Development of a scintillation proximity assay for beta-ketoacyl-acyl carrier protein synthase III

Assays of beta-ketoacyl-acyl carrier protein synthases III (KASIII; FabH), a key enzyme initiating bacterial type II fatty acid biosynthesis, usually involve incubation of radiolabeled acetyl-coenzyme A and malonyl-acyl carrier protein (MACP). The radiolabeled acetoacetyl-ACP product is precipitated and separated from the substrate before quantitation. We have developed a scintillation proximity assay (SPA) where use of biotinylated MACP (BMACP) allows the generation of a biotinylated acetoacetyl-ACP. This product, when captured by the streptavidin-coated scintillant-impregnated microspheres, generates an SPA signal. A BMACP K(m) of 7.1 microM was determined using this SPA with the Streptomyces glaucescens FabH. A similar MACP K(m) (6 microM) was determined in a precipitation assay, demonstrating that BMACP is an effective substrate for FabH. IC(50) values of 15.2 microM (SPA) and 24.8 microM were obtained with iodoacetamide and the S. glaucescens FabH. Comparable IC(50) values of 160 microM (SPA) and 125 microM were also obtained with the antibiotic thiolactomycin and the Escherichia coli FabH. These observations demonstrate that FabH inhibitors can be readily detected using a SPA with BMACP and that the effectiveness of inhibitors in the SPA is comparable to that obtained using MACP and a standard TCA precipitation assay. A FabH SPA adaptable to high-throughput screening should facilitate the discovery of potential novel antibiotics.     

Development of a scintillation proximity assay for histone deacetylase using a biotinylated peptide derived from histone-H4

Measurement of histone deacetylase activity is usually accomplished by incubation of the enzyme(s) with acetate-radiolabeled histones or synthetic peptides based on histone sequences, followed by extraction and quantification of released radiolabeled acetic acid. Consequently, this assay is both time consuming and extremely limiting when large numbers of samples are involved. We have now developed a simple, two-step histone deacetylase assay that is based on the scintillation proximity assay (SPA) principle. A biotinylated [3H]acetyl histone H4 peptide substrate was synthesized and shown to generate a radioactive signal upon binding to streptavidin-coated SPA beads. Incubation of biotinylated [3H]acetyl peptide with HeLa nuclear extract (source of histone deacetylase) resulted in a time- and protein-dependent decrease in the SPA signal, providing a measure of enzyme activity. The histone deacetylase-mediated decrease in SPA counts was accompanied by a proportional appearance in free 3H-labeled acetate in the assay mixture. Histone deacetylase activity measured by SPA was concordant with that determined via the traditional ethyl acetate extraction procedure. Furthermore, a broad range of histone deacetylase inhibitors was demonstrated to have comparable effects on the catalytic activity of the HeLa nuclei enzyme using both assays. The histone deacetylase SPA system described here should be readily applicable for automated high-throughput screening and therefore facilitate the discovery of new inhibitors of histone deacetylases.     

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.     

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.     

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.     

High-throughput scintillation proximity assay for transglutaminase activity measurement

Members of the transglutaminase enzyme family are involved in a broad range of biological phenomena, including haemostasis, apoptosis, semen coagulation, skin formation, and wound healing. A new and rapid method for measurement of transglutaminase activity is described in this article. The enzyme links tritium-labeled putrescine to biotinylated oligoglutamine, and the tritiated peptide is bound to a streptavidin-coated microtiter plate permanently covered by a thin layer of scintillant. Only the radioisotope incorporated into the peptide substrate is close enough to the scintillant molecules for photons to be produced. The signal generation depends on the transglutaminase activity, and it can be detected by appropriate light-measuring instrumentation without separation steps. The assay is sensitive, specific, linear at concentrations of tissue transglutaminase between 0.05 and 1.6m U/ml, and suitable for high-throughput measurements.     

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.     

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.     

