Scintillation proximity assay as a high-throughput method to identify slowly dissociating nonpeptide ligand binding to the GnRH receptor

Many nonpeptide antagonists of the gonadotropin-releasing hormone (GnRH) receptor, as well as other drug targets, possess a broad range of dissociation kinetic rate constants. Current methods to accurately define kinetic rate parameters such as K(on) and K(off) are time and labor intensive, prompting the development of a screening assay to identify slowly dissociating compounds for follow-up rate constant determination. The authors measured inhibition binding constants (K(i)) for GnRH receptor antagonists after 30 min and 10 h of incubation and observed several compounds with markedly decreased K(i) values over time (Ki(30 min)/Ki(10 h) > 6). They used scintillation proximity assay technology to perform these binding experiments because this homogeneous assay does not have a fixed termination end point as does filtration binding, permitting successive readings to be taken from the same assay plate over an extended period of time. They also used a quantitative method of kinetic rate analysis to confirm that a large disparity between a compound's K(i) value at 30 min and 10 h could identify compounds that dissociate slowly. Thus, the K(i) ratio can be used to screen for and select compounds to test using more quantitative, albeit lower throughput methods to accurately define kinetic rate constants.     

Optimisation of Conditions for Solubilisation of the Bovine Dopamine D2 Receptor

Solubilisation of the dopamine D2 receptor from a membrane preparation of bovine corpus striatum using cholate and NaCl was independently optimised with regard to cholate (0.2%, wt/vol), NaCl (1.5 M), and membrane protein (4 mg/ml) concentrations. A maximum solubilisation yield of 58% was obtained and receptors were measured using a [3H]spiperone binding assay incorporating a polyethylene glycol precipitation step. Solubilisation was confirmed by ultracentrifugation studies, passage of the receptor through fine-pore filters, increased thermolability, and by retention of the prelabelled receptor on gel filtration. The soluble receptor showed saturability and reversibility of binding. Displacement of [3H]spiperone from the soluble receptor by competing compounds correlated closely with displacement from the membrane-bound receptors. [3H]Spiperone binding was found to be pH-dependent, with maximum binding occurring at pH 7.8. A comparison of solubilisation was made with six other agents both with and without added NaCl and it was concluded that the cholate/NaCl solubilisation system provides an efficient, inexpensive, and reliable method for the preparation of functional bovine dopamine D2 receptors.     

State-dependent compound inhibition of Nav1.2 sodium channels using the FLIPR Vm dye: on-target and off-target effects of diverse pharmacological agents

Voltage-gated sodium channels (NaChs) are relevant targets for pain, epilepsy, and a variety of neurological and cardiac disorders. Traditionally, it has been difficult to develop structure-activity relationships for NaCh inhibitors due to rapid channel kinetics and state-dependent compound interactions. Membrane potential (Vm) dyes in conjunction with a high-throughput fluorescence imaging plate reader (FLIPR) offer a satisfactory 1st-tier solution. Thus, the authors have developed a FLIPR Vm assay of rat Nav1.2 NaCh. Channels were opened by addition of veratridine, and Vm dye responses were measured. The IC50 values from various structural classes of compounds were compared to the resting state binding constant (Kr)and inactivated state binding constant (Ki)obtained using patch-clamp electrophysiology (EP). The FLIPR values correlated with Ki but not Kr. FLIPRIC50 values fell within 0.1-to 1.5-fold of EP Ki values, indicating that the assay generally reports use-dependent inhibition rather than resting state block. The Library of Pharmacologically Active Compounds (LOPAC, Sigma) was screened. Confirmed hits arose from diverse classes such as dopamine receptor antagonists, serotonin transport inhibitors, and kinase inhibitors. These data suggest that NaCh inhibition is inherent in a diverse set of biologically active molecules and may warrant counterscreening NaChs to avoid unwanted secondary pharmacology.     

