Type II Inhibitors Targeting CDK2

Kinases can switch between active
and inactive conformations of
the ATP/Mg2+ binding motif DFG, which has been explored
for the development of type I or type II inhibitors. However, factors
modulating DFG conformations remain poorly understood. We chose CDK2
as a model system to study the DFG in–out transition on a target
that was thought to have an inaccessible DFG-out conformation. We
used site-directed mutagenesis of key residues identified in structural
comparisons in conjunction with biochemical and biophysical characterization
of the generated mutants. As a result, we identified key residues
that facilitate the DFG-out movement, facilitating binding of type
II inhibitors. However, surprisingly, we also found that wild type
CDK2 is able to bind type II inhibitors. Using protein crystallography
structural analysis of the CDK2 complex with an aminopyrimidine-phenyl
urea inhibitor (K03861) revealed a canonical type II binding mode
and the first available type II inhibitor CDK2 cocrystal structure.
We found that the identified type II inhibitors compete with binding
of activating cyclins. In addition, analysis of the binding kinetics
of the identified inhibitors revealed slow off-rates. The study highlights
the importance of residues that may be distant to the ATP binding
pocket in modulating the energetics of the DFG-out transition and
hence inhibitor binding. The presented data also provide the foundation
for a new class of slow off-rate cyclin-competitive CDK2 inhibitors
targeting the inactive DFG-out state of this important kinase target.