Development of a scintillation proximity assay for analysis of Na+/Cl- -dependent neurotransmitter transporter activity

Human placental choriocarcinoma (JAR) cells endogenously expressing glycine transporter type 1a (GlyT1a) have been cultured in 96-well scintillating microplates to develop a homogenous screening assay for the detection of GlyT1 antagonists. In these microplates uptake of [14C]glycine was time dependent and saturable with a Michaelis-Menten constant (Km) of 27+/-3 microM. The GlyT1 transport inhibitors sarcosine, ALX-5407, and Org-24598 were tested and shown to block [14C]glycine uptake with expected IC50 values of 37.5+/-4.6 microM, 2.8+/-0.6 nM, and 6.9+/-0.9 nM, respectively. The [14C]glycine uptake process was sensitive to membrane Na+ gradient as blockade of membrane Na+/K+-ATPase by ouabain or Na+ exchanger by benzamil-disrupted glycine accumulation in JAR cells. Glycine influx was not affected by concentration of dimethyl sulfoxide up to 2%. The versatility of this technological approach was further confirmed by the characterization of a saturable [14C]taurine uptake in JAR cells. Taurine transport was of high affinity with a Km of 10.2+/-1.7 microM and fully inhibited by ALX-5407 (IC50=522 +/-83 nM). The developed assay is homogenous, rapid, versatile and amenable to automation for the discovery of new neurotransmitter transporter 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 high-throughput scintillation proximity assay for the identification of C-domain translational initiation factor 2 inhibitors

Translational initiation factor 2 (IF2) is the largest of the 3 factors required for translation initiation in prokaryotes and has been shown to be essential in Escherichia coli. It stimulates the binding of fMet-tRNA(f)(Met) to the 30S ribosomal subunit in the presence of GTP. The selectivity is achieved through specific recognition of the tRNA(f)(Met) blocked alpha-amino group. IF2 is composed of 3 structural domains: N-domain, whose function is not known; G-domain, which contains the GTP/GDP binding site and the GTPase catalytic center; and C-domain, which recognizes and binds fMet-tRNA(f)(Met). Its activity is strictly bacteria specific and highly conserved among prokaryotes. So far, antibiotics targeting IF2 function are not known, and this makes it an ideal target for new drugs with mechanisms of resistance not yet developed. A few assays have been developed in the past, which allow the detection of IF2 activity either directly or indirectly. In both instances, the assays are based on radioactive detection and do not allow for high throughput because of the need for separation or solvent extraction steps. The authors describe a novel biochemical assay for IF2 that exploits the molecular recognition of fMet-tRNA(f)(Met) by the C-domain. The assay is based on the incubation of biotinyl-IF2 with fMet-tRNA(f)(Met) and the subsequent capture of the radiolabeled complex by streptavidin-coated beads, exploiting the scintillation proximity assay (SPA) technology. The assay has been designed in an automatable, homogeneous, miniaturized fashion suitable for high-throughput screening and is rapid, sensitive, and robust to dimethyl sulfoxide (DMSO) up to 10% v/v. The assay, used to screen a limited chemical collection of about 5000 compounds and a subset of compounds originated by a 2-D substructural search, has shown to be able to detect potential IF2 inhibitors.     

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.     

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.     

Development of a plate-based scintillation proximity assay for the mycobacterial AftB enzyme involved in cell wall arabinan biosynthesis

A number of mycobacterial arabinosyltransferases, such as the Emb proteins, AftA, AftB, AftC, and AftD have been characterized and implicated to be involved in the cell wall arabinan assembly. These arabinosyltransferases are essential for the viability of the organism and are logically valid targets for developing new anti-tuberculosis agents. For instance, Ethambutol, a first line anti-tuberculosis drug, targets the Emb proteins involved in the formation of the arabinan of cell wall arabinogalactan. Among these arabinosyltransferases, the terminal β-(1→2) arabinosyltransferase activity has been associated with AftB. The predicted topology of AftB in Mycobacterium tuberculosis has 10 N terminal transmembrane domains and a C terminal hydrophilic domain similar to the Emb proteins. It has a conserved GT-C motif and is difficult to express. In a cell free assay, synthetic disaccharide, α-d-Araf-(1→5)-α-d-Araf-octyl, has been used as a substrate to explore the function of AftB. In our work, the disaccharide was synthesized in its pentenylated and biotinylated form, and the enzymatic product formed was identified as the β-(1→2) arabinofuranose adduct. When synthetic tri- and tetra-saccharides were used as substrates, a mixture of products containing both β-(1→2) and α-(1→5) linkages were formed. Therefore, the biotinylated disaccharide was selected to develop a scintillation proximity assay.     

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.     

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.     

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 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.