Assays and Target Based Applications of HCS

These assays/applications can be grouped into two classes, namely target-orientated or mechanism-orientated. Target-based approaches seek the activity or localization of a defined protein within the cells, whereas mechanism-based approaches measure general cellular parameters such as intercellular communication or multi-faceted phenomena, such as apoptosis.

Today, GPCRs are becoming increasingly accepted as excellent targets in HCS because of the availability of appropriate assay techniques for drug screening.

The majority of known GPCRs desensitize and internalize into the cytoplasm upon ligand binding to the receptor. Desensitization (receptor aggregation on the cell surface) is accompanied by receptor internalization via clathrin-coated pits into an endosomal compartment [95]. This internalization phenomenon was quantitatively measured using a fusion protein consisting of the parathyroid hormone receptor (PTH) and GFP in mammalian cells. Upon PTH binding, the receptor-GFP internalization, visualized as punctuate spots within the cells, was both time- and dose-dependent, and shown to be selective for the ligand PTH [96]. Pharmacological characterization of the PTH receptor-GFP construct revealed no differences in and Vmax values, as well as in a functional Ca + release assay, between the wild-type PTH receptor and the recombinant receptor-GFP construct expressed in cells, thus validating the internalization assay for this receptor [97].

Another generic assay format for HCS is to measure the translocation of GPCR-interacting proteins. Beta-arrestins interact with GPCRs which have been phosphorylated in response to agonists. This interaction is central to the internalization of GPCRs via clathrin-coated pits, owing to the ability of beta-arrestins to interact with clathrin adapter proteins [95]. Measurement of the internalized GPCR is accomplished by using a beta-arrestin2 fused to GFP as a biosensor which binds with high affinity to ligand-occupied phosphorylated

GPCRs [98]. The universality of this approach has been demonstrated for many GPCRs, whether they couple to different G proteins (Gs, GQ/11 or Gi/o), are classified by ligand (biogenic amines, peptides or lipids), or by the receptor sequences (class A rhodopsin-like, class B secretin-like family or class C metabotropic-like subfamily) [99].

As GPCRs are sequestered into acidic compartments during the internalization process, the use of a receptor-tagged dye, Cypher 5, which is fluorescent only at acidic pH, greatly expands the options for GPCR assays in HCS. On internalization into cells, the dye is protonated and becomes highly fluorescent in the acidic endosomal compartments. The dye can be bound to the receptor via dye-labeled antibodies or ligands. This approach has been used successfully to monitor the agonist-mediated internalization of GPCRs that couple to Gs-, Gi- and GQ/11-mediated signaling pathways [100].

To date, HCS approaches have been used in secondary screening approaches for lead identification or optimization. However, one of the first applications of HCS in primary screening was conducted at Hoffmann-La Roche Inc., USA. A primary cell-based HTS was carried out to identify agonists of a novel orphan GPCR using an assay based on receptor internalization measuring GFP-tagged beta-arrestin. A library of 800 000 compounds was screened, and approximately 800 hits were initially identified. Follow-up studies were carried out to filter out fluorescent compounds and compounds inducing morphological changes. Some compounds were identified that induced beta-arrestin relocalization independently of GPCR binding, possibly acting as kinase inhibitors [101].

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