using the catalytically inactive (C215S) mutant

Protein tyrosine phosphatases are a class of enzymes that function to modulate tyrosine phosphorylation of cellular proteins and play an essential role in regulating cell function. PTP1B has been implicated in the negative regulation of the insulin signaling pathway by dephosphorylating the activated insulin receptor. Inhibiting this phosphatase and preventing the insulin-receptor downregulation has been suggested as a target for the treatment of Type II diabetes. A high-throughput screen for inhibitors of PTP1B was developed using a scintillation proximity assay (SPA) with GST-- or FLAG--PTP1B((1-320)) and a potent [(3)H]-tripeptide inhibitor. The problem of interference from extraneous oxidizing and alkylating agents which react with the cysteine active-site nucleophile was overcome by the use of the catalytically inactive C215S form of the native enzyme (GST--PTP1B(C215S)). The GST--PTP1B was linked to the protein A scintillation bead via GST antibody. The radiolabeled inhibitor when bound to the enzyme gave a radioactive signal that was competed away by the unknown competitive compounds. Further utility of PTP1B(C215S) was demonstrated by mixing in the same well both the catalytically inactive GST--PTP1B(C215S) and the catalytically active FLAG--CD45 with an inhibitor. Both a binding and kinetic assay was then performed in the same 96-well plate with the inhibition results determined for the PTP1B(C215S) (binding assay) and CD45 (activity assay). In this way inhibitors could be differentiated between the two phosphatases under identical assay conditions in one 96-well assay plate. The use of a mutant to reduce interference in a binding assay and compare with activity assays is also amenable for most cysteine active-site proteases.     

Development of a scintiplate assay for recombinant human alpha(2B)-adrenergic receptor

A high-throughput solid-phase platform for ligand-binding assays using microtiter plates (Scintiplates) has been developed using the scintillation proximity assay principle. The system has been developed using human alpha(2B)-adrenergic receptor (alpha(2B)-AR) expressed from Semliki Forest virus vectors in CHO cells. Alpha(2B)-AR bind natural (adrenaline and noradrenaline) and synthetic ligands with different affinities to mediate a variety of physiological and pharmacological responses. Antagonist radioligands were used for the binding experiments, and the values obtained for the binding constants with the Scintiplate system are in good agreement with those obtained by the traditional filter-binding assay system. The Scintiplate assay offers the advantages of a high-throughput format over the filter-binding assay and is amenable for screening many compounds rapidly for generation of leads.     

A comparison of hydraulic and laser capture microdissection methods for collection of single B cells,PCR, and sequencing of antibody VDJ

A new procedure for measuring ATPase activity in which gamma-(33)P-labeled inorganic orthophoshate is detected by addition of ammonium molybdate followed by selective adsorption of the resulting phosphomolybdate to scintillation proximity beads in the presence of cesium chloride is described. This method is shown to give accurate and reproducible results over a wide range of detection conditions and product concentrations. It requires no separation or filtration steps and is highly compatible with automated high-throughput screening. Rates of hydrolysis are easily and accurately determined over a wide range, and thus the method is useful for kinetic studies also. We show that this scintillation proximity assay is useful for the study of the E1 helicase of human papillomavirus, but it is a general procedure which could also be applied to any ATPase or other nucleotide triphosphate-hydrolyzing enzyme or any other enzyme which generates orthophosphate as a reaction product.     

A FlashPlate assay for the identification of PARP-1 inhibitors

A novel FlashPlate scintillation proximity assay has been developed for the high-throughput screening (HTS) of large compound libraries to identify inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1), an important enzyme involved in DNA repair. The assay was originally developed for the 96-well FlashPlate but is easily transferred to a 384-well format. Moreover, the authors demonstrate that the assay is sufficiently sensitive to determine accurate IC(50) values and adaptable for kinetic evaluation of lead molecules. The mechanism of action of the assay requires the binding of PARP-1 to a double-stranded DNA oligonucleotide leading to the active enzyme. Using NAD(+) and (3)H-NAD(+) as substrate, activated PARP-1 synthesizes labeled poly(ADP-ribose) chains. Once the reaction is stopped, ADP-ribose polymers are brought into proximity with the pretreated FlashPlate walls, resulting in signal amplification. This signal is then detected by a TopCount scintillation plate reader. The developed assay is a robust and reproducible method of screening for PARP-1 inhibitors that is low maintenance and cost-effective and can easily be automated.     

