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Biolayer Interferometry Assay for Cyclin-Dependent Kinase-Cyclin Association Reveals Diverse Effects of Cdk2 Inhibitors on Cyclin Binding Kinetics
Cyclin-dependent kinases (CDKs) are key mediators of
cell proliferation
and have been a subject of oncology drug discovery efforts for over
two decades. Several CDK and activator cyclin family members have
been implicated in regulating the cell division cycle. While it is
thought that there are canonical CDK-cyclin pairing preferences, the
extent of selectivity is unclear, and increasing evidence suggests
that the cell-cycle CDKs can be activated by a pool of available cyclins.
The molecular details of CDK-cyclin specificity are not completely
understood despite their importance for understanding cancer cell
cycles and for pharmacological inhibition of cancer proliferation.
We report here a biolayer interferometry assay that allows for facile
quantification of CDK binding interactions with their cyclin activators.
We applied this assay to measure the impact of Cdk2 inhibitors on
Cyclin A (CycA) association and dissociation kinetics. We found that
Type I inhibitors increase the affinity between Cdk2 and CycA by virtue
of a slowed cyclin dissociation rate. In contrast, Type II inhibitors
and other small-molecule Cdk2 binders have distinct effects on the
CycA association and dissociation processes to decrease affinity.
We propose that the differential impact of small molecules on the
cyclin binding kinetics arises from the plasticity of the Cdk2 active
site as the kinase transitions between active, intermediate, and inactive
states