Development of an intact cell reporter gene β-lactamase assay for G protein-coupled receptors for high-throughput screening

Kunapuli, Priya; Ransom, Richard W.; Murphy, Kathy; Pettibone, D. J.; Kerby, Julie; Grimwood, Sarah; Zuck, Paul; Hodder, Peter; Lacson, Raul; Hoffman, Ira; Inglese, James; Strulovici, Berta

doi:10.1016/s0003-2697(02)00587-0

Cited by 80 publications

(65 citation statements)

References 35 publications

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“…To establish that Cpd 1 is able to suppress glucagon-induced gene expression, we examined the agent's effect on the induction of a cAMP-responsive ␤-lactamase reporter gene in CHO cells. The ␤-lactamase reporter gene cell line was prepared by introducing the hGCGR into cells expressing a ␤-lactamase reporter gene under the control of a cAMPresponsive promoter element (24). Glucagon increased ␤-lactamase activity in these cells with an EC 50 of 250 pmol/l.…”

Section: Resultsmentioning

confidence: 99%

“…To study changes in cAMP-dependent transcription in these cells, 5,000 cells per well were plated onto 96-well, clear-bottomed plates and allowed to adhere to the plate for ϳ18 -24 h. Subsequently, the medium was removed and each well was washed with IMDM containing 0.1% FBS and incubated with one of the compounds or 1% DMSO (as a control) in the same medium for 30 min. Cells were stimulated with 250 pmol/l glucagon, 1 mol/l forskolin, or 100 pmol/l GIP (for cells expressing the hGIPR) for 4 h and then loaded with CCF2/AM (a ␤-lactamase substrate dye) to detect ␤-lactamase activity, as previously described (24). Glycogen synthesis assay.…”

Section: Methodsmentioning

confidence: 99%

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A Novel Glucagon Receptor Antagonist Inhibits Glucagon-Mediated Biological Effects

et al. 2004

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Glucagon maintains glucose homeostasis during the fasting state by promoting hepatic gluconeogenesis and glycogenolysis. Hyperglucagonemia and/or an elevated glucagon-to-insulin ratio have been reported in diabetic patients and animals. Antagonizing the glucagon receptor is expected to result in reduced hepatic glucose overproduction, leading to overall glycemic control. Here we report the discovery and characterization of compound 1 (Cpd 1), a compound that inhibits binding of 125 I-labeled glucagon to the human glucagon receptor with a half-maximal inhibitory concentration value of 181 ؎ 10 nmol/l. In CHO cells overexpressing the human glucagon receptor, Cpd 1 increased the half-maximal effect for glucagon stimulation of adenylyl cyclase with a K DB of 81 ؎ 11 nmol/l. In addition, Cpd 1 blocked glucagon-mediated glycogenolysis in primary human hepatocytes. In contrast, a structurally related analog (Cpd 2) was not effective in blocking glucagon-mediated biological effects. Real-time measurement of glycogen synthesis and breakdown in perfused mouse liver showed that Cpd 1 is capable of blocking glucagoninduced glycogenolysis in a dosage-dependent manner. Finally, when dosed in humanized mice, Cpd 1 blocked the rise of glucose levels observed after intraperitoneal administration of exogenous glucagon. Taken together, these data suggest that Cpd 1 is a potent glucagon receptor antagonist that has the capability to block the effects of glucagon in vivo. Diabetes 53

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A Novel Glucagon Receptor Antagonist Inhibits Glucagon-Mediated Biological Effects

et al. 2004

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“…These CREBs bind to the CRE of the EGFP promoter, which results in increased EGFP transcription and green fluorescence emission [9,10] . The measurement of CRE-regulated reporter gene activity provides a sensitive assay for assessing intracellular changes in cAMP levels [11][12][13] in order to study ligand affinity and efficacy for β 2 -AR.…”

Section: Introductionmentioning

confidence: 99%

Identification of anti-asthmatic compounds in Pericarpium citri reticulatae and evaluation of their synergistic effects

et al. 2009

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Aim:To investigate the anti-asthmatic mechanisms of the traditional Chinese medicine Pericarpium citri reticulatae (PCR). Methods: The alkaloid section (AS) of PCR was extracted using an ion exchange resin, separated, and purified into different fractions by semi-preparative HPLC. These fractions were screened for beta2-adrenergic receptor (β 2 AR) agonistic activity using rat β 2 AR-transfected CHO-CRE-EGFP cells. AS and its isolated components were characterized by ultra-performance liquid chromatography/quadrupole time-of-flight MS (UPLC/Q-Tof MS) and were evaluated for their spasmolytic and antitussive activities both in vitro and in vivo in a guinea pig model. Results: We demonstrated that the AS component responsible for activating β 2 AR signaling was synephrine. Both AS and synephrine showed significant spasmolytic effects on acetylcholine chloride (ACh)-induced contractions in isolated guinea pig trachea, and they protected against histamine-induced experimental asthma by prolonging the latent period. We further identified stachydrine as the antitussive component that could significantly reduce citric acid-induced coughing. The combination of these two bioactive compounds had a more potent spasmolytic activity in comparison with the single use of synephrine or stachydrine. Conclusion: We conclude that synephrine and stachydrine are the key components of AS that mediate asthma relief due to their synergism when used in combination.

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“…There are different reporter genes available and their application is very broad. The most common systems are based on b-galactosidase (2,3), alkaline phosphatase (4,5), luciferase (6,7), green fluorescence protein (8,9), and b-lactamase (10)(11)(12)(13).…”

mentioning

confidence: 99%

“…The b-lactamase gene reporting system has characteristics such as no endogenous activity in mammalian cells, high sensitivity, rapid reporting, reproducibility, nonradioactive materials, and no sensitivity to miniaturization that are optimal for the development of HTS assays (10,11).…”

mentioning

confidence: 99%

Preparation and characterization of calibration beads for sorting cells expressing a β‐lactamase gene reporter

et al. 2005

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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 b-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 b-lactamase gene using the CCF2 substrate. Methods: To model F€ orster 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.

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Development of an intact cell reporter gene β-lactamase assay for G protein-coupled receptors for high-throughput screening

Cited by 80 publications

References 35 publications

A Novel Glucagon Receptor Antagonist Inhibits Glucagon-Mediated Biological Effects

A Novel Glucagon Receptor Antagonist Inhibits Glucagon-Mediated Biological Effects

Identification of anti-asthmatic compounds in Pericarpium citri reticulatae and evaluation of their synergistic effects

Preparation and characterization of calibration beads for sorting cells expressing a β‐lactamase gene reporter

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