A homogeneous assay of kinase activity that detects phosphopeptide using fluorescence polarization and zinc

Homogeneous antibody-free assays of protein kinase activity have great utility in high-throughput screening in support of drug discovery. In an effort to develop such an assay, we have used a pair of fluorescein-labeled peptides of identical amino acid sequence with and without phosphorylation on serine to mimic the substrate and product, respectively, of a kinase. Using fluorescence polarization (FP), we have demonstrated that a mixture of zinc sulfate, phosphate-buffered saline, and bovine serum albumin added to the peptides dramatically and differentially increased the fluorescence polarization of the phosphorylated peptide over its nonphosphorylated derivative. A similar FP differential was observed using different peptide pairs, though the magnitude varied. The FP values obtained using this method were directly proportional to the fraction of phosphopeptide present. Therefore, an FP assay was developed using a proprietary kinase. Using this FP method, linear reaction kinetics were obtained in enzyme titration and reaction time course experiments. The IC(50) values for a panel of inhibitors of kinase activity were determined using this FP method and a scintillation proximity assay. The IC(50) values were comparable between the two methods, suggesting that the zinc FP assay may be useful as an inexpensive high-throughput assay for identifying inhibitors of kinase activity.     

Fluorometric assay for quantitation of biotin covalently attached to proteins and nucleic acids

As a component of the (strept)avidin affinity system, biotin is often covalently linked to proteins or nucleic acids. We describe here a microplate-based high-throughput fluorometric assay for biotin linked to either proteins or nucleic acids based on fluorescence resonance energy transfer (FRET). This assay utilizes a complex of Alexa Fluoro 488 dye-labeled avidin with a quencher dye, 2-(4'-hydroxyazobenzene) benzoic acid (HABA), occupying the biotin binding sites of the avidin. In the absence of biotin, HABA quenches the fluorescence emission of the Alexa Fluor 488 dyes via FRET HABA is displaced when biotin binds to the Alexa Fluor 488 dye-labeled avidin, resulting in decreased FRET efficiency. This mechanism results in an increase in fluorescence intensity directly related to the amount of biotin present in the sample. The assay is able to detect as little as 4 pmol biotin in a 0.1 mL volume within 15 min of adding sample to the reagent, with a Z-factor > 0.9.     

Detection of p56(lck) kinase activity using scintillation proximity assay in 384-well format and imaging proximity assay in 384- and 1536-well format

p56(lck) is a lymphocyte-specific tyrosine kinase that plays an important role in both T-cell maturation and activation. We have developed a homogeneous assay in which p56(lck) catalyzes the transfer of the gamma-phosphate group from [gamma-(33)P]ATP to a biotinylated peptide substrate. The labeled peptide is then captured on a streptavidin-coated scintillation proximity assay (SPA) bead or imaging proximity bead. The SPA is counted in a microplate scintillation counter and the imaging proximity assay is counted in a charge-coupled device-based imaging system called LEADseekertrade mark, recently launched as a homogeneous imaging system by Amersham Pharmacia Biotech. We show, via time-dependence assays and inhibitor studies, that this assay can be performed in 1536-well microplate format using imaging proximity as the method of detection. The results compare favorably with the same assay performed in 384-well microplate format using both SPA and imaging proximity as the detection methods. From this study, we conclude that a kinase assay can be performed in 384- and 1536-well format using imaging as the detection method, with significant time savings over standard scintillation counting. In addition, we show cost saving advantages of 1536- over 384-well format in terms of reagent usage, higher throughput, and waste disposal.     

Scintillation proximity assay of arginine methylation

Methylation of arginine residues, catalyzed by protein arginine methyltransferases (PRMTs), is one important protein posttranslational modification involved in epigenetic regulation of gene expression. A fast and effective assay for PRMT can provide valuable information for dissecting the biological functions of PRMTs, as well as for screening small-molecule inhibitors of arginine methylation. Currently, among the methods used for PRMT activity measurement, many contain laborious separation procedures, which restrict the applications of these assays for high-throughput screening (HTS) in drug discovery. The authors report here a mix-and-measure method to measure PRMT activity based on the principle of scintillation proximity assay (SPA). In this assay, (3)H-AdoMet was used as methyl donor, and biotin-modified histone H4 peptide served as a methylation substrate. Following the methylation reaction catalyzed by PRMTs, streptavidin-coated SPA beads were added to the reaction solution, and SPA signals were detected by a MicroBeta scintillation counter. No separation step is needed, which simplifies the assay procedure and greatly enhances the assay speed. Particularly, the miniaturization and robustness suggest that this method is suited for HTS of PRMT inhibitors.     

