Type II Inhibitors Targeting CDK2.

Area of Science:

• Biochemistry and structural biology
• Drug discovery and kinase inhibition

Background:

• Kinases exhibit active and inactive conformations involving the DFG motif, crucial for type I/II inhibitor design.
• Factors controlling DFG conformations, particularly the DFG-out state, are not fully understood.
• CDK2 was selected as a model to investigate DFG transitions, previously thought inaccessible in the DFG-out state.

Purpose of the Study:

• To identify residues modulating the DFG in-out transition in CDK2.
• To characterize the binding of type II inhibitors to CDK2.
• To explore the potential for developing novel CDK2 inhibitors targeting the DFG-out conformation.

Main Methods:

• Site-directed mutagenesis of CDK2 based on structural comparisons.
• Biochemical and biophysical characterization of CDK2 mutants.
• Protein crystallography to determine the structure of CDK2-inhibitor complexes.
• Kinetic analysis of inhibitor binding.

Main Results:

• Identified key residues that promote the DFG-out transition, enhancing type II inhibitor binding.
• Demonstrated that wild type CDK2 binds type II inhibitors, contrary to previous assumptions.
• Obtained the first co-crystal structure of CDK2 with a type II inhibitor (K03861), revealing a canonical binding mode.
• Showed that identified type II inhibitors compete with cyclin binding and exhibit slow off-rates.
• Highlighted the role of distant residues in modulating DFG-out transition energetics and inhibitor binding.

Conclusions:

• Residues distant from the ATP pocket can significantly influence DFG-out transition energetics and inhibitor binding.
• Wild type CDK2 binds type II inhibitors, suggesting a broader applicability than previously thought.
• Provides a foundation for developing a new class of slow off-rate, cyclin-competitive CDK2 inhibitors targeting the inactive DFG-out state.