Kinases

Over the past few years, apart from GPCRs, kinases have been the most important drug targets in the pharmaceutical industry. In fact, the activation of kinase-mediated signaling pathways has emerged as an area that is particularly suitable for HCS. The activation of many signaling proteins involves either phosphorylation on specific tyrosine, threonine or serine residues, and/or intracellular trafficking of proteins within the pathways. These phenomena can be detected by immunostaining using phospho-specific antibodies or, in the case of protein relocalization, GFP-tagged proteins.

One example of a HCS approach was recently carried out at Merck KgaA [102], whereby a cellular assay was set up which determined the autophosphorylation of a membrane-bound receptor tyrosine kinase (RTK). Activation of the RTK with ligand induced RTK autophosphorylation which was detected with a phospho-specific antibody, while a second anti-primary antibody was labeled with Alexa 488 (Molecular Probes). Cellular counterstaining was carried out with the red fluorescent dye Syto 64, which enabled the toxic compounds to be identified. Using this approach, the activation (autophosphorylation) status of the RTK could be measured independently ofwhether the cells were treated with a kinase inhibitor, or not. The assay was run on the Acumen Explorer; although this platform is a laserscanning cytometer without microscopic optics, the resolution was comparable to that obtained with a fluorescence microscope (Fig. 5.2).

Fig. 5.2 High-content screening (HCS) assay for receptor tyrosine kinase (RTK) activation. Green objects reflect cells with phosphorylated RTK, red objects the total cell number. Screenshots are shown from the reader (a) and microscopic pictures (b).

The activation of a signaling pathway by a specific receptor can also be measured using a multiplexed assay for HCS. Activation of the MAPK by the epidermal growth factor (EGF) receptor was quantitated using the nuclear localization of phosphorylated ERK by immunofluorescence, together with internalization of the receptor ligand EGF, which was labeled with Texas Red. This assay allowed the identification of compounds able to block activation of the MAPK pathway by a specific relevant oncology target [103].

Using a protein translocation as readout, a HCS screen for inhibitors of p38 kinase was performed at Eli Lilly & Co, USA. The assay was based on the inhibition of anisomycin-induced nuclear import of MAPKAP-K2, fused to GFP. Among 32 000 compounds tested, 48 were identified with an IC50 < 10 ^M. Two of the identified compounds were found to be novel p38 inhibitors [101].

HCS can significantly accelerate lead optimization, resulting in the faster selection of a clinical candidate. This was demonstrated using a multiplexed assay by the Oncology group at Astra Zeneca, who replaced three different individual assays for aurora kinase by a single multiplexed assay that simultaneously measured histone H3 phosphorylation, nuclear area change (a marker for failed cell division), and cell count [101].

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