Abstract

Transition through the mammalian cell cycle is co-ordinated by distinct
cyclin/cyclin dependent kinase (CDK) heterodimeric complexes. These complexes
regulate the function of effector proteins including transcription factors that
promote cell cycle phase transition. Specific cyclin/CDKs regulate distinct
phases of the cell cycle at particular times to ensure DNA synthesis and the
transfer of DNA to daughter cells is regulated. Because of the central role
cyclin/CDKs have in controlling the cell cycle, their regulators and substrates
are often deregulated in cancer. Although CDKs have been targeted by drug
discovery for many years, identification of specific cyclin/CDK inhibitors has
been a longstanding challenge.

Here we describe the development of a multiparametric
high-throughput cell-based imaging assay that can be used to assess compound
effects on cell cycle phase, cell viability and phosphorylation of specific CDK
substrates. Nuclear staining intensity is used to determine cell cycle phase, allowing
phosphorylation of CDK substrates to be quantified at different phases of cell
cycle progression. A number of previously described CDK inhibitors have been validated
using this imaging algorithm, with important differences in potency against key
CDK substrates. This work has facilitated the identification and
characterisation of specific cell cycle inhibitors.