Photoproteins: Important New Tools in Drug Discovery

Eglen, Richard M.; Reisine, Terry

doi:10.1089/adt.2008.160

Cited by 28 publications

(16 citation statements)

References 83 publications

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“…The use of fluorescent dyes can also be replaced by the use of Ca 2+ -sensitive biosensors. Recombinant expression of the jellyfish photoprotein aequorin, which provides an intense luminescent signal in response to elevated intracellular Ca 2+ in the presence of a coelenterazine derivative, has also been developed for functional screens of GPCRs 31 . With the overexpression of promiscuous G-proteins, the Ca 2+ assay does not require the prior knowledge of G protein coupling conditions and signaling pathways of the receptor, so it is widely used to de-orphan GPCRs 32, 33 .…”

Section: G-protein Dependent Functional Assaysmentioning

confidence: 99%

Tools for GPCR drug discovery

2012

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G-protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic targets for a broad spectrum of diseases. The design and implementation of high-throughput GPCR assays that allow the cost-effective screening of large compound libraries to identify novel drug candidates are critical in early drug discovery. Early functional GPCR assays depend primarily on the measurement of G-protein-mediated 2nd messenger generation. Taking advantage of the continuously deepening understanding of GPCR signal transduction, many G-protein-independent pathways are utilized to detect the activity of GPCRs, and may provide additional information on functional selectivity of candidate compounds. With the combination of automated imaging systems and label-free detection systems, such assays are now suitable for high-throughput screening (HTS). In this review, we summarize the most widely used GPCR assays and recent advances in HTS technologies for GPCR drug discovery.

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Section: G-protein Dependent Functional Assaysmentioning

confidence: 99%

Tools for GPCR drug discovery

2012

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“…These use enzymes such as luciferases, and the complementary luciferin photon-emitting substrates [93, 94]. The most widely used enzymes are firefly luciferase, Renilla luciferase , and aequorin [95–97].…”

Section: General Concepts Underlying the Common Deployed Screening Comentioning

confidence: 99%

Epigenetic assays for chemical biology and drug discovery

Gul

2017

Clin Epigenet

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The implication of epigenetic abnormalities in many diseases and the approval of a number of compounds that modulate specific epigenetic targets in a therapeutically relevant manner in cancer specifically confirms that some of these targets are druggable by small molecules. Furthermore, a number of compounds are currently in clinical trials for other diseases including cardiovascular, neurological and metabolic disorders. Despite these advances, the approved treatments for cancer only extend progression-free survival for a relatively short time and being associated with significant side effects. The current clinical trials involving the next generation of epigenetic drugs may address the disadvantages of the currently approved epigenetic drugs.The identification of chemical starting points of many drugs often makes use of screening in vitro assays against libraries of synthetic or natural products. These assays can be biochemical (using purified protein) or cell-based (using for example, genetically modified, cancer cell lines or primary cells) and performed in microtiter plates, thus enabling a large number of samples to be tested. A considerable number of such assays are available to monitor epigenetic target activity, and this review provides an overview of drug discovery and chemical biology and describes assays that monitor activities of histone deacetylase, lysine-specific demethylase, histone methyltransferase, histone acetyltransferase and bromodomain. It is of critical importance that an appropriate assay is developed and comprehensively validated for a given drug target prior to screening in order to improve the probability of the compound progressing in the drug discovery value chain.

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“…They do, however, suffer from the same type of interference described above for the fluorescent dyes. See Eglen and Reisine (2009) for a more thorough review on the variety of calcium‐sensitive photoproteins available.…”

Section: Calcium Indicatorsmentioning

confidence: 99%

Elusive equilibrium: the challenge of interpreting receptor pharmacology using calcium assays

Charlton

Vauquelin

2010

British J Pharmacology

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Calcium is a key intracellular signal that controls manifold cellular processes over a wide temporal range. The development of calcium-sensitive fluorescent dyes and proteins revolutionized our ability to visualize this important second messenger and its complex signalling characteristics. The subsequent advent of high throughput plate-based fluorescence readers has resulted in the calcium assay becoming the most widely utilized assay system for the characterization of novel receptor ligands. In this review we discuss common approaches to calcium assays, paying particular attention to the potential issues associated with interpretation of receptor pharmacology using this system. Topics covered include dye saturation and forced-coupling of receptors to the calcium pathway, but special consideration is given to the influence of non-equilibrium conditions in this rapid signalling system. Modelling the calcium transient in a kinetic mode allows the influence of ligand kinetics, receptor reserve and read time to be explored. This demonstrates that observed ligand pharmacology at very early time points can be quite different to that determined after longer incubations, even resulting in reversal of agonist potency orders that may be misinterpreted as agonist biased signalling. It also shows that estimates of antagonist affinity, whether by Schild analysis or inhibition curves, are similarly affected by hemi-equilibrium conditions. Finally we end with a discussion on practical approaches to accurately estimate the affinity of insurmountable antagonists using calcium assays. LINKED ARTICLESThis article is part of a themed section on Analytical Receptor Pharmacology in Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2010.161.issue-6 Abbreviations AUC, area under the curve; BC50, ligand concentration producing half-maximal receptor occupancy; CHO cells, Chinese hamster ovary cells; CHO-M3, Chinese hamster ovary cells recombinantly expressing the M3 muscarinic receptor; DR, dose ratio; EC50, agonist concentration producing half-maximal response; FLIPR, fluorescence imaging plate reader; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; HEK cells, human embryonic kidney cells; InsP3, inositol 1,4,5-trisphosphate; Kd, equilibrium dissociation constant, equivalent to the concentration of ligand required to bind 50% of the available receptor sites at equilibrium; kobs, pseudo first-order rate constant for ligand-receptor association. Other kinetic constants are specified in Scheme 1; KE, parameter describing the efficiency by which receptor occupancy is transduced to a biological effect; nH, hill coefficient; PLC, phospholipase C enzyme; t, [R]total/KE where [R]total is the total receptor concentration Historically, drug discovery was performed using tissue preparations and whole animal models. This had the clear advantage that drugs were studied in a physiologically relevant system, but co-expression of related family members made the task of characterizing the ph...

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Photoproteins: Important New Tools in Drug Discovery

Cited by 28 publications

References 83 publications

Tools for GPCR drug discovery

Tools for GPCR drug discovery

Epigenetic assays for chemical biology and drug discovery

Elusive equilibrium: the challenge of interpreting receptor pharmacology using calcium assays

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