Matthew A. Sills

[3H]CGP 39653: a new N-methyl-D-aspartate antagonist radioligand with low nanomolar affinity in rat brain

CGP 39653 (D,L-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid) was initially discovered to inhibit the binding of [3H]L-glutamate and [3H]3-[+/-)2-carboxypiperazin-4-yl)-propyl-1- phosphonic acid [( 3H]CPP) with Ki values of 230 and 5 nM, respectively. The radiolabeled compound [3H]CGP 39653 binds to rat frontal cortical membranes in a saturable and reversible manner. Analysis of saturation experiments revealed that the ligand labels one binding site with a Kd value of 6 nM. Competition experiments indicated that the order of potency of a number of competitive excitatory amino acid agonist and antagonist compounds was similar to that found previously for other N-methyl-D-aspartate (NMDA) receptor ligands. In contrast to these competitive inhibitors, which produced steep inhibition curves, glycine inhibited binding in a complex manner. When the functional activity of the unlabeled compound was explored, CGP 39653 blocked NMDA-evoked depolarizations in the rat cortical wedge in vitro and inhibited L-glutamate stimulated [3H]N(1-[2-thienyl]cyclohexyl)3,4-piperidine [( 3H]TCP) binding in cortical membranes. These results suggest that [3H]CGP 39653 selectively binds to the NMDA receptor as an antagonist with high affinity and is currently the ligand of choice for labeling the NMDA receptor.

Characterization of the binding of [3H]-CGS 19755: a novel N-methyl-d-aspartate antagonist with nanomolar affinity in rat brain

1 CGS 19755 (cis‐4‐phosphonomethyl‐2‐piperidine carboxylic acid), a rigid analogue of 2‐amino‐5‐phosphonopentanoic acid (AP5), is one of the most potent competitive N‐methyl‐d‐aspartate (NMDA) antagonists described. Using Triton‐treated crude synaptic membranes from rat brain, binding studies indicated that [3H]‐CGS 19755 bound with high affinity and selectivity to the NMDA‐type excitatory amino acid receptor. 2 [3H]‐CGS 19755 binding was saturable, reversible, heat‐labile, pH‐dependent and linear with protein concentration. Specific binding represented 80–85% of the total amount bound. 3 Using a centrifugation assay, saturation experiments revealed two distinct binding components with Kd values of 9 and 200 nM, and corresponding Bmax values of 0.55 and l.00 pmol mg−1 protein. In contrast, a single binding component with a Kd value of 24 nM and an apparent value of 0.74 pmol mg−1 protein was observed with a filtration assay. 4 Competition experiments in which both assay techniques were used, showed that [3H]‐CGS 19755 selectively labels the NMDA receptor. The most active inhibitors of [3H]‐CGS 19755 binding were l‐glutamate and CGS 19755 (IC50 values = 100 nm). 5 In the centrifugation assay, a number of excitatory amino acids were found to generate shallow inhibition curves, and computer analysis indicated the presence of two binding components. The quisqualate receptor ligand AMPA (D,L‐α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate), kainic acid and the non‐competitive NMDA antagonists, such as phencyclidine, tiletamine and MK‐801, were without activity. 6 The high affinity binding obtained with [3H]‐CGS 19755 by use of filtration techniques thus permits the more rapid evaluation of compounds as potential NMDA antagonists and agonists. Therefore, this rigid analogue of AP5 is a more suitable radioligand for NMDA receptors than [3H]‐CPP (3‐(±)−(2‐carboxypiperazin‐4‐yl)propyl‐l‐phosphonic acid), the corresponding analogue of 2‐amino‐7‐phosphonoheptanoic acid (AP7).

