A Hierarchical Free Energy Framework for Ranking CDK Inhibitors: From PMF Barriers to Alchemical Precision

Ganga Grazielia Chrisna; Qu Chen

doi:10.63313/AERpc.2021

Authors

Ganga Grazielia Chrisna Zhejiang University of Science and Technology, Hangzhou 310000, Zhejiang, China Author
Qu Chen Zhejiang University of Science and Technology, Hangzhou 310000, Zhejiang, China Author

DOI:

https://doi.org/10.63313/AERpc.2021

Keywords:

Free Energy Calculation, Alchemical Methods, Umbrella Sampling, MM/PBSA, CDK Inhibitors, Thermodynamic Integration, MBAR, Kinase Selectivity

Abstract

Accurate prediction of binding free energies for structurally homologous kinase targets remains one of the most challenging tasks in computational drug discovery. CDK1 and CDK2 provide an exceptionally stringent benchmark: their active sites differ by only 0.72 Å backbone RMSD, yet experimental ITC data reveal up to 170-fold selectivity differences for certain inhibitors, corresponding to a ΔΔG of only 2.87 kcal/mol, which is smaller than the typical error of most computational methods. Here we establish and validate a three-tier hierarchical free energy protocol applied to five CDK–inhibitor complexes (Dinaciclib, AZD5438, and CGP74514A bound to CDK1 and CDK2). Tier 1 (MM/PBSA endpoint calculations) achieves a qualitative affinity ranking but fails quantitatively, yielding R ≈ 0.45 and a critical rank inversion for CDK2-Dinaciclib. Tier 2 (umbrella sampling with 56 harmonically restrained windows and WHAM reconstruction) correctly identifies the primary free energy barrier to dissociation in the 6-12 Å center-of-mass distance regime and correctly rank-orders all complexes, though absolute values are systematically underestimated. Tier 3 (alchemical double-decoupling via 62 λ-states with thermodynamic integration and MBAR cross-validation converged to within 0.2 kcal/mol) achieves near-chemical accuracy with a mean absolute error of ~1.11 kcal/mol and R ≈ 1.0.

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