Scintillation proximity assay of inositol phosphates in cell extracts: high-throughput measurement of G-protein-coupled receptor activation

The phosphatidylinositol turnover assay is used widely to measure activation, and inhibition, of G(q)-linked G-protein-coupled receptors. Cells expressing the receptor of interest are labeled by feeding with tritiated myo-inositol. The label is incorporated into cellular phosphatidylinositol 4,5-bisphosphate, which, upon agonist binding to the receptor, is hydrolyzed by phospholipase C to inositol 1,4,5-trisphosphate (IP(3)) and diacylglycerol. In the presence of Li(+), dephosphorylation of IP(3) to inositol is blocked, and the mass of soluble inositol phosphates is a quantitative readout of receptor activation. Current protocols for this assay all involve an anion-exchange chromatography step to separate radiolabeled inositol phosphates from radiolabeled inositol, making the assay cumbersome and difficult to automate. We now describe a scintillation proximity assay to measure soluble inositol phosphate mass in cell extracts, thus obviating the need for the standard chromatography step. The method uses positively charged yttrium silicate beads that bind inositol phosphates, but not inositol. We have used this assay to measure activation of recombinant and endogenous muscarinic acetylcholine receptors and activation of recombinant neuropeptide FF2 receptor coupled to IP(3) production by coexpression of a chimeric G protein. Further, we demonstrate the use and functional validity of this assay in a semiautomated, 384-well format, by characterizing the muscarinic receptor antagonists pirenzepine and atropine.     

A miniaturized high-throughput screening assay for fucosyltransferase VII

Fucosyltransferase VII (FucTVII) is a very promising drug target for treatment of inflammatory skin diseases. Its activity is required for synthesis of the sialyl-Lewis X glycoepitopes on the E- and P-selectin ligands, necessary for lymphocyte migration into the skin. High-throughput screening (HTS) of large chemical libraries has become the main source of novel chemical entities for the pharmaceutical industry. The screening of very large compound collections requires the use of specialized assay techniques that minimize time and costs. We describe the development of a miniaturized scintillation proximity assay for human FucTVII based on a oligosaccharide acceptor substrate that is identical to the glycosylation of the physiological substrate. In addition to assay development, the assay performance in a HTS campaign is shown. We screened 798,131 compounds from the Schering AG HTS library and identified 233 IC50 hits; 229 hits were FucTVII specific in so far as they did not inhibit either alpha-fucosidase or galactosyltransferase. In addition to screening a drug-like small-molecule collection, we worked on rational approaches to develop inhibitors or glycosidic decoys based on oligosaccharide-substrate analogues. The structure-activity relationship observed thereby is very narrow and shows strict requirements that are consistent with the described substrate specificity of FucTVII.     

A high-throughput assay for the adenylation reaction of bacterial DNA ligase

DNA ligase catalyzes the closure of single-strand nicks in double-stranded DNA that arise during replication and recombination. Inhibition of bacterial ligase is expected to cause chromosome degradation and cell death, making it an attractive target for new antibacterials. The prototypical bacterial ligase couples the hydrolysis of NAD(+) to phosphodiester bond formation between an adjacent 3'OH and 5'-terminal phosphate of nicked duplex DNA. The first step is the reversible formation of a ligase-adenylate from the reaction between apoenzyme and NAD(+). Inhibitors that compete with NAD(+) are expected to be bacterial specific because eukaryotic DNA ligases use ATP and differ in the sequence composition of their adenylation domain. We report here a high-throughput assay that measures the adenylation reaction specifically by monitoring ligase-AMP formation via scintillation proximity technologies. Escherichia coli DNA ligase was biotinylated in vivo; after reaction with radiolabeled NAD(+), ligase-[(3)H]AMP could be captured onto the streptavidin-coated surface of the solid scintillant. The method was ideal for high-throughput screening because it required minimal manipulations and generated a robust signal with minimal scatter. Certain adenosine analogs were found to inhibit the adenylation assay and had similar potency of inhibition in a DNA ligation assay.     