Characterization of Dopamine and β-Adrenergic Receptors Linked to Cyclic AMP Formation in Intact Cells of the Clone D384 Derived from a Human Astrocytoma

3,4-Dihydroxyphenylethylamine (dopamine) and beta-adrenergic receptor agonists and antagonists were assessed for their effects on cyclic AMP accumulation in human astrocytoma derived clone D384 cells. Dopamine, SKF 38393, and 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene increased cyclic AMP content with Ka values of 2.0, 0.2, and 1.6 microM. The D1-selective antagonists SCH 23390 (Ki, 1.2 nM) and SKF 83566 (Ki, 0.8 nM) were over 5,000-fold more potent than the D2-selective antagonist domperidone (Ki, 6.7 microM) at inhibiting dopamine stimulation of cyclic AMP formation. SCH 23388 (Ki, 560 nM; the S-enantiomer of SCH 23390) was 400-fold less potent than SCH 23390. Isoprenaline, adrenaline, salbutamol, and noradrenaline increased cyclic AMP content with Ka values of 0.13, 0.12, 0.22, and 7.60 microM. The beta 2-selective antagonist ICI 118,551 (Ki,0.8 nM) was almost 8,000-fold more potent than the beta 1-selective antagonist practolol (Ki, 5.9 microM) at inhibiting isoprenaline stimulated cyclic AMP accumulation. These results demonstrate that D384 cells express D1-dopamine and beta 2-adrenergic receptors linked to adenylate cyclase. Furthermore, the dopamine receptor expressed by D384 cells exhibits a pharmacological profile typical of a mammalian striatal D1-receptor and therefore the use of this clone represents another approach to studying central D1-receptors.     

The Metabolism of Dopamine by Both Forms of Monoamine Oxidase in the Rat Brain and Its Inhibition by Cimoxatone

In the rat brain, dopamine is metabolised by both A and B forms of monoamine oxidase (MAO), although the A form of the enzyme is the major component. The Km of MAO-A toward dopamine (120 microM) is lower than the Km of MAO-B toward this substrate (340 microM). The activity of MAO-A was lower in old rats than in young rats, and the same degree of decrease was found for 5-hydroxytryptamine as for dopamine as substrates for this enzyme form. The activity of MAO-B was higher in the old rats, the degree of increase being the same for dopamine as for beta-phenethylamine as substrates for this enzyme form. The Ki values of the inhibition of MAO-A by cimoxatone and MD770222 (the principal plasma metabolite of cimoxatone) were independent of the substrate used to assay for activity, but were lower than the Ki values for the inhibition of MAO-B by these compounds.     

Benzamides derived from 1,2-diaminocyclopropane as novel ligands for human D2 and D3 dopamine receptors

Benzamides (3a-f) derived from 4-amino-5-chloro-2-methoxybenzoic acid and either cis or trans 1,2-diaminocyclopropane were synthesised and were evaluated in binding assays employing, bovine striatal D2 receptors, recombinant human hD2 and hD3 receptors expressed in CHO cells and rat, cortical 5-HT3 and striatal 5-HT4 receptors. The cis and trans isomers of the derivatives were isolated and characterised. The results demonstrated the superiority of the cis conformers over the trans conformers in dopamine receptor binding assays (Ki hD2 = 13.4 and 6.9 nM and Ki hD3 = 17.7 and 4.5 nM for the cis-3b and cis-3f compounds, respectively; Ki hD2 = 816 and >l000 nM and Ki hD3 = 469 and >1000 nM for the corresponding trans-3b and trans-3f compounds respectively). The cis compounds are folded: the benzamide group and the basic nitrogen atom were in a syn relationship. Compound 3f can be superimposed with a conformation of the tropane derivative, BRL 25594, having the benzyl group in an axial position to give a suitable fit, indicating that both compounds may have a common binding site in the dopamine receptor.     

Adaptation of the Nelson-Somogyi reducing-sugar assay to a microassay using microtiter plates

The Nelson-Somogyi assay for reducing sugars was adapted to microtiter plates. The primary advantages of this modified assay are (i) smaller sample and reagent volumes, (ii) elimination of boiling and filtration steps, (iii) automated measurement with a dual-wavelength scanning TLC densitometer, (iv) increased range and reproducibility, and (v) automated colorimetric readings by reflectance rather than absorbance.     

Simultaneous high-performance liquid chromatographic determination of urinary metabolites of benzene, nitrobenzene, toluene, xylene and styrene

A high-performance liquid chromatographic method is described for the simultaneous determination of six urinary metabolites of several aromatic chemicals: phenol (from benzene), hippuric acid (from toluene), 3-methylhippuric acid (from xylene), mandelic and phenylglyoxylic acid (from styrene) and 4-nitrophenol (from nitrobenzene). Reversed-phase liquid chromatography was performed in an isocratic mode at 1 ml/min on a 5-μm C₁₈ column using two mobile phases: (A) acetonitrile—1% phosphoric acid (10:90); (B) acetonitrile—1% phosphoric acid (30:70). Phase A separates the six metabolites well, but phase B allows to a more rapid and reproducible simultaneous determination of phenolic compounds than phase A. For these compounds a prior enzymic hydrolysis step using Helix pomatia juice is performed to hydrolyse their sulphate and glucuronate conjugates. The reproducibility and the specificity are both excellent. Furthermore, the method is rapid, economical and easily automated. The proposed method appears very suitable for the routine monitoring of workers exposed to these chemicals on the basis of the biological threshold limit values.     

