Identification of small molecule antagonists of the human mas-related gene-X1 receptor

The recently identified mas-related-gene (MRG) family of receptors, located primarily in sensory neurons of the dorsal root ganglion, has been implicated in the perception of pain. Thus, antagonists of this class of receptors have been postulated to be useful analgesics. Toward this end, we developed a cell-based beta-lactamase (BLA) reporter gene assay to identify small molecule antagonists of the human MRG-X1 receptor from a library of compounds. Single-cell clones expressing functional receptors were selected using the BLA reporter gene technology. The EC50 for the MRG agonist peptide, BAM15, appeared to be comparable between the BLA assay and the intracellular Ca2+ transient assays in these cells. Ultra high-throughput screening of approximately 1 million compounds in a 1.8-microl cell-based BLA reporter gene assay was conducted in a 3456-well plate format. Compounds exhibiting potential antagonist profile in the BLA assay were confirmed in the second messenger Ca2+ transient assay. A cell-based receptor trafficking assay was used to further validate the mechanism of action of these compounds. Several classes of compounds, particularly the 2,3-disubstituted azabicyclo-octanes, appear to be relatively potent antagonists at the human MRG-X1 receptors, as confirmed by the receptor trafficking assay and radioligand binding studies. Furthermore, the structure-activity relationship reveals that within this class of compounds, the diphenylmethyl moiety is constant at the 2-substituent, whereas the 3-substituent is directly correlated with the antagonist activity of the compound.     

Miniaturization of whole live cell-based GPCR assays using microdispensing and detection systems

Cell-based beta-lactamase reporter gene assays designed to measure the functional responses of G-protein-coupled receptors (GPCRs) were miniaturized to less than 2 microL total assay volume in a 3456-well microplate. Studies were done to evaluate both receptor agonists and antagonists. The pharmacology of agonists and antagonists for target GPCRs originally developed in a 96-well format was recapitulated in a 3456-well microplate format without compromising data quality or EC(50)/IC(50) precision. These assays were employed in high-throughput screening campaigns, allowing the testing of more than 150,000 compounds in 8 h. The instrumentation used and practical aspects of the assay development are discussed.     

An improved beta-lactamase reporter assay: multiplexing with a cytotoxicity readout for enhanced accuracy of hit identification

A problem inherent to the use of cellular assays for drug discovery is their sensitivity to cytotoxic compounds, which can result in false hits from certain compound screens. To alleviate the need to follow-up hits from a reporter assay with a separate cytotoxicity assay, the authors have developed a multiplexed assay that combines the readout of a beta-lactamase reporter with that of a homogeneous cytotoxicity indicator. Important aspects to the development of the multiplexed format are addressed, including results that demonstrate that the IC(50) values of 40 select compounds in a beta-lactamase reporter assay for nuclear factor kappa B and SIE pathway antagonists are not affected by the addition of the cytotoxicity indicator. To demonstrate the improvement in hit confirmation, the multiplexed assay was used to perform a small-library screen (7728 compounds) for serotonin 5HT1A receptor antagonists. Hits identified from analysis of the beta-lactamase reporter data alone were compared to those hits determined when the reporter and cytotoxicity data generated from the multiplexed assay were combined. Confirmation rates were determined from compound follow-up using dose-response analysis of the potential antagonist hits identified by the initial screen. In this representative screen, the multiplexed assay approach yielded a 19% reduction in the number of compounds flagged for follow-up, with a 37% decrease in the number of false hits, demonstrating that multiplexing a beta-lactamase reporter assay with a cytotoxicity readout is a highly effective strategy for reducing false hit rates in cell-based compound screening assays.     

A fluorescent reporter assay for the detection of ligands acting through Gi protein-coupled receptors

Accompanying the advances in basic biology of G protein-coupled receptors (GPCRs) is the practical need among biopharmaceutical companies for sensitive assays to assess GPCR function, particularly formats that are compatible with high-throughput drug screening. Here we describe a novel cell-based assay format for the high-throughput detection of ligands for Gi protein-coupled receptors. Two Gi-GPCRs, mu-opioid receptor (mu-OPR) and 5-hydroxytryptamine receptor la (5HT1aR) are employed as model receptor targets. The key feature of this assay system is the isolation of stable, clonal Chinese hamster ovary (CHO) cell lines that carry three separate expression plasmids: (1) a chimeric Gq/i5 protein (which re-directs a negative Gi-type signal to a positive Gq-type response), (2) a given Gi-GPCR, and (3) a beta-lactamase (beta1a) reporter gene responsive to Gi-GPCR signaling. Cell-based assays built using this format show appropriate rank order of potency among a reference set of receptor agonist and antagonist compounds. Such assays are also robust, reliable, and can be used for industrial-scale applications such as high-throughput screening for drug leads.     

