Objective

Cell-based assays are becoming increasingly popular as a powerful approach to uncover novel biological therapeutic targets as well as pharmacological candidates for clinical therapeutic use. By focussing on the smallest unit at which a cellular-relevant biological pathway can be recapitulated, cell-based screening interrogates the diversity of interactions and complexity of cellular-regulatory networks to dissect biology. Providing there is a clear understanding of the underlying pathophysiology for the design and development of a high confidence hit finding screening cascade, biologically active small molecules can be utilised for molecular target identification of new ‘on pathway’ targets. These principles were utilised to deconvolute the highly homologous CDC2L1 and CDC2L2 kinases as critical cancer cell survival targets identified from phenotypic cell-screening. CDC2L1 and CDC2L2 are members of the p34Cdc2 protein kinase family and are near identical with 100% sequence homology in their catalytic ATP binding sites. Knockdown experiments in multiple cancer cell lines have shown decreased viability, induction of apoptosis and the appearance of cells with an aberrant nuclear morphology and these results are phenocopied by compound treatment at low nM concentrations. Proposed literature functions of CDC2L1/2 include a role in the G2/M phase of cell cycle, autophagy, RNA splicing and apoptosis.