Towards automated production and drug sensitivity testing using scaffold-free spherical tumor microtissues

Although the relevance of three-dimensional (3-D) culture has been recognized for years and exploited at an academic level, its translation to industrial applications has been slow. The development of reliable high-throughput technologies is clearly a prerequisite for the industrial implementation of 3-D models. In this study the robustness of spherical microtissue production and drug testing in a 96-well hanging-drop multiwell plate format was assessed on a standard 96-well channel robotic platform. Microtissue models derived from six different cell lines were produced and characterized according to their growth profile and morphology displaying high-density tissue-like reformation and growth over at least 15 days. The colon cancer cell line HCT116 was chosen as a model to assess microtissue-based assay reproducibility. Within three individual production batches the size variations of the produced microtissues were below 5%. Reliability of the microtissue-based assay was tested using two reference compounds, staurosporine and chlorambucil. In four independent drug testings the calculated IC(50) values were benchmarked against 2-D multiwell testings displaying similar consistency. The technology presented here for the automated production of a variety of microtissues for efficacy testing in a standard 96-well format will aid the implementation of more organotypic models at an early time point in the drug discovery process.     

A selective dopamine D4 receptor antagonist, NRA0160: a preclinical neuropharmacological profile

NRA0160, 5 - [2- ( 4- ( 3 - fluorobenzylidene) piperidin-1-yl) ethyl] - 4 -(4-fluorophenyl) thiazole-2-carboxamide, has a high affinity for human cloned dopamine D4.2, D4.4 and D4.7 receptors, with Ki values of 0.5, 0.9 and 2.7 nM, respectively. NRA0160 is over 20,000fold more potent at the dopamine D4.2 receptor compared with the human cloned dopamine D2L receptor. NRA0160 has negligible affinity for the human cloned dopamine D3 receptor (Ki=39 nM), rat serotonin (5-HT)2A receptors (Ki=180 nM) and rat alpha1 adrenoceptor (Ki=237 nM). NRA0160 and clozapine antagonized locomotor hyperactivity induced by methamphetamine (MAP) in mice. NRA0160 and clozapine antagonized MAP-induced stereotyped behavior in mice, although their effects did not exceed 50% inhibition, even at the highest dose given. NRA0160 and clozapine significantly induced catalepsy in rats, although their effects did not exceed 50% induction even at the highest dose given. NRA0160 and clozapine significantly reversed the disruption of prepulse inhibition (PPI) in rats produced by apomorphine. NRA0160 and clozapine significantly shortened the phencyclidine (PCP)-induced prolonged swimming latency in rats in a water maze task. These findings suggest that NRA0160 may have unique antipsychotic activities without the liability of motor side effects typical of classical antipsychotics.     

Development of a homogeneous binding assay for histamine receptors

Histamine is critically involved in a wide range of physiological and pathological processes through its actions at different receptors. Thus, histamine receptors have been actively pursued as therapeutic targets in the pharmaceutical industry for the treatment of a variety of diseases. There are currently four histamine receptors that have been cloned, all of which are G protein-coupled receptors. Studies from both academia and pharmaceutical companies have identified compounds that modulate the function of specific histamine receptors. These efforts led to the successful introduction of histamine H(1) and H(2) receptor antagonists for the treatment of allergy and excess gastric acid secretion, respectively. Histamine H(3) receptor ligands are currently under investigation for the treatment of obesity and neurological disorders. The recently identified histamine H(4) receptor is preferentially expressed in the immune tissues, suggesting a potential role in normal immune functions and possibly in the pathogenesis of inflammatory diseases. Even with the long history of histamine research and the important applications of histamine receptor ligands, assays to measure the affinity of compounds binding to histamine receptors are still routinely analyzed using a filtration assay, a very low-throughput assay involving washing and filtration steps. This article describes a simple, robust, and homogeneous binding assay based on the scintillation proximity assay (SPA) technology that provides results equivalent to those obtained using the more complex filtration assay. The SPA format is easily adapted to high-throughput screening because it is amenable to automation. In summary, this technique allows high-throughput screening of compounds against multiple histamine receptors and, thus, facilitates drug discovery efforts.     