Preparation and characterization of calibration beads for sorting cells expressing a beta-lactamase gene reporter.

BACKGROUND: Modern drug discovery has been based on high-throughput screening using whole-cell assays. A prominent role has been assigned to the reporter gene technology based on a beta-lactamase and the fluorogenic substrate CCF2. Successful application of this technology requires fluorescence-activated cell sorting. We describe the preparation and characterization of calibration beads for sorting cells expressing the beta-lactamase gene using the CCF2 substrate. METHODS: To model Forster resonance energy transfer (FRET) between the coumarin donor and the fluorescein acceptor of the CCF2 reporting dye, we used activated polystyrene beads with primary amino groups. Donor and acceptor fluorophores were attached to the beads at different ratios via succinimidyl esters. The beads were characterized with a fluorescence plate reader and a flow cytometer. RESULTS: We prepared polystyrene beads with five different ratios of donor and acceptor fluorophores and beads that carried a donor or a receptor fluorophore alone. Fluorescence measurements demonstrated that the prepared beads well represent the FRET of CCF2 substrate. CONCLUSION: We have demonstrated that the prepared beads can be successfully used for the setup of fluorescence-activated cell sorting to sort cells with CCF2 reporter substrate and the beta-lactamase reporter gene.     

Receptors for bradykinin and kallidin.

In order to establish if bradykinin (BK) and Lys-bradykinin (Lys-BK), alias kallidin, act on the same or on different receptors, experiments were performed on strips of cat terminal ileum and of rabbit aorta. The first preparation contains receptor B2 and the second has the newly identified receptor B1. The criterion used for establishing the identity of receptors for BK and Lys-BK in the cat ileum (receptor B2) was desensitization, while for the rabbit aorta (receptor B1) we measured the apparent affinity (pA2 value) of a competitive and specific inhibitor of BK, [Leu-OMe3,des-Arg9]-BK. Since cat ileum desensitized with BK or Lys-BK shows a significant decrease or a complete disappearance of the response to the other agent, while maintaining full sensitivity for histamine, and since the pA2 values of [Leu-OMe8,des-Arg9]-BK against BK and Lys-BK are identical in the rabbit aorta, we conclude that the two kinins act on the same types of receptors.     

Fluorescent and biotinylated probes for B2 bradykinin receptors: agonists and antagonists.

Peptide ligands carrying additional reporter groups are valuable research tools to facilitate biochemical and pharmacological studies of G protein-coupled receptors. B2 bradykinin receptors, widely distributed in mammalian tissues, regulate many physiological systems and are therapeutic targets. Acylation of the amino-terminus of bradykinin (BK) and a B2a-selective antagonist produced ligands derivatized with biotinamidocaproate or 7-Amino-4-methylcoumarin-3-acetate. These fluorescent and biotinylated peptides bound with high affinity to bovine and rodent B2 receptors. Analysis of second messenger production confirmed that fluorescent and biotinylated analogs of BK were B2 receptor agonists whereas derivatives of DArg0[Hyp3,DPhe7,Leu8]BK were BK receptor antagonists. The complimentary properties of these selective receptor probes will be useful in studying B2 receptor localization, expression and desensitization.     

Development of a generic dual-reporter gene assay for screening G-protein-coupled receptors

Multiple assay formats have been developed for the pharmacological characterization of G-protein-coupled receptors (GPCRs) and for screening orphan receptors. However, the increased pace of target identification and the rapid expansion of compound libraries present the need to develop novel assay formats capable of screening multiple GPCRs simultaneously. To address this need, the authors have developed a generic dual-reporter gene assay that can detect ligand activity at 2 GPCRs within the same assay. Two stable HEK293 cell lines were generated expressing either a firefly (Photinus) luciferase gene under the control of multiple cAMP-response elements (CREs) or a Renilla luciferase gene under the control of multiple 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive elements (TREs). Coseeded reporter cells were used to assess ligand binding activity at both Galphas-and Galphaq-coupled receptors. By selectively coexpressing receptors with a chimeric G-protein, agonist activity was assessed at Galphai/o-coupled receptors in combination with either Galphas-or Galphaq-coupled receptors. The dual-reporter gene assay was shown to be capable of simultaneously performing duplexed screens for a variety of agonist and/or antagonist combinations. The data generated from the duplexed reporter assays were pharmacologically relevant, and Z' factor analysis indicated the suitability of both agonist and antagonist screens for use in high-throughput screening.     

Molecular cloning, functional expression and pharmacological characterization of a human bradykinin B2 receptor gene.

