Second Messenger Assays

To be more specific and closer to the molecular target of interest, receptor-targeted screens have been developed which directly monitor second messengers such as cAMP or changes in intracellular Ca +-concentration. These assays have been well established to investigate the functional effects of compounds targeting GPCRs. A number of different detection technologies exist for cAMP-detection which are mostly based on an immunochemical detection of the cyclic nucleotide (for a review, see [69, 70]). In principle, measurements of cAMP concentration in cells are mostly performed using competitive immunoassays. Here, cAMP-specific antibodies bind either labeled cAMP analogues, or unlabeled, free cAMP generated by the cells. In HTS applications, the read-out will usually be homogeneous and can use (for example) Flashplate™, AlphaScreen, LANCE (PerkinElmer), or HTRF (Cisbio) technologies. The activation of a Gaq-coupled GPCR leads to phospholipase C stimulation, turnover of inositol phosphates (IPs), and a subsequent increase in intracellular Ca +-concentration. Whereas early studies used laborious radioactive methods for 45Ca2+-uptake and [3H]-IP3 turnover, methods based on the fluorescence of cell-permeable Ca +-chelators such as Fura2-AM, Fluo3-AM or Ca-mediated bioluminescence of aequorin, are widely used today [71, 72]. Due to the transient nature of the signal, these assays were not HTS-compatible until the introduction of the Fluorescence Imaging Plate Reader (FLIPR®), produced by Molecular Devices [73]. This instrument has not only stimulated cellular approaches in HTS, but also initiated the development of new generations of instruments designed specifically to measure cellular fluorescence or luminescence signals [74]. Today, these instruments can be integrated into robotic systems [71], and can perform assays in 1536-well format [75]. Recently, a HTRF-based IP1 immunoassay was described which can measure IP-signaling using an endpoint assay [76]. The above-mentioned instruments have addressed the need of functional assays for the important target class of GPCRs.

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