Characterization of a scintillation proximity assay to detect modulators of transforming growth factor alpha (TGF alpha) binding.

A scintillation proximity assay (SPA) for transforming growth factor alpha (TGF alpha) using SPA beads coated with A431 membranes has been studied. Binding of TGF alpha to the beads was characteristic of a receptor interaction. A class of high-affinity receptors for [125I]-TGF alpha (Kd = 0.10-0.26 nM) was detected by competition studies between [125I]TGF alpha and cold TGF alpha and by analysis of association and dissociation rate constants. An antibody to the epidermal growth factor receptor (clone 528) inhibited binding of [125I]TGF alpha (IC50 = 0.20 micrograms/ml), but an anti-TGF alpha antibody (clone 134A-2B3) (less than 25 micrograms/ml) did not block binding. Suramin inhibited [125I]-TGF alpha binding (IC50 = 0.20 mM). The ether lipids 1-O-hexadecyl-2-O-methyl-sn-glycero-3-phosphocholine, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine, and rac-lyso-platelet activating factor inhibited TGF alpha binding (IC50 values of 49, 69, and 57 microM, respectively). SPA is a convenient method for identifying agents that may act by interfering with TGF alpha binding.     

A simple method for measurement of phosphoramidon-sensitive endothelin converting enzyme activity.

We have developed a rapid and convenient assay for measurement of the action of endothelin (ET) converting enzyme (ECE) using the scintillation proximity assay (SPA) principle. On incubation of [125I]big ET-1 at 37 degrees C for 0.5-6 hr with an enzyme preparation, the reaction was terminated by the addition of an ET-1-specific antibody formulated in a buffer designed to shift the pH to alkaline. The antibody was allowed to come to equilibrium for 1 hr at room temperature and the amount of ET-1 produced, detected in a single step by the addition of protein A SPA beads. Using this assay, ECE activities of enzyme preparations obtained from porcine cultured endothelial cells and rat lung were clearly detected. These activities were inhibited by phosphoramidon in a concentration-dependent manner. The SPA based assay is homogeneous requiring no separation steps and takes a half day to complete. This method is therefore suitable for the high throughput screening of potential ECE inhibitors.     

An immobilized metal ion affinity adsorption and scintillation proximity assay for receptor-stimulated phosphoinositide hydrolysis

A novel approach to measuring receptor-stimulated phosphoinositide hydrolysis was developed based on the principles of immobilized metal ion affinity chromatography (IMAC) and scintillation proximity assay (SPA). Hard Lewis metal ions, such as Zr(4+), Ga(3+), Al(3+), Fe(3+), Lu(3+), and Sc(3+), were immobilized on SPA beads via metal chelate and utilized as affinity ligands to entrap inositol phosphates. [3H]Inositol phosphates bound to IMAC-SPA beads through the strong interaction of their phosphate group with the immobilized metal ions. The binding brought [3H]inositol phosphates in close proximity to the scintillant embedded in the SPA beads, thereby allowing the radioactivity to be quantified. Quantification of [3H]inositol phosphate production in cells preincubated with [3H]inositol provided a highly sensitive measurement of phosphoinositide hydrolysis. The utility of this approach was demonstrated in measuring the response mediated by the G-protein-coupled neurokinin NK1 receptor and the tyrosine kinase-linked platelet-derived growth factor (PDGF) receptor. Substance P stimulated phosphoinositide hydrolysis concentration-dependently in CHO cells expressing NK1 receptors with a maximal 12-fold increase in inositol phosphate production. Similarly, PDGF-BB stimulated a 5-fold increase in phosphoinositide hydrolysis in quiescent Swiss 3T3 cells. This new approach is highly sensitive, fast, simple, easily performed on 96-well plates, and amenable for high-throughput screening.     

