Screening
and reproducible in the final phase of the experi- ment. Primary hits are identified by their position on the microarray and then sequenced to confirm. Expression vectors encoding these primary hits, plus positive and negative controls are spotted on to custom slides and the HEK293s cells are reverse transfected. Binding of the phenotypic molecule is then confirmed and non-specific hits weeded out using appropriate controls and competition exper- iments. Each stage of the cell microarray screening involves duplication at multiple levels, for example each vector is spotted in duplicate on each slide and each phase is tested on duplicate slides. This rigorous procedure maximises the chance of identifying specific, reproducible membrane interactor(s) for the test molecule and generates a very low incidence of false positive results. This makes it relatively straight-forward to follow up and validate the cell microarray results using orthogonal approaches.
Can knowing the target alter the route taken? Recently a study by Sandercock et al1 demonstrat- ed how powerful the combination of effective phe- notypic screening coupled with efficient target deconvolution could be in opening up new avenues for drug development. The group focused on ScFv antibodies and designed ankyrin repeat proteins
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(DARPins), isolating functional candidates by phage display selection against primary cells from non-small cell lung cancer patients. Cells were grown in multiple formats, including 3-D cultures, and were monitored to detect the anti-proliferative and pro-apoptotic activity of each molecule. Multiple antibodies that demonstrated a desired activity were isolated and the specific antigens that they targeted were identified using cDNA-expres- sion cell microarray technology. Notably, a subset of antibodies that had distinct phenotypic effects all bound to CUB-domain containing protein 1 (CDCP1) – a cell surface transmembrane protein that is upregulated in many tumour cells and is associated with invasive and metastatic pheno- types. These strong functional effects led the group to test an anti-CDCP1 IgG antibody from their panel for the potential to inhibit tumour growth in vivo using a mouse xenograft model. Although the antibody treatment alone was not efficacious, sig- nificant enhancement of tumour growth inhibition and increased survival times were observed when the antibody was co-administered with cisplatin, compared to cisplatin alone at the same dose. This identified CDCP1 as a potential target for cisplatin combination therapy.
cDNA expression microarray technology has been used in other phenotypic screening pro- grammes to identify new, critical receptors. A study
Drug Discovery World Fall 2017
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