Development of a Ca2+-Activated Photoprotein, Photina®, and Its Application to High-Throughput Screening

Bovolenta, Silvia; Foti, Maria; Lohmer, Stefan; Corazza, Sabrina

doi:10.1177/1087057107301497

Cited by 38 publications

(34 citation statements)

References 36 publications

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“…One of these relates to the relatively low sensitivity of the assay stemming from cellular background fluorescence of dye-loaded cells. 38 As a result, the levels of Ca 2ϩ transients following GPCR activation is not as robust as one would desire, particularly with those GPCRs poorly coupled to PLC and PI turnover. Although this may not be such a problem when identifying GPCR agonists, it creates difficulties in identifying partial agonists, inverse agonists, and antagonists for which small responses must be discerned against high background signals.…”

Section: Fluorescent Calcium Dyes To Measure Changes In Intracellularmentioning

confidence: 98%

Photoproteins: Important New Tools in Drug Discovery

Eglen

Reisine

2008

ASSAY and Drug Development Technologies

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The G protein-coupled receptor (GPCR) family is a major target for drug discovery, and most, if not all, GPCRs can couple to Ca2+ signaling. Consequently, there are a number of cellbased, primary, high-throughput screening (HTS) assays used for drug discovery that assess changes in intracellular Ca2+ as a functional readout of GPCR activation. Historically, changes in intracellular Ca2+ levels have been readily detected using fluorescent dyes that emit light in proportion to changes in intracellular Ca2+ concentration. An alternative approach to indirectly measure changes in Ca2+ concentrations involves the use of recombinantly expressed biosensor photoproteins, of which aequorin is a prototypic example. These biosensors have the advantage that they provide an intense luminescent signal in response to elevations in intracellular Ca2+. This exquisite sensitivity, the high signal-to-noise ratios, and the ability to target expression to discrete subcellular sites (in order to detect Ca2+ microdomains) have made photoproteins a principal choice in a wide range of GPCR drug discovery programs. Photoproteins are also finding increasing use in detecting activation of other molecular target classes such as ligand-gated ion channels and transporters. This review focuses upon the use of calcium photoproteins principally for use in GPCR drug discovery and HTS.

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Section: Fluorescent Calcium Dyes To Measure Changes In Intracellularmentioning

confidence: 98%

Photoproteins: Important New Tools in Drug Discovery

Eglen

Reisine

2008

ASSAY and Drug Development Technologies

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“…Besides high levels of receptor expression achieved in the insect cells (30,34) through the use of plasmid-based expression vectors employing genetic control elements of the silkmoth and its baculovirus (31,32), the major functional component of the assay system is a construct directing robust expression of Photina, a Ca 2ϩ -activated photoprotein (36). The activation of this photoprotein provides a luminescence-based readout reporting quantitatively on increases of intracellular Ca 2ϩ ions; hence, in this study, ion channel activity upon administration of cognate olfactory receptor agonists could be studied.…”

Section: Discussionmentioning

confidence: 99%

“…To establish the functionality of the expressed receptors in this system and develop a platform suitable for quantitative assessments of receptor activity, we introduced into the cells a reporter construct for a Ca 2ϩ -activated photoprotein, Photina (36) (Fig. 1A), which functions similarly to aequorin, but has enhanced quantum yield (36,40). Upon activation of OR ligand-gated cation channels, which cause Ca 2ϩ influx into the cells, Photina undergoes a conformational change leading to oxidation of coelenterazine and emission of blue light proportional to the Ca 2ϩ influx (Fig.…”

Section: Mosquito Ors Produce Orco-dependent and Odorant-specificmentioning

confidence: 99%

Inhibition of Anopheles gambiae Odorant Receptor Function by Mosquito Repellents

Tsitoura

Koussis

Iatrou

2015

Journal of Biological Chemistry

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Background:The mode of action of insect repellents on odorant receptor (OR) function remains unclear. Results: Anopheles gambiae OR function in vitro is inhibited by specific repellents. Conclusion: The identified inhibitory effects are due to functional blocking of Orco, the common subunit of OR heteromers. Significance: The specific mechanism of action is distinct from the proposed modes of DEET function.

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“…To overcome current limitations with fluorescence applications, use of photoproteins to sense intracellular Ca 2＋ concentrations more effectively have been developed (Hedley et al, 1996;Stables et al, 1997;Dupriez et al, 2002;Le Poul et al, 2002;Bovolenta et al, 2007). Most recently, the aequorin parental cell line was licensed from PerkinElmer (ES-000-A30) through previous collaborative research program with Euroscreen S.A. (Brussels, Belgium).…”

Section: Discussionmentioning

confidence: 99%

Aequorin Based Functional Assessment of the Melanin Concentrating Hormone Receptor by Intracellular Calcium Mobilization

Lee¹

2010

Biomolecules and Therapeutics

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-Melanin concentrating hormone is a neuropeptide highly expressed in the brain that regulates several physiological functions mediated by receptors in the G-protein coupled receptor family, especially plays an important role in the complex regulation of energy balance and body weight mediated by the melanin concentrating hormone receptor subtype 1 (MCH1). Compelling pharmacological evidence implicating MCH1 signaling in the regulation of food intake and energy expenditure has generated a great deal of interest by pharmaceutical companies as MCH1 antagonists may have potential therapeutic benefit in the treatment of obesity and metabolic syndrome. Although fluorescence-based calcium mobilization assay platform has been one of the most widely accepted tools for receptor research and drug discovery, fluorescence interference and shallow assay window limit their application in high throughput screening and have led to a growing interest in alternative, luminescence-based technologies. Herein, a luminescence-based functional assay system for the MCH1 receptor was developed and validated with the mitochondrial targeted aequorin. Aequorin based functional assay system for MCH1 presented excellent Z' factor (0.8983) and high signal-to-noise ratio (141.9). The nonpeptide MCH1 receptor antagonist, SNAP 7941 and GSK 803430, exhibited IC50 values of 0.62 ± 0.11 and 12.29 ± 2.31 nM with excellent correlation coefficient. These results suggest that the aequorin based assay system for MCH1 is a strong alternative to the traditional GPCR related tools such as radioligand binding experiments and fluorescence functional determinations for the compound screening and receptor research.

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Development of a Ca2+-Activated Photoprotein, Photina®, and Its Application to High-Throughput Screening

Cited by 38 publications

References 36 publications

Photoproteins: Important New Tools in Drug Discovery

Photoproteins: Important New Tools in Drug Discovery

Inhibition of Anopheles gambiae Odorant Receptor Function by Mosquito Repellents

Aequorin Based Functional Assessment of the Melanin Concentrating Hormone Receptor by Intracellular Calcium Mobilization

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