A high-throughput assay for a human telomerase protein-human telomerase RNA interaction

The rapid rate at which cancer cells divide necessitates a mechanism for telomere maintenance, and in approximately 90% of all cancer types the enzyme telomerase is used to maintain the length of telomeric DNA. Telomerase is a multi-subunit enzyme that minimally contains a catalytic protein subunit, hTERT, and an RNA subunit, hTR. Proper assembly of telomerase is critical for its enzymatic activity and therefore is a requirement for the proliferation of most cancer cells. We have developed the first high-throughput screen capable of identifying small molecules that specifically perturb human telomerase assemblage. The screen uses a scintillation proximity assay to identify compounds that prevent a specific and required interaction between hTR and hTERT. Rather than attempting to disrupt all of the individual hTR-hTERT interactions, we focused the screen on the interaction of the CR4-CR5 domain of hTR with hTERT. The screen employs a biotin-labeled derivative of the CR4-CR5 domain of hTR that independently binds [(35)S]hTERT in a functionally relevant manner. The complex between hTERT and biotin-labeled RNA can be captured on streptavidin-coated scintillation proximity beads. Use of 96-well filter plates and a vacuum manifold enables rapid purification of the beads. After optimization, statistical evaluation of the screen generated a Z' factor of 0.6, demonstrating the high precision of the assay.     

A homogeneous and nonisotopic assay for phosphatidylinositol 4-kinases

Phosphatidylinositol 4-kinases (PI 4-kinases) catalyze the conversion of phosphatidylinositol to phosphatidylinositol 4-phosphate (PtdIns4P). The four known mammalian PI 4-kinases, PI4KA, PI4KB, PI4K2A, and PI4K2B have roles in intracellular lipid and protein trafficking. PI4KA and PI4KB also assist in the replication of several positive-sense RNA viruses. The identification of selective inhibitors of these kinases would be facilitated by assays suitable for high-throughput screening. We describe a homogeneous and nonisotopic assay for PI 4-kinase activity based on the bioluminescent detection of the ADP produced by kinase reactions. We have evaluated this assay with known nonselective inhibitors of PI 4-kinases and show that it performs similar to radiometric assay formats previously described in the literature. In addition, this assay generates Z-factor values of >0.7 for PI4KA in a 384-well format, demonstrating its suitability for high-throughput screening applications.     

Development of a scintillation proximity assay binding method for the human 5-hydroxytryptamine 6 receptor using intact cells

We describe the first validated scintillation proximity assay (SPA) binding method for quantitation of ³H-labeled d-lysergic acid diethylamide (LSD) binding to recombinant human 5-hydroxytryptamine 6 (5-HT₆) receptors expressed in Chinese hamster ovary (CHO)-Dukx and HeLa cells. The assay was developed using intact cells as a receptor source because membrane fractions derived from these cells failed to discern specific binding from a high level of nonspecific binding. The pharmacological binding profile of seven 5-HT₆ agonists and antagonists using intact CHO-Dukx/5-HT₆ cells in the SPA format was similar to data obtained from a filtration binding assay using HeLa/5-HT₆ membranes. K_i values and rank order of potencies obtained in the SPA format were consistent with published filtration data as follows: SB-271046 (K_i = 1.9 nM) > methiothepin (K_i = 6.2 nM) > mianserin (K_i = 74.3 nM) > 5-methoxytryptamine (5-MeOT, K_i = 111 nM) > 5-HT (K_i = 150 nM) > ritanserin (K_i = 207 nM) > 5-carboxamidotryptamine (5-CT, K_i = 704 nM). Additional evaluation with four antipsychotics demonstrated strong agreement with previous literature reports. A high specific binding signal and low assay variability, as determined by Z′ = 0.81 ± 0.017, make the SPA format amenable to automation and higher throughput; hence, this assay can be a viable alternative to the more labor-intensive filtration and centrifugation methods.     

Development of a scintillation proximity assay for human insulin-like growth factor-binding protein 4 compatible with inhibitor high-throughput screening

