Screening
Figure 1
Generalised overview of the phenotypic screening
approach. Target deconvolution is the critical step following identification of molecules that induce a desired phenotype in a disease-relevant screen
cells) there is a high likelihood that the critical target receptors for newly-discovered phenotypic biologics will be cell surface proteins. Although there are various approaches for target deconvo- lution, the most challenging and poorly-served area is the human cell surface protein. Standard protein arrays involve generating proteins (often in yeast or insect cells), purifying these and then spotting them on to substrates to then probe with the molecule of interest. This provides a high throughput and relatively low-cost way of screen- ing for targets. However, the arrayed proteins are not localised in a plasma membrane, are not like- ly to be appropriately folded (and are therefore not presenting conformational epitopes for bind- ing) and have not been subject to the normal post-translational modifications that would occur in human cells. This results in low rates of success in identifying biologically-relevant ligand-recep- tor interactions.
The other standard approach to target deconvo- lution is the use of pull-down assays coupled with mass spectrometry. This provides a source of pro- teins from a relevant cell or tissue type, however, solubilising these proteins from membranes will
Figure 2 Schematic of the cDNA expression microarray process
have its own disruptive effect. In addition, the screening process can be time- and resource-inten- sive, again, with limited rates of success.
Using physiologically-relevant receptors as prey for phenotypic ‘baits’ The development of cDNA expression arrays now provides a powerful way to deconvolute the targets of both phenotypic antibodies and small molecules by focusing exclusively on cell surface, plasma membrane targets. Membrane protein-encoding cDNA expression vectors in a lipid complex are arrayed on to specialised slides. Human cells are then seeded on the slides. Cells that grow directly over each of the vector ‘spots’ become reverse- transfected resulting in over-expression of the membrane protein in a cellular context in situ. A phenotypic molecule or other ligand of inter- est is added to the slides and allowed to bind to its over-expressed target (Figure 2). The primary receptor and any potential secondary target bind- ing of the phenotypic molecule is detected using fluorescence imaging, typically after the applica- tion of a fluorescent secondary antibody (Figures 3A and B). Radiolabelled small molecules can also
46 Drug Discovery World Fall 2017
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