Enhanced endothelin-1 response and receptor expression in small mesenteric arteries of insulin-resistant rats

Hyperinsulinemia, a primary feature of insulin resistance, is associated with increased endothelin-1 (ET-1) activity. This study determined the vascular response to ET-1 and receptor binding characteristics in small mesenteric arteries of insulin-resistant (IR) rats. Rats were randomized to control (C) (n = 32) or IR (n = 32) groups. The response to ET-1 was assessed (in vitro) in arteries with (Endo+) and without (Endo-) endothelium. In addition, arteries (Endo+) were pretreated with the ET(B) antagonist A-192621 or the ET(A) antagonist A-127722. Finally, binding characteristics of [(125)I]ET-1 were determined. Results showed that in Endo+ arteries the maximal relaxation (E(max)) to ET-1 was similar between C and IR groups; however, the concentration at 50% of maximum relaxation (EC(50)) was decreased in IR arteries. In Endo- arteries, the E(max) to ET-1 was enhanced in both groups. Pretreatment with A-192621 enhanced the E(max) and EC(50) to ET-1 in both groups. In contrast, A-127722 inhibited the ET-1 response in all arteries in a concentration-dependent manner; however, a greater ET-1 response was seen at each concentration in IR arteries. Maximal binding of [(125)I]ET-1 was increased in IR versus C arteries although the dissociation constant values were similar. In conclusion, we found the vasoconstrictor response to ET-1 is enhanced in IR arteries due to an enhanced expression of ET receptors and underlying endothelial dysfunction.     

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.     

Homogeneous proximity tyrosine kinase assays: scintillation proximity assay versus homogeneous time-resolved fluorescence

Two homogeneous proximity assays for tyrosine kinases, scintillation proximity assay (SPA) and homogeneous time-resolved fluorescence (HTRF), have been developed and compared. In both formats, the kinase assay was performed using biotinylated peptide substrate, ATP ([33P]ATP in the case of SPA), and tyrosine kinase in a 96-well assay format. After the kinase reaction was stopped, streptavidin-coated SPA beads or europium cryptate-labeled anti-phosphotyrosine antibody and streptavidin-labeled allophycocyanin were added as detection reagents for SPA or HTRF assays, respectively. Since the assay signal was detected only when the energy donor (radioactivity for SPA, Eu for HTRF) and the energy acceptor molecules (SPA beads for SPA, allophycocyanin for HTRF) were in close proximity, both assays required no wash or liquid transfer steps. This homogeneous ("mix-and-measure") nature allows these assays to be much simpler, more robust, and easier to automate than traditional protein kinase assays, such as a filter binding assay or ELISA. Both assays have been miniaturized to a 384-well format to reduce the assay volume, thereby saving the valuable screening samples as well as assay reagents, and automated using automated pipetting stations to increase the assay throughput. Several advantages and disadvantages for each assay are described.     

A novel scintillation proximity assay for fatty acid amide hydrolase compatible with inhibitor screening

A binding assay for human fatty acid amide hydrolase (FAAH) using the scintillation proximity assay (SPA) technology is described. This SPA uses the specific interactions of [3H]R(+)-methanandamide (MAEA) and FAAH expressing microsomes to evaluate the displacement activity of FAAH inhibitors. We observed that a competitive nonhydrolyzed FAAH inhibitor, [3H]MAEA, bound specifically to the FAAH microsomes. Coincubation with an FAAH inhibitor, URB-597, competitively displaced the [3H]MAEA on the FAAH microsomes. The released radiolabel was then detected through an interaction with the SPA beads. The assay is specific for FAAH given that microsomes prepared from cells expressing the inactive FAAH-S241A mutant or vector alone had no significant ability to bind [3H]MAEA. Furthermore, the binding of [3H]MAEA to FAAH microsomes was abolished by selective FAAH inhibitors in a dose-dependent manner, with IC50 values comparable to those seen in a functional assay. This novel SPA has been validated and demonstrated to be simple, sensitive, and amenable to high-throughput screening.     