The gene encoding a putative G protein-coupled receptor (HG10) was cloned from human genomic DNA by low stringency PCR and found to be homologous to the recently described rat bradykinin B2 receptor. The receptor was expressed in xenopus oocytes and stably transfected CHO cell lines. Binding studies demonstrated that HG10 encodes a high affinity BK receptor with an apparent Kd of 150 pM. Displacement by BK agonists and antagonists allowed the characterization of the receptor as a B2 subtype. Functional coupling to the Ca(2+)-phosphatidylinositol cascade was demonstrated in transfected CHO cells where inositol phosphates accumulation and intracellular calcium concentration were elevated in response to BK stimulation. The agonistic and antagonistic properties of BK analogs do not match strictly the pharmacological profile described for the rat or guinea pig B2 receptor subtypes or the putative B3 subtype. This discrepancy is attributed either to species variability or to differences in the coupling efficiency of receptors to the transduction cascade in different cell types. From our results, the existence of B3 receptors and of B2 subtypes appears questionable.     

Engineered yeasts as reporter systems for odorant detection

The functional expression of olfactory receptors (ORs) is a primary requirement to utilize olfactory detection systems. We have taken advantage of the functional similarities between signal transduction cascades in the budding yeast Saccharomyces cerevisiae and mammalian cells. The yeast pheromone response pathway has been adapted to allow ligand-dependent signaling of heterologous expressed G-protein coupled receptors (GPCRs) via mammalian or chimeric yeast/mammalian Galpha proteins. Two different strategies are reported here which offer a positive screen for functional pairs. The OR and Galpha protein are introduced into the modified yeast cells such that they hijack the pheromone response pathway usually resulting in cell cycle arrest. The first strategy utilizes ligand-induced expression of a FUS1-HIS3 reporter gene to permit growth on a selective medium lacking histidine; the second to induce ligand-dependent expression of a FUSI-Hph reporter gene, conferring resistance to hygromycin. Validation of the systems was performed using the rat 17 receptor response to a range of aldehyde odorants previously characterized as functional ligands. Of these only heptanal produced a positive growth response in the concentration range 5 x 10(-8) to 5 x 10(-6) M. Induction conditions appear to be critical for functional expression, and the solvents of odorants have a toxic effect for the highest odorant concentrations. The preference of rat 17 receptor for the ligand heptanal in yeast has to be compared to concurrent results obtained with mammalian expression systems.     

Mammalian PSP24s (alpha and beta isoforms) are not responsive to lysophosphatidic acid in mammalian expression systems

Xenopus PSP24 (xPSP24) is a G-protein-coupled receptor which was originally identified as a functional lysophosphatidic acid (LPA) receptor. We obtained two different types (alpha and beta) of mammalian homologues of xPSP24 and found that these receptors are highly expressed in the brain (Kawasawa et al., Biochem. Biophys. Res. Commun. 276, 952-956, 2000). These receptor did not respond to LPA by GTPgammaS binding assays, while Edg2 or Edg4 showed responses to LPA under the same assay conditions. Furthermore, a sensitive reporter gene assay using PC12 cells with serum response element promoter failed to detect any response of mammalian PSP24s to various concentrations of LPA. Thus, unlike xPSP24, we conclude that PSP24s are not functional LPA receptors in mammalian systems.     

Agonistic effects of the opioid buprenorphine on the nociceptin/OFQ receptor

The nociceptin/orphanin FQ (N/OFQ) receptor (e.g. the human ortholog ORL1) has been shown to be pharmacologically distinct from classic opioid receptors. Recently, we have identified buprenorphine as a full ORL1 agonist using a reporter gene assay. For further functional analysis, buprenorphine's effects on ORL1 receptors were investigated using a K(+) channel (GIRK1) assay in Xenopus oocytes and GTPgammaS assay in CHO-K1 membrane preparations. In both assays, buprenorphine behaved as a partial agonist compared to nociceptin itself. The N/OFQ agonism of buprenorphine might contribute to actions of buprenorphine in pain models in vivo beside its mu- or kappa-opioid receptor mediated effects.     

Odorant response assays for a heterologously expressed olfactory receptor

Odorant responsiveness of a mouse olfactory receptor, mOR-EG, was investigated in various heterologous cells using a variety of detection methods. Odorant-induced Ca(2+) response was observed in HEK293 cells that coexpressed mOR-EG and the promiscuous G protein, G alpha 15. Without G alpha 15, a robust increase in cAMP level was observed upon odorant-stimulation in various mammalian cells. A luciferase reporter gene assay using zif268 promoter was adopted to amplify the cAMP signals. In Xenopus laevis oocytes, odorant-stimulated currents were recorded when mOR-EG cRNA was co-injected with either G alpha 15 or cAMP-dependent channel. These results suggest that odorant responsiveness can be monitored via a signaling pathway mediated by endogenous G alphas or transfected G alpha 15 in heterologous cell systems. Various functional assays for a heterologously expressed olfactory receptor reported in this study, are potentially useful for high-throughput ligand screening and functional analyses of hundreds of olfactory receptors.