A Comparison of ALPHAScreen, TR-FRET, and TRF as Assay Methods for FXR Nuclear Receptors

New developments in detection technologies are providing a variety of biomolecular screening strategies from which to choose. Consequently, we performed a detailed analysis of both separation-based and non-separation-based formats for screening nuclear receptor ligands. In this study, time-resolved fluorescence resonance energy transfer (TR-FRET), ALPHAScreen, and time-resolved fluorescence (TRF) assays were optimized and compared with respect to sensitivity, reproducibility, and miniaturization capability. The results showed that the ALPHAScreen system had the best sensitivity and dynamic range. The TRF assay was more time consuming because of the number of wash steps necessary. The TR-FRET assay had less interwell variation, most likely because of ratiometric measurement. Both the ALPHAScreen and the TR-FRET assays were miniaturized to 8-μl volumes. Of the photomultiplier tube-based readers, the ALPHAScreen reader (ALPHAQuest) presented the advantage of faster reading times through simultaneous reading with four photomultiplier tubes.

Comparison of Assay Technologies for a Tyrosine Kinase Assay Generates Different Results in High Throughput Screening

In todays high-throughput screening (HTS) environment, an increasing number of assay detection technologies are routinely utilized in lead finding programs. Because of the relatively broad applicability of several of these technologies, one is often faced with a choice of which technology to utilize for a specific assay. The aim of this study was to address the question of whether the same compounds would be identified from screening a set of samples in three different versions of an HTS assay. Here, three different versions of a tyrosine kinase assay were established using scintillation proximity assay (SPA), homogeneous time-resolved fluorescence resonance energy transfer (HTR-FRET), and fluorescence polarization (FP) technologies. In this study, 30,000 compounds were evaluated in each version of the kinase assay in primary screening, deconvolution, and dose-response experiments. From this effort, there was only a small degree of overlap of active compounds identified subsequent to the deconvolution experiment. When all active compounds were then profiled in all three assays, 100 and 101 active compounds were identified in the HTR-FRET and FP assays, respectively. In contrast, 40 compounds were identified in the SPA version of the kinase assay, whereas all of these compounds were detected in the HTR-FRET assay only 35 were active in the FP assay. Although there was good correlation between the IC50 values obtained in the HTR-FRET and FP assays, poor correlations were obtained with the IC50 values obtained in the SPA assay. These findings suggest that significant differences can be observed from HTS depending on the assay technology that is utilized, particularly in assays with high hit rates.

The novel anticonvulsant MK-801 interacts with central phencyclidine recognition sites in rat brain.

The novel anticonvulsant MK-801 ([(+)-5methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten5,10-imine maleate]; Clineschmidt et al., 1982) has been described as a non-competitive antagonist of the excitatory amino acid neurotransmitter, Nmethyl-D-aspartate (NMDA) on the basis of binding and electrophysiological experiments (Wong et al., 1986). Consistent with this finding was the observation (Murphy et al., in press) that at concentrations up to 10 /xM, MK-801 was without significant effect on the binding of the radiolabeled selective NMDA antagonist, CPP [(3-(2carboxypiperazin-4-yl)-propyl-1 -phosphonic acid]. Binding of the phencylidine (PCP) analog, [3H]TCP (1-(2-thienyl)-cyclohexylpiperidine) to EDTA-washed rat brain membranes can be markedly stimulated (+265%) by 10 /xM glutamate (Loo et al. 1986). Furthermore, Anis et al. (1983) have shown that dissociative anesthetics such as phencyclidine and ketamine can inhibit NMDA-induced neuronal responses. Together, these findings provide evidence for the possible existence of an N M D A / P C P receptor complex in rat brain. In the present study, the possibility that MK801 might be producing its NMDA antagonist effects via a direct interaction with PCP sites was investigated. [3H]TCP binding was measured as described previously (Loo et al., 1986) using

Interaction of the D1 Receptor Antagonist Sch 23390 with the Central 5-HT System: Radioligang Binding Studies, Measurements of Biochemical Parameters and Effects on L-5-HTP Syndrome