The insulin-like growth factor-binding protein 4 (IGFBP-4), which exists in many different tissues and biological fluids, modulates insulin-like growth factor 1 (IGF-1) bioavailability in part by competitive sequestration and prevention of interaction with cell membrane IGF-1 receptors. Accordingly, small molecules that inhibit the ability of IGF-1 to associate with IGFBP-4 may have clinical utility as regulators of cellular proliferation, survival, and differentiation. Currently, a polyethylene glycol-based precipitation of [(125)I]IGF-1 bound to IGFBP-4 is used to quantify selective IGFBP-4 ligand interactions. We have developed a novel 96-well plate scintillation proximity assay (SPA) for measuring small molecule interactions at IGFBP-4 using a biotinylated form of IGFBP-4 coupled to streptavidin-coated polyvinyltoluene (PVT) SPA microbeads and using [(125)I]IGF-1 as the endogenous ligand. Dose-displacement curves with unlabeled IGF-1 exhibited a mean K(d) value of 0.46 nM. Parallel studies using the nonselective IGFBP inhibitor, NBI-31772, generated a K(i) value of 47 nM. Under optimized conditions, the IGFBP-4 SPA was stable for up to 24h at room temperature and was unaffected by dimethyl sulfoxide (DMSO,<0.5%). This homogeneous binding assay is simple, stable, sensitive, and amenable to automation. The good signal/noise ratio (10:1) and Z' factor (0.7-0.8) make it compatible with high-throughput screening platforms for the identification of IGFBP-4 inhibitors. The IGFBP-4 binding assay may be expanded to other IGFBP members, in biotinylated form, to provide a powerful tool amenable to drug screening and the design of therapeutics to treat a variety of IGF-responsive diseases.     

A homogeneous,nonradioactive high-throughput fluorogenic protein kinase assay

Protein kinases play an important role in many cellular processes and mediate cellular responses to a variety of extracellular stimuli. They have been identified by many pharmaceuticals as valid targets for drug discovery. Because of the large number of protein kinases, and the large number of compounds to be screened, it is important to develop assay systems that are not only sensitive but also homogeneous, fast, simple, nonradioactive, and cost-effective. Here we present a novel, rapid, robust assay to measure the enzyme activity of low concentrations of several serine/threonine and tyrosine protein kinases. It is based on the use of fluorogenic peptide substrates (Rhodamine 110, bis peptide amide) that are cleaved before phosphorylation to release the free Rhodamine 110; upon phosphorylation, cleavage is hindered, and the compound remains as a nonfluorescent peptide conjugate. The assay can be carried out in single- as well as multiwell plate formats such as 96- and 384-well plates. The signal-to-noise ratio is very high (40), the Z(') is over 0.8, and the signal is stable for at least 4h. Finally, the assay is easily adapted to a robotic system for drug discovery programs targeting protein kinases.     

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.     

SWTY--a general peptide probe for homogeneous solution binding assay of 14-3-3 proteins

Dimeric 14-3-3 proteins exert diverse functions in eukaryotes by binding to specific phosphorylated sites on diverse target proteins. Critical to the physiological function of 14-3-3 proteins is the wide range of binding affinity to different ligands. The existing information of binding affinity is mainly derived from nonhomogeneous-based methods such as surface plasmon resonance and quantitative affinity precipitation. We have developed a fluorescence anisotropy peptide probe using a genetically isolated 14-3-3-binding SWTY motif. The synthetic 5-(and-6)-carboxyfluorescein(FAM)-RGRSWpTY-COOH peptide, when bound to 14-3-3 proteins, exhibits a seven-fold increase in fluorescence anisotropy. Different from the existing assays for 14-3-3 binding, this homogeneous assay tests the interaction directly in solution. Hence it permits more accurate determination of the dissociation constants of 14-3-3 binding molecules. Protocols for a simple mix-and-read format have been developed to evaluate 14-3-3 protein interactions using either purified recombinant 14-3-3 fusion proteins or native 14-3-3s in crude cell lysate. Optimal assay conditions for high-throughput screening for modulators of 14-3-3 binding have been determined.     

Development of a highly sensitive,high-throughput assay for glycosyltransferases using enzyme-coupled fluorescence detection

Glycosyltransferases catalyze transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds. Identification of selective modulators of glycosyltransferases is important both to provide new tools for investigating pathophysiological roles of glycosylation reactions in cells and tissues, and as new leads in drug discovery. Here we describe a universal enzyme-coupled fluorescence assay for glycosyltransferases, based on quantification of nucleotides produced in the glycosyl transfer reaction. GDP, UDP, and CMP are phosphorylated with nucleotide kinase in the presence of excess ATP, generating ADP. Via coupled enzyme reactions involving ADP-hexokinase, glucose-6-phosphate dehydrogenase, and diaphorase, the ADP is utilized for conversion of resazurin to resorufin, which is determined by fluorescence measurement. The method was validated by comparison with an HPLC method, and employed to screen the LOPAC1280 library for inhibitors in a 384-well plate format. The assay performed well, with a Z′-factor of 0.80. We identified 12 hits for human galactosyltransferase B4GALT1 after elimination of false positives that inhibited the enzyme-coupled assay system. The assay components are all commercially available and the reagent cost is only 2 to 10 US cents per well. This method is suitable for low-cost, high-throughput assay of various glycosyltransferases and screening of glycosyltransferase modulators.     

