Development of a facile method for high throughput screening with reporter gene assays.

This report describes a facile methodology for high throughput screening with stable mammalian cell reporter gene assays. We have adapted a 96-well adherent cell method to an assay in which cells propagated in suspension are dispensed into 96- or 384-well plates containing test compounds in 100% DMSO. The validation of a stable CHO cell line that expresses 6xCRE-luciferase for use as a reporter gene host cell line is described. The reporter gene, when expressed in this particular CHO cell line, appears to respond specifically to modulation of cAMP levels, thus the cell line is appropriate for screening and pharmacological analysis of Galpha(s)- and Galpha(i)-coupled seven-transmembrane receptors. The development of the new suspension cell assay in both 96- and 384-well formats was performed using a derivative of the CHO host reporter cell line that was stably transfected with human melanocortin-1 receptor. The response of this cell line to NDP-alpha-melanocyte-stimulating hormone and forskolin was nearly identical between the adherent and suspension methods. The new method offers improvements in cost, throughput, cell culture effort, compound stability, accuracy of compound delivery, and hands-on time. The 384-well assay can be performed at high capacity in any laboratory without the use of expensive automation systems such that a single person can screen 100 plates per day with 3.5-4 h hands-on time. Although the system has been validated using Galpha(s)-coupled receptor-mediated activation of a cAMP response element, the method can be applied to other types of targets and/or transcriptional response elements.

Development of scintillation-proximity assays for alpha adrenoceptors.

Binding assays have long been used to determine compound affinity and selectivity for various seven-transmembrane receptors. Over time, the degree of complexity has significantly reduced, whereas the throughput of the various assays has greatly increased. In this article, we detail the development of a filter-binding assay and a scintillation-proximity assay (SPA) designed to quantify a compound's affinity for the three alpha1-adrenoceptor subtypes, alpha1A, alpha1B, and alpha1D. The various components of the assays such as ease of assay performance, robustness, cost, and generation of radioactive waste are compared and contrasted. On the basis of the results, the SPA offers many advantages of high-throughput assay formats over the traditional filter-binding assay. To follow up on the success of the alpha1-adrenoceptor SPA, SPAs for the three alpha2-adrenoceptors were developed and are detailed in this article. Affinity data generated for a select number of alpha2 compounds agree with reported literature values. These assays, like those for alpha1 subtypes, are very amenable to high-throughput screening campaigns. In conclusion, scintillation-proximity assays offer significant advantages over filter-binding assays.

A combination assay for simultaneous assessment of multiple signaling pathways.

We have developed an assay in which modulation of two or more signaling pathways can be assessed concurrently by combining reporter gene systems with fluorescent probe technology. The validation of this method was achieved by indirect analysis of adenylyl cyclase activation with the use of a cyclic AMP response element (CRE)-luciferase reporter system in combination with the measurement of calcium mobilization by Calcium Green-1 AM fluorescence on a fluorescent imaging plate reader. To demonstrate the utility of the method in studying the pharmacology of receptors that couple to more than one G protein, Chinese hamster ovary (CHO) cells, which stably expressed both the CRE-luciferase reporter gene and the human pituitary adenylyl cyclase-activating peptide (PACAP) receptor, were treated with PACAP 1-27 and 1-38. Calcium mobilization and the induction of adenylyl cyclase activity in response to each concentration of peptide were assessed in individuals wells. This assay may also be used to screen for ligands of two or more unrelated receptors simultaneously without compromising the assessment of either signaling pathway. To illustrate this point, Rat-1 fibroblasts, which expressed human alpha1A receptors, were cocultured with CRE-luciferase CHO cells, which expressed human GLP-1 receptors. Calcium mobilization elicited by phenylephrine agonism of the alpha1A receptor was assessed in the same assay as GLP-1-induced activation of adenylyl cyclase. The pEC(50) for each agonist was similar to that observed when the cell lines were not cocultured. The number of different receptors that can be screened per well is limited only by the ability to distinguish different reporter gene signals and fluorescent indicators.

Alpha 1-adrenergic receptor subtype determinants for 4-piperidyl oxazole antagonists.