A scintillation proximity assay for human interleukin-5 (hIL-5) high-affinity binding in insect cells coexpressing hIL-5 receptor alpha and beta subunits

The high-affinity receptor for human interleukin-5 (hIL-5) is composed of alpha and beta subunits. A baculovirus expression system was established in Sf9 cells capable of expressing hIL-5 receptor alpha and beta subunits simultaneously. By using wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads to capture 125I-labeled hIL-5-bound Sf9 cells, a SPA was developed and used to measure hIL-5 high-affinity binding. The hIL-5 receptors expressed in the Sf9 cells represented a single class of high-affinity binding sites with a dissociation constant (Kd) of 0. 24 nM and a density of 2.95 x 10(5) sites/cell. This is the first study in which the high-affinity Kd value similar to that for hIL-5 binding to human eosinophils was achieved using a recombinant expression system. The SPA compared favorably with the filter binding assay with regard to various binding parameters. We also found that several lectins, when coated on SPA beads, were even more effective than WGA-coated SPA beads for capturing the insect cells. We conclude that the baculovirus expression system was highly efficient in producing the high-affinity hIL-5 receptors and that the SPA was a simple and sensitive assay that could be readily adapted into a high-throughput screening format. The SPA described here could be a prototype for binding assays for other multimeric receptors.     

Scintillation proximity assay for DNA binding by human p53

Many DNA binding proteins are known to regulate gene expression. When that binding is altered, a disease state can result. A common method for measuring DNA binding, namely electrophoretic mobility shift assay (EMSA) is often used but it is not amenable to rapid screening of many samples. As an alternative method, we have developed a DNA binding assay for the tumor suppressor protein p53 in a 96-well microtiter plate format using scintillation proximity assay (SPA) beads. We have shown this assay to be sensitive (as little as 0.5 ng p53 can be detected), quick (assay completed in as little as 15 min), and easily quantitated using a microtiter plate scintillation counter We also used the assay to analyze the kinetics of the DNA binding to p53. The specificity of this p53 DNA binding SPA was confirmed using competition by oligonucleotides either from the same gene or from mutated versions of this sequence. Thus, SPA is a good alternative to gel shift assays for DNA binding and may be useful for the analysis of multiple tumor cell samples or for high-throughput screens for compounds affecting DNA binding by proteins of interest.     

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.     

High-throughput screening for N-type calcium channel blockers using a scintillation proximity assay

N-type calcium channels located on presynaptic nerve terminals regulate neurotransmitter release, including that from the spinal terminations of primary afferent nociceptors. Accordingly, N-type calcium channel blockers may have clinical utility as analgesic drugs. A selective N-type calcium channel inhibitor, ziconotide (Prialt), is a neuroactive peptide recently marketed as a novel nonopioid treatment for severe chronic pain. To develop a small-molecule N-type calcium channel blocker, the authors developed a 96-well plate high-throughput screening scintillation proximity assay (SPA) for N-type calcium channel blockers using [125I]-labeled omega-conotoxin GVIA as a channel-specific ligand. Assay reagents were handled using Caliper's Allegro automation system, and bound ligands were detected using a PerkinElmer TopCount. Using this assay, more than 150,000 compounds were screened at 10 microM and approximately 340 compounds were identified as hits, exhibiting at least 40% inhibition of [125I]GVIA binding. This is the 1st demonstration of the use of [125I]-labeled peptides with SPA beads to provide a binding assay for the evaluation of ligand binding to calcium channels. This assay could be a useful tool for drug discovery.     

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.     

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.     

Serum immunoassay of a small cell lung cancer associated ganglioside: development of a sensitive scintillation proximity assay

