PP-1 (University of Illinois)

Cyclin-dependent kinase 2 (CDK2) is a key regulatory protein, controlling cell cycle progression through its conformational and catalytic mechanisms. Dysregulation of this enzyme contributes to uncontrolled cell proliferation in several human malignancies. In this study, the macromolecular dynamics and inhibition mechanism of CDK2 were explored in complex with pyrazolopyrimidine (PP) analogues of roscovitine. Quantum chemical analysis of the PP analogues revealed enhanced electronic-level reactivity relative to roscovitine. Molecular docking and a total of approximately 1 μs molecular dynamics (MD) simulations for each protein-ligand system, performed in three independent replicates of 300 ns production MD runs, provided insights into the stabilizing interactions, conformational compactness, and dynamic stability of the macromolecular assembly of CDK2 upon inhibitor binding. Notably, the CDK2-PP1 and CDK2-PP2 complexes exhibited improved structural stability, a stronger hydrogen bonding network, and reduced conformational fluctuations compared with the reference inhibitor complex. Markov state model (MSM) analysis was performed for the conformational dynamics of the CDK2 protein, which identified five major metastable conformational states of the protein structure in each system and their transition pathways to convert from one state to another. Alchemical binding free energy calculations corroborated the high inhibitory potency of PP1 and PP2. These findings offer molecular-level insights into structural dynamics and inhibition of CDK2 protein, contributing to the understanding of kinase regulation and supporting future macromolecule-focused drug design efforts.