Development of an intact cell reporter gene beta-lactamase assay for G protein-coupled receptors for high-throughput screening

G protein-coupled receptors (GPCRs) are involved in a large variety of physiological disorders, and are thus important pharmaceutical drug targets. Here, we describe the development and characterization of a beta-lactamase reporter gene assay as a functional readout for the ligand-induced activation of the human bradykinin B1 receptor, expressed recombinantly in CHO cells. The beta-lactamase reporter gene assay provides high sensitivity due to the absence of endogenous beta-lactamase activity in mammalian cells. The cell-permeable fluorogenic substrate allows single-cell cloning of cells expressing functional BK1 receptors. Pharmacological characterization reveals comparable sensitivity and potency of known BK1 receptor agonists and antagonists between the beta-lactamase assay, competition-binding assay, and other direct measurements of second messengers. The beta-lactamase assay has been optimized for cell density, time of agonist stimulation, and DMSO sensitivity. This CHO-hBK1-beta-lactamase assay is well suited to automation and miniaturization required for high-throughput screening.     

Development of a novel assay for human tyrosyl DNA phosphodiesterase 2

Tyrosyl DNA phosphodiesterase 2 (TDP2), a newly discovered enzyme that cleaves 5′-phosphotyrosyl bonds, is a potential target for chemotherapy. TDP2 possesses both 3′- and 5′-tyrosyl-DNA phosphodiesterase activity, which is generally measured in a gel-based assay using 3′- and 5′-phosphotyrosyl linkage at the 3′ and 5′ ends of an oligonucleotide. To understand the enzymatic mechanism of this novel enzyme, the gel-based assay is useful, but this technique is cumbersome for TDP2 inhibitor screening. For this reason, we have designed a novel assay using p-nitrophenyl-thymidine-5′-phosphate (T5PNP) as a substrate. This assay can be used in continuous colorimetric assays in a 96-well format. We compared the salt and pH effect on product formation with the colorimetric and gel-based assays and showed that they behave similarly. Steady-state kinetic studies showed that the 5′ activity of TDP2 is 1000-fold more efficient than T5PNP. Tyrosyl DNA phosphodiesterase 1 (TDP1) and human AP-endonuclease 1 (APE1) could not hydrolyze T5PNP. Sodium orthovanadate, a known inhibitor of TDP2, inhibits product formation from T5PNP by TDP2 (IC₅₀ = 40 mM). Our results suggest that this novel assay system with this new TDP2 substrate can be used for inhibitor screening in a high-throughput manner.     

A novel binding assay identifies high affinity ligands to the rosiglitazone binding site of mitoNEET

A novel outer mitochondrial membrane protein containing [2Fe-2S] clusters, mitoNEET was first identified through its binding to the anti-diabetic drug pioglitazone. Pioglitazone belongs to a family of drugs that are peroxisome proliferator-activated receptor (PPAR) gamma agonists, collectively known as glitazones. With the lack of pharmacological tools available to fully elucidate mitoNEET's function, we developed a binding assay to probe the glitazone binding site with the aim of developing selective and high affinity compounds. We used multiple thiazolidine-2,4-dione (TZD), 2-thioxothiazolidin-4-one (TTD), and 2-iminothiazolidin-4-one (ITD) compounds to establish several trends to enhance ligand development for the purpose of elucidating mitoNEET function.     