Mutational studies in conjunction with ligand binding assays were used to examine the basis of alpha1-adrenergic receptor subtype selectivity for a series of 4-piperidyloxazole antagonists. A set of chimeric alpha 1A receptors were created by systematically substituting individual transmembrane domains from alpha 1D adrenergic receptors. The oxazole antagonists exhibited significant reductions in affinity against the receptor construct alpha 1A/D(TM2), and moderate reductions in affinity versus constructs alpha 1A/D(TM5), alpha 1A/B(TM5), and alpha 1A/D(TM6). Antagonist affinities for these chimeras exceeded those found for wild type alpha 1D and alpha 1B. Site-directed mutagenesis methods were then used to explore the role that individual residues in TM2 and TM5 play in ligand binding affinity and selectivity. These studies revealed that mutations at position 86 in the second transmembrane domain and position 185 in the fifth transmembrane domain of the alpha 1A receptor have a major impact on receptor subtype selectivity.

Structure activity relationships of a series of buspirone analogs at alpha-1 adrenoceptors: further evidence that rat aorta alpha-1 adrenoceptors are of the alpha-1D-subtype.

The activity of a series of busprione analogs at recombinant and rat thoracic aorta alpha-1 adrenoceptors was investigated. Compound affinity for recombinant alpha-1A, alpha-1B and alpha-1D adrenoceptors from human and animal sources was determined by radioligand binding assays using membranes prepared from rat-1 fibroblasts expressing recombinant receptors with ( +/- )-[125l]iodo-4-hydroxyphenyl)-ethyl-aminomethyl-tetralone as the radioligand. Compound affinity and functional activity at rat aortic alpha-1 adrenoceptors were determined using endothelium denuded rings contracted with phenylephrine. BMY 7378 ¿8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]-ehtyl)-8-azaspiro [4,5]decane-7,9-dione dihydrochloride¿ and MDL 73005EF ¿8-[2-(1,4-benzodioxan-2-ylmethylamino) ethyl]-8-azaspiro[4,5]decane-7,9-dione hydrochloride¿ were found to have significant selectivity for the alpha-1D-subtype and were high affinity antagonists of the alpha-1 adrenoceptors in the rat aorta. Leverage plot analysis of affinities of the buspirone analogs and a series of structurally diverse alpha-1 antagonists for recombinant alpha-1 adrenoceptors and rat aorta alpha-1 adrenoceptors demonstrate that the alpha-1 adrenoceptors in the rat aorta are predominantly of the alpha-1D subtype.

BMY 7378 is a selective antagonist of the D subtype of alpha 1-adrenoceptors.

BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8- azaspiro[4.5]decane-7,9-dione dihydrochloride), a 5-HT1A receptor partial agonist, also binds to alpha 1-adrenoceptors. Competition assays were performed using (+/-)-beta-([125I]iodo-4-hydroxyphenyl)-ethyl-aminomethyl-tetralone ([125I]HEAT), and membranes prepared from Rat-1 fibroblasts expressing hamster alpha 1b-, bovine alpha 1c-, or rat alpha 1d-adrenoceptor, or their respective human homologues. Results indicate that BMY 7378 is selective for the alpha 1D-adrenoceptor subtype (pKi: hamster alpha 1b-adrenoceptor 6.2 +/- 0.03, human alpha 1b-adrenoceptor 7.2 +/- 0.05; bovine alpha 1c-adrenoceptor 6.1 +/- 0.02, human alpha 1c-adrenoceptor 6.6 +/- 0.20; rat alpha 1d-adrenoceptor 8.2 +/- 0.06, human alpha 1d-adrenoceptor 9.4 +/- 0.05) and has high affinity (pA2, 8.9 +/- 0.1) for rat aorta alpha 1-adrenoceptor.

Characterization of alpha-1 adrenoceptor subtypes in human and canine prostate membranes.

Experiments were designed to characterize the predominant subtype of alpha-1 adrenoceptors in human and canine prostate tissue. The chemical (+/-)-beta-([125I]iodo-4-hydroxyphenyl)-ethyl- aminomethyl-tetralone bound in a specific, saturable manner to a single class of binding sites in membranes that expressed recombinant hamster alpha-1B, bovine alpha-1C and rat alpha-1D receptors expressed in rat-1 fibroblasts and to those from prostate tissue. Competition assays with human and canine prostate membranes revealed only a single class of binding sites. Binding affinity in both human and canine prostate most significantly correlated with binding affinity for the recombinant bovine alpha-1C receptor (r = .98 human, .95 canine). Further analysis with leverage plots demonstrated that binding affinity in human and canine prostate tissue is best predicted by binding affinity to recombinant bovine alpha-1C (P < .01 human and P < .001 canine). These data are consistent with a single class of alpha-1 adrenoceptors in human and canine prostate tissue, which is best represented as the alpha-1C subtype.