We here report an enzyme linked immunosorbent assay (ELISA) and a scintillation proximity assay (SPA) for detection of the ganglioside FucGM₁ in sera from small cell lung cancer (SCLC) patients. The SPA was more sensitive and reproducible than the ELISA. In this assay, monoclonal antibodies specific for FucGM₁ were bound to SPA particles and incubated with labelled FucGM₁ and 100 µl test-serum overnight, and counted in a -counter. The sensitivity was 0.2 ng. Seven out of twenty sera from SCLC patients were positive, whereas none of twenty sera from healthy individuals were positive for FucGM₁. The SPA was more sensitive than the previously reported HPTLC as well as a direct ELISA.Abbreviations MAb monoclonal antibody - SPA scintillation proximity assay - HPTLC high performance thin layer chromatography - SCLC small cell lung cancer - FucGM₁ Fuc1-2Gal1-3GalNAc1-4(NeuAc2-3)-Gal1-4Glc1-1Cer - ELISA enzyme linked immunosorbent assay - FCS foetal calf serum - PBS phosphate buffered saline     

Compounds capable of generating singlet oxygen represent a source of artifactual data in scintillation proximity assays measuring phosphopeptide binding to SH2 domains.

We developed scintillation proximity assays (SPA) to discover compounds which inhibit phosphopeptide binding to Src homology 2 (SH2) domain proteins Grb2 and Syk. An assay artifact is reported here as a caveat to others. The SPA used an antibody to couple glutathione-S-transferase SH2 domain fusion proteins to scintillant beads coated with protein A. A pyrazoloquinolone and indolocarbazole inhibited [3H]phosphopeptide binding in both assays. Their potency in the SPA increased with prolonged (2 to 24 h) assay exposure to ambient light. They were inactive in absence of light and in an alternate binding assay. Both compounds absorbed visible light and generated singlet oxygen based on 2-methylfuran-trapping experiments. Their inhibitory activity was suppressed by the singlet oxygen scavengers sodium azide and dithiothreitol. The results suggest that compounds, not previously considered photosensitizers, generated enough singlet oxygen to damage oxidant-sensitive SPA components. Therefore, this SPA should be protected from light to minimize occurrence of false positives.     

Measuring substrate binding and affinity of purified membrane transport proteins using the scintillation proximity assay

The scintillation proximity assay (SPA) is a rapid radioligand binding assay. Upon binding of radioactively labeled ligands (here L-[(3)H]arginine or D-[(3)H]glucose) to acceptor proteins immobilized on fluoromicrospheres (containing the scintillant), a light signal is stimulated and measured. The application of SPA to purified, detergent-solubilized membrane transport proteins allows substrate-binding properties to be assessed (e.g., substrate specificity and affinity), usually within 1 d. Notably, the SPA makes it possible to study specific transporters without interference from other cellular components, such as endogenous transporters. Reconstitution of the target transporter into proteoliposomes is not required. The SPA procedure allows high sample throughput and simple sample handling without the need for washing or separation steps: components are mixed in one well and the signal is measured directly after incubation. Therefore, the SPA is an excellent tool for high-throughput screening experiments, e.g., to search for substrates and inhibitors, and it has also recently become an attractive tool for drug discovery.     

A homogeneous method to measure aminoacyl-tRNA synthetase aminoacylation activity using scintillation proximity assay technology

A new method to measure the aminoacylation of tRNA based upon the use of the scintillation proximity assay (SPA) technology has been developed. The assay detects incorporation of radiolabeled amino acids into cognate tRNA, catalyzed by a specific aminoacyl-tRNA synthetase (aaRS). Under acidic conditions, uncoated yttrium silicate SPA beads were found to bind tRNA aggregates, while the radiolabeled amino acid substrate remains in solution, resulting in good signal discrimination of these two species in the absence of any separation steps. The usefulness of this approach was demonstrated by measurement of steady-state kinetic constants and inhibitor binding constants for a range of aaRS enzymes in comparison with data from standard, trichloroacetic acid-precipitation-based assays. In all cases, the data were quantitatively comparable. Although the radioisotopic counting efficiency of the SPA method was less than that of standard liquid scintillation counting, the statistical performance (i.e., signal to background, variability, stability) of the SPA assays was at least equivalent to the separation-based methods. The assay was also shown to work well in miniaturized 384-well microtiter plate formats, resulting in considerable reagent savings. In summary, a new method to characterize aaRS activity is described that is faster and more amenable to high-throughput screening than traditional methods.