PBAN receptor: Employment of anti-receptor antibodies for its characterization and for development of a microplate binding assay

This study describes generation of an anti-PBAN receptor (PBAN-R) antiserum and its employment for the characterization of the PK/PBAN-R(s). The antiserum recognized, in a specific and dose-dependent manner, the presence of PBAN-R in pheromone gland membrane preparations of three female moths: Heliothis peltigera, Helicoverpa armigera and Spodoptera littoralis. It also reacted specifically with the S. littoralis larval receptor in vivo, most likely by competing with the ligand on the binding site and consequently inhibiting cuticular melanization. Despite its ability to react with the receptor of H. peltigera in dot blot experiments, the antiserum did not react with the receptor in vivo and failed to inhibit sex pheromone biosynthesis. The antiserum was also used to develop two microplate binding assays. The Ab described in this study is the first raised against an insect neuropeptide (Np) receptor to be used in vivo, and its employment for characterization of the PK/PBAN-R(s) may thus provide important information on the mode of action of this Np family. The present study adds important information on the difference between the receptors in the two moth species, hints at the possible existence of receptor subtypes, and provides a platform for the development of a high-throughput assay (HTA) for screening of PK/PBAN agonists and antagonists.     

A single-vector EYFP reporter gene assay for G protein-coupled receptors

We here present an improved and simplified assay to study signal transduction of the G_s class of G protein-coupled receptors (GPCRs). The assay is based on a single plasmid combining the genes for any G_s protein-coupled GPCR and the cAMP response element-related expression of enhanced yellow fluorescent protein. On transfection, stable human embryonic kidney 293 (HEK293) cell lines presented high assay sensitivity and an unprecedented signal-to-noise ratio of up to 300, even in the absence of trichostatin A. The robustness of the assay was demonstrated through the cloning of reporter gene cell lines with melanocortin 4 receptor (MC4R), the human type I pituitary adenylate cyclase-activating polypeptide receptor (hPAC1), and the two vasoactive intestinal peptide receptors (VPAC1 and VPAC2).     

Development of a scintillation proximity binding assay for high-throughput screening of hematopoietic prostaglandin D2 synthase

Prostaglandin D₂ synthase (PGDS) catalyzes the isomerization of prostaglandin H₂ (PGH₂) to prostaglandin D₂ (PGD₂). PGD₂ produced by hematopoietic prostaglandin D₂ synthase (H-PGDS) in mast cells and Th2 cells is proposed to be a mediator of allergic and inflammatory responses. Consequently, inhibitors of H-PGDS represent potential therapeutic agents for the treatment of inflammatory diseases such as asthma. Due to the instability of the PGDS substrate PGH₂, an in-vitro enzymatic assay is not feasible for large-scale screening of H-PGDS inhibitors. Herein, we report the development of a competition binding assay amenable to high-throughput screening (HTS) in a scintillation proximity assay (SPA) format. This assay was used to screen an in-house compound library of approximately 280,000 compounds for novel H-PGDS inhibitors. The hit rate of the H-PGDS primary screen was found to be 4%. This high hit rate suggests that the active site of H-PGDS can accommodate a large diversity of chemical scaffolds. For hit prioritization, these initial hits were rescreened at a lower concentration in SPA and tested in the LAD2 cell assay. 116 compounds were active in both assays with IC₅₀s ranging from 6 to 807 nM in SPA and 82 nM to 10 μM in the LAD2 cell assay.     

A direct fluorometric assay for tissue transglutaminase

Herein we report the design of a direct and continuous fluorometric assay for determining tissue transglutaminase (TGase) activity. The progress of the TGase-catalyzed reaction of 4-(N-carbobenzoxy-l-phenylalanylamino)-butyric acid coumarin-7-yl ester was monitored as an increase of fluorescence (lambda(exc) 330 nm, lambda(em) 460 nm) due to the release of 7-hydroxycoumarin. Using this assay, we determined the K(m) of two acceptor substrates, N-acetyl-L-lysine methyl ester and aminoacetonitrile. We also determined the K(m) of 4-(N-carbobenzoxy-L-phenylalanylamino)-butyric acid coumarin-7-yl ester for its TGase-mediated hydrolysis and for its enzymatic reaction with the acyl acceptor substrates N-acetyl-L-lysine methyl ester and aminoacetonitrile. We ascertained that the fluorescent substrate was selective toward tissue TGase by testing it with different enzymes, namely microbial transglutaminase (mTGase), Factor XIIIa, papain, and gamma-glutamyl transpeptidase. 4-(N-carbobenzoxyglycinylamino)-butyric acid coumarin-7-yl ester, lacking the benzyl side chain, was also found to be an efficient fluorogenic substrate of tissue TGase. Finally, we have shown that this method is applicable to 96-well microtiter plate format.