Use of cellular glucose-6-phosphate dehydrogenase for cell quantitation: applications in cytotoxicity and apoptosis assays

A fluorescence-based microplate assay was developed to quantify cell death based upon the measurement of glucose-6-phosphate dehydrogenase (G6PD) activity. G6PD is a cytosolic enzyme and leaks from cells when plasma membrane integrity is compromised. In this assay, cell death is measured by correlating the activity of extracellular G6PD to the reduction of resazurin to the fluorescent product, resorufin, via a coupled-enzyme reaction. The coupled-enzyme reaction permits rapid signal amplification from small amounts of G6PD, an advantage over assays based on resazurin alone. This assay is rapid, nontoxic, and amenable to high-throughput screening. The assay has a Z' factor of 0.78.     

Direct determination of endothelin receptor antagonist levels in plasma using a scintillation proximity assay

An assay using scintillation proximity bead technology has been developed suitable for the quantitation of endothelin (ET) receptor antagonists in preclinical and clinical samples of plasma. The assay measures the competitive inhibition of radiolabelled ET-1 binding to ET(A) receptor membranes bound to wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads in the presence of plasma containing A-127722, a potent orally active, ET(A) selective ET antagonist. The assay requires as little as 50 microl plasma and no extraction procedure is needed. The SPA methodology eliminates the need for the separation of bound from free ligand. Using this method, A-127722 could be directly quantified in rat plasma with a detection limit of 1 ng/ml.     

A high-throughput fluorescence polarization assay for inhibitors of gyrase B

DNA gyrase, a type II topoisomerase that introduces negative supercoils into DNA, is a validated antibacterial drug target. The holoenzyme is composed of 2 subunits, gyrase A (GyrA) and gyrase B (GyrB), which form a functional A(2)B(2) heterotetramer required for bacterial viability. A novel fluorescence polarization (FP) assay has been developed and optimized to detect inhibitors that bind to the adenosine triphosphate (ATP) binding domain of GyrB. Guided by the crystal structure of the natural product novobiocin bound to GyrB, a novel novobiocin-Texas Red probe (Novo-TRX) was designed and synthesized for use in a high-throughput FP assay. The binding kinetics of the interaction of Novo-TRX with GyrB from Francisella tularensis has been characterized, as well as the effect of common buffer additives on the interaction. The assay was developed into a 21-μL, 384-well assay format and has been validated for use in high-throughput screening against a collection of Food and Drug Administration-approved compounds. The assay performed with an average Z' factor of 0.80 and was able to identify GyrB inhibitors from a screening library.     

Development of a high throughput scintillation proximity assay for hepatitis C virus NS3 protease that reduces the proportion of competitive inhibitors identified

A screening assay has been developed for hepatitis C virus (HCV) NS3 protease using the scintillation proximity assay (SPA) technology. The sequence of the peptide substrate used was taken from the site cleaved by the enzyme in the mature nonstructural protein of HCV. The peptide was biotinylated at the N-terminus and tritiated at the C-terminus so that a decrease in signal was detected as a result of enzyme activity. IC(50) values were calculated for the cleaved product, and it was shown that the value obtained was dependent on the substrate concentration used. The effect of substrate concentration on the inhibition of HCV NS3 protease was further highlighted in a mock screening assay, using colored natural product samples, in which the hit rate was altered by a change in substrate concentration. An increase in substrate concentration reduced the proportion of competitive inhibitors identified. This study highlighted the importance of optimizing the components used in SPA assays in order to obtain an assay format valid for high throughput screening.     

Cellular solid-phase binding assay and mass spectrometry for screening of alpha 4 beta 7 integrin antagonists.

A qualitative cellular solid-phase binding assay for screening alpha 4 beta 7 integrin antagonists attached via photolinker to TentaGel Macrobeads has been developed. An activation of the integrins with Mn(2+) was necessary to achieve binding to the bead bound antagonists. The identification of the resin bound compounds was done by mass spectrometry.