The interaction of SCH 23390 with dopamine (DA) and serotonin (5-HT) systems has been examined in vivo and in vitro. Like selective 5-HT2 blockers, SCH 23390 inhibited in vivo [3H]spiperone binding in the rat frontal cortex (ID50: 1.5 mg/kg) without interacting at D2 sites. SCH 23390 was equipotent to cinanserin and methysergide. In vitro, SCH 23390 inhibited [3H]ketanserin binding to 5-HT2 sites (IC50 = 30 nM). Biochemical parameters linked to DA and 5-HT were not changed excepted in striatum where SCH 23390 increased HVA and DOPAC. In the L-5-HTP syndrome model, SCH 23390 clearly showed antagonism of 5-HT2 receptors. SCH 23390 had weak affinity for 5-HT1B (IC50 = 0.5 microM), 5-HT1A (IC50 = 2.6 microM) and alpha 1-adrenergic receptors (IC50 = 4.4 microM).

High affinity interkakin-6 binding sites in bovine hypothalamus

The cytokine interleukin-6 (IL-6) has been proposed to interact with the hypothalamic-pituitary axis. The present data provide the first reported evidence of high affinity binding sites for [125I]IL-6 in brain tissue, specifically bovine hypothalamic membranes. Binding was saturable and represented a single site, with a Kd of 316 +/- 49 pM and receptor density of 15.8 +/- 3.2 fmol/mg protein. Other cytokines tested did not interact with this site, but a neutralizing monoclonal anti-IL-6 antibody blocked specific binding. These findings support the proposed involvement of IL-6 in communication between neural and immune systems.

Further Comparison of Primary Hit Identification by Different Assay Technologies and Effects of Assay Measurement Variability

High-throughput screening (HTS) has grown rapidly in the past decade, with many advances in new assay formats, detection technologies, and laboratory automation. Recently, several studies have shown that the choice of assay technology used for the screening process is particularly important and can yield quite different primary screening outcomes. However, because the screening assays in these previous studies were performed in a single-point determination, it is not clear to what extent the difference observed in the screening results between different assay technologies is attributable to inherent assay variability and day-to-day measurement variation. To address this question, a nuclear receptor coactivator recruitment assay was carried out in 2 different assay formats, namely, AlphaScreen™ and time-resolved fluorescence resonance energy transfer, which probed the same biochemical binding events but with different detection technologies. For each assay format, 4 independent screening runs in a typical HTS setting were completed to evaluate the run-to-run screening variability. These multiple tests with 2 assay formats allow an unambiguous comparison between the discrepancies of different assay formats and the effects of the variability of assay and screening measurements on the screening outcomes. The results provide further support that the choice of assay format or technology is a critical factor in HTS assay development.

Probing the Primary Screening Efficiency by Multiple Replicate Testing: A Quantitative Analysis of Hit Confirmation and False Screening Results of a Biochemical Assay

Despite a large body of references on assay development, assay optimization, strategies, and methodologies for high-throughput screening (HTS), there have been few reports on investigations of the efficiency of primary screening in a systematic and quantitative manner for a typical HTS process. Recently, the authors investigated the primary hit comparison and the effect of measurement variability by screening a library of approximately 25,000 random compounds in multiple replicate tests in a nuclear receptor recruitment assay with 2 different assay detection technologies. In this report, we utilized these sets of multiple replicate screening data from a different perspective and conducted a systematic data analysis in order to gain some insights into the hit-finding efficiency of a typical primary screening process. Specifically, hit confirmation, false-positive (declaration) rates, and false-negative rates at different hit cutoff limits were explored and calculated from the 2 different assay formats. Results and analyses provided some quantitative estimation regarding the reliability and efficiency of the primary screening process. For the 2 assay formats tested in this report, the confirmation rate (activity repeated at or above a certain hit limit) was found to be 65% or above. It was also suggested that, at least in this case, applying some hit-selection strategies, it is possible to decrease the number of false-negative or false-positive hits without significantly increasing the efforts in primary screening.

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

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