A microtechnique for quantification of detergent-solubilized muscarinic and nicotinic acetylcholine receptors using a semi-automated cell harvestor

A specific method for the rapid assay of muscarinic acetylcholine receptors (mAChR), either detergent-solubilized or in neuroblastoma cells, is described. This method is also applicable to the assay of nicotinic acetylcholine receptors. The procedure employs a cell harvestor and microtiter plates, and has the advantage of requiring small quantities of radioligand, microgram quantities of detergent-solubilized cholinergic receptor or less cells. The binding parameters such as the equilibrium dissociation constants (Kd) of mAChR and nicotinic acetylcholine receptor (nAChR) and inhibition constants (Ki) for antagonists determined by the present method are in excellent agreement with values determined by other methods. This assay procedure for mAChR and nAChR should facilitate the rapid screening of cholinergic agonists/antagonists and also the further purification and characterization of mAChR.     

D1 Dopamine Receptors of NS20Y Neuroblastoma Cells Are Functionally Similar to Rat Striatal D1 Receptors

Dopamine or agonists with D1 receptor potency stimulated cyclic AMP (cAMP) accumulation in whole cell preparations of NS20Y neuroblastoma cells. The accumulation of cAMP after D1 stimulation was rapid and linear for 3 min. Both dopamine and the novel D1 receptor agonist dihydrexidine stimulated cAMP accumulation two- to three-fold over baseline. The pseudo-Km for dopamine was approximately 2 microM, whereas for dihydrexidine it was approximately 30 nM. The effects of both drugs were blocked by either the D1-selective antagonist SCH23390 (Ki, 0.3 nM) or the nonselective antagonist (+)-butaclamol (Ki, 5 nM). Both (-)-butaclamol and the D2-selective antagonist (-)-sulpiride were ineffective (Ki greater than 3 microM). Forskolin (10 microM), prostaglandin E1 (1 microM), and adenosine (10 microM) also stimulated cAMP accumulation, but none were antagonized by SCH23390 (1 microM). Finally, muscarinic receptor stimulation (100 microM carbachol) inhibited both D1- and forskolin-stimulated increases in cAMP accumulation by 80%. The present results indicate that NS20Y neuroblastoma cells have D1 receptors that are coupled to adenylate cyclase, and that these receptors have a pharmacological profile similar to that of the D1 receptor(s) found in rat striatum.     

Tyrosine hydroxylase. Activation by protein phosphorylation and end product inhibition.

Protein phosphorylation activates tyrosine hydroxylase in crude extracts of rat striatum, hypothalamus, and adrenal glands by a reduction in the apparent Km value for 6-methyltetrahydropterin. Removal of endogenous catecholamines by gel filtration or cation exchange results in a similar activation. Phosphorylation causes only a small additional reduction in the apparent Km for reduced pterin in striatal extracts from which catecholamines have been removed. Kinetic analysis indicates that protein phosphorylation causes a significant increase in the Ki for end product dopamine, whereas gel filtration or cation exchange treatment has little effect on the dopamine Ki value. None of the above treatments appears to change the molecular weight of the enzyme. At physiological concentrations of dopamine, the increase in Ki by phosphorylation would effectively release tyrosine hydroxylase from end product feedback inhibition.     

The use of real-time cell analyzer technology in drug discovery: defining optimal cell culture conditions and assay reproducibility with different adherent cellular models

The use of impedance-based label-free technology applied to drug discovery is nowadays receiving more and more attention. Indeed, such a simple and noninvasive assay that interferes minimally with cell morphology and function allows one to perform kinetic measurements and to obtain information on proliferation, migration, cytotoxicity, and receptor-mediated signaling. The objective of the study was to further assess the usefulness of a real-time cell analyzer (RTCA) platform based on impedance in the context of quality control and data reproducibility. The data indicate that this technology is useful to determine the best coating and cellular density conditions for different adherent cellular models including hepatocytes, cardiomyocytes, fibroblasts, and hybrid neuroblastoma/neuronal cells. Based on 31 independent experiments, the reproducibility of cell index data generated from HepG2 cells exposed to DMSO and to Triton X-100 was satisfactory, with a coefficient of variation close to 10%. Cell index data were also well reproduced when cardiomyocytes and fibroblasts were exposed to 21 compounds three times (correlation >0.91, p < 0.0001). The data also show that a cell index decrease is not always associated with cytotoxicity effects and that there are some confounding factors that can affect the analysis. Finally, another drawback is that the correlation analysis between cellular impedance measurements and classical toxicity endpoints has been performed on a limited number of compounds. Overall, despite some limitations, the RTCA technology appears to be a powerful and reliable tool in drug discovery because of the reasonable throughput, rapid and efficient performance, technical optimization, and cell quality control.     

