Historically, drug discovery focused on native tissues which involved assays such as neurotransmitter or hormone release measurements and radioactive binding assays. Researchers now have a plethora of reagents and assays available to screen for selective lead molecules. As sources of receptors, researchers can choose native tissue, recombinant or nonrecombinant cell lines (see Chapter 8). Issues such as specificities of native tissues due to expression of multiple receptor subtypes, difficulties in obtaining large amounts of native tissues, inabilities to achieve sufficiently high expression levels, and nonhuman pharmacologies have led most screening laboratories to utilize recombinant cell lines.5
In most cases, receptor expression levels in nonrecombinant cell lines will be too low for effective use in screening assays (<1 pmol/mg). Because recombinant cell lines usually express receptors of interest at high levels, they are more suitable for HTS. For pharmacological reasons, some researchers prefer utilizing human cell lines instead of nonhuman cell lines [e.g., human embryonic kidney (HEK293) versus Chinese hamster ovary (CHO cells)]. The use of insect of cell lines in a functional assay for GPCRs has also been reported.6
If the receptor of interest is unavailable, screening with a native tissue or non-recombinant cell line as a receptor source may be the most practical or only alternative. To overcome the inability to screen with nonrecombinant cell lines due to low endogenous expression levels, certain techniques have been developed to allow for boosting receptor expression without the use of cDNA. Zinc finger proteins (ZFPs) that recognize novel DNA sequences serve as the basis of a technology platform developed by Sangamo Biosciences (Richmond, California) that can be used to upregulate GPCR gene expression at a level that allows the cell line to be used for screening ligands without the use of exogenous cDNA.7 GeneDriver® technology (Invitrogen, Carlsbad, California) is based on homologous recombination between the endogenous target GPCR gene and a synthesized corresponding DNA fragment that contains a strong cytomegalovirus (CMV) promoter.89
Whether to use a cell-based or membrane-based assay is a matter of personal choice that depends predominantly on the equipment availability for HTS and on the assay technology. Ligand binding should be measured in membranes, whereas calcium signaling is primarily measured in live cells using kinetic readouts [(e.g., fluorescence imaging plate reader (FLIPR)] or gene reporter assays. For pharmacological reasons, it is important to note that differences in potency can be seen when utilizing membrane- or cell-based assays.10 These differences can be explained by changes in the environment of the receptor during preparation of membranes from whole cells. Also, compound interferences are different in membranes compared to cell-based assays; especially when compound concentration is reduced due to avid uptake in the cells or binding to serum components. Other compounds can elicit cytotoxic cell responses and/or interfere with downstream signaling when utilizing a detection method distant from receptor binding.
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