Screening
be screened using the arrays, with target binding detected by phosphorimaging (Figure 3C). Currently, the largest library of cDNA expres- sion vectors that encode full-length, untagged, unfused human plasma membrane proteins for receptor identification exceeds 4,500 clones. This is estimated to cover around 75% of the plasma membrane proteome. Importantly, there is no bias within the library towards any particular sub-class of human membrane protein. As a result there is representation from all major sub-classes including GPCRs, receptor kinases, Ig superfamily receptors, ion channels and GPI-anchor proteins, among oth- ers. As the technology uses both human proteins and human cells it provides the necessary environ- ment for proteins to be appropriately localised in the plasma membrane in the correct conformation, having also been subject to the normal post-trans- lational modifications which can be critical in mediating interactions3. Use of the highly-trans- fectable human HEK293 cell line results in good levels of over-expression, providing favourable ratios for exogenous:endogenous expression that
allows background binding to untransfected cells not to cause an issue for the vast majority of molecules screened.
The utility of cell microarrays for target deconvolution
A cDNA expression microarray target deconvolu- tion screen typically involves three distinct phases of activity. The first phase ensures that there is low or negligible binding of the phenotypic molecule to the host cells (in this case, untransfected HEK293 cells). If levels of endogenous binding are high enough to compete with the interaction signal then the screen becomes unfeasible. However, more than 90% of molecules that are tested have low enough levels of background binding to be deemed compatible with the technology.
Phenotypic molecules with sufficiently low back- ground binding are progressed into the second phase where they are screened for binding against cell microarrays that are over-expressing the full library of membrane proteins. This identifies pri- mary ‘hits’ which need to be confirmed as specific
Fluorescence
2ug/ml anti-CD52 (hIgG1) + AlexaFluor647 anti-hIgG Fc
Fluorescence 2ug/ml CTLA4-Fc + AlexaFluor647 anti-hIgG Fc 20nM 3H-Naloxone Drug Discovery World Fall 2017 47
Phosphorescence μ-opioid receptor
CD86 CD52
Figure 3 cDNA-expression microarray results (in duplicate) showing: A) an antibody:target interaction between over- expressed CD52 and an anti- CD52 antibody detected using fluorescently-labelled anti-hIgG Fc; B) the interaction between over-expressed CD86 and a CTLA4-Fc fusion protein, detected using fluorescently- labelled anti-hIgG Fc; and C) example of small
molecule:target interaction between 3H-labelled naloxone and over-expressed mu-opioid receptor detected by phosphorescence
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