In vitro and in vivo binding of (E)- and (Z)-N-(iodoallyl)spiperone to dopamine D2 and serotonin 5-HT2 neuroreceptors

Apparent affinities (Ki) of (E)- and (Z)-N-(iodoallyl)spiperone [E)- and (Z)-NIASP) for dopamine D2 and serotonin 5-HT2 receptors were determined in competition binding assays. (Z)-NIASP (Ki 0.35 nM, D2; Ki 1.75 nM, 5-HT2) proved slightly more potent and selective for D2 sites in vitro than (E)-NIASP (Ki 0.72 nM, D2; Ki 1.14 nM, 5-HT2). In vivo, radioiodinated (E)- and (Z)-[125I]-NIASP showed regional distributions in mouse brain which are consonant with prolonged binding to dopamine D2 receptors accompanied by a minor serotonergic component of shorter duration. Stereoselective, dose-dependent blockade of (E)-[125I]-NIASP uptake was found for drugs binding to dopamine D2 sites, while drugs selective for serotonin 5-HT2, alpha 1-adrenergic and dopamine D1 receptors did not inhibit radioligand binding 2 hr postinjection. Specific binding in striatal tissue was essentially irreversible over the time course of the study, and (E)-[125I]-NIASP gave a striatal to cerebellar tissue radioactivity concentration of 16.9 to 1 at 6 hr postinjection. Thus, (E)-[125I]-NIASP binds with high selectivity and specificity to dopamine D2 sites in vivo.     

Direct plasma injection for high-performance liquid chromatographic–mass spectrometric quantitation of the anxiolytic agent CP-93 393

A direct plasma injection method has been developed for the rapid analysis of drugs in biological fluids. A new generation restricted access media column specifically designed to accommodate direct injection of plasma and other fluids is utilized for on-line HPLC–ESI-MS analysis. For rapid analysis the on-line extraction column is linked to a HPLC–ESI-MS system. Good results are obtained for the quantitation of CP-93 393 and deuterated internal standard over the range of 10–1000 ng/ml. The lower limit of detection for the assay was 58 pg injected on column. Accuracy and precision values are 9.0% or better over the entire range of the assay. In addition, more than 200 injections (100 μl) were performed per column with unattended, automated analysis.     

Purification and kinetic characterization of a dopamine-sulfating form of phenol sulfotransferase from human brain

The kinetic and biochemical properties of a purified, monoamine-sulfating form of phenol sulfotransferase (M-PST) from human brain are described. M-PST activity was separated and purified from phenol-sulfating activity by anion-exchange chromatography on DEAE-cellulose and subsequently purified on AffiGel Blue and Sephacryl S-200, routinely giving a final purification of over 20 000-fold, with approximately a 3% yield. The molecular weight of the active species, as estimated by gel filtration chromatography, was 250 000. The purified enzyme was inhibited by NaCl (50% at 325 mM) and showed an optimum for dopamine sulfation at pH 7.0. Of the monoamine substrates examined, 4-methoxytyramine was the most extensively sulfated at 20 microM, while at higher substrate concentrations (200 microM), tyramine was the apparent preferred substrate. Kinetic analysis demonstrated that sulfation by M-PST proceeds via an ordered, bisubstrate reaction mechanism, where 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is the leading substrate. True Km values for dopamine and PAPS were 2.9 and 0.35 microM, respectively. The product inhibitor 3'-phosphoadenosine 5'-phosphate possessed a Ki of 0.07 microM, while the dead-end inhibitor ATP exhibited a Ki of 170 microM.     

Adaptation of an enzymatic polyfructosan assay to clinical practice

Inulin or polyfructosan clearance is regarded as the most accurate method of assessing the glomerular filtration rate. We propose an enzymatic method of polyfructosan determination based on the hydrolysis of polyfructosan into fructose by inulinase and the elimination of the interfering quantity of glucose by glucose oxidase. This spectrophotometric microplate formatted assay, which demonstrated very good specificity and reproducibility (within-run precision <1% and between-run precision <3.5%), is cheap and simple to perform and can be used by all analytical laboratories and in all clinical conditions.