Design and Benchmarking of CK2α′-Preferring Inhibitors for Huntington’s Disease: The Limits of Structure-Based Isoform Selectivity

Protein kinase CK2 acts through two catalytic isoforms, CK2α (A1) and CK2α′ (A2), whose ATP-binding sites differ at only two hinge positions, making isoform-selective inhibition difficult. We tested whether a standard structure-based pipeline can design and rank CK2α′-preferring compounds. In a paired benchmark of 140 compounds measured against both isoforms, none of the five docking-derived criteria, computed with GNINA and convolutional neural network rescoring, correlated with measured selectivity (ρ = −0.19 to +0.12), and for matched pairs differing by a single linker methylene, the scoring reversed the experimental order. The only reliable determinant of preference, a two-carbon linker within the benzo[c][2,6]naphthyridine scaffold, was obtained from the measured activities rather than from any computed score. Molecular-dynamics ensembles of the two isoforms overlapped (0.88 Å between versus 0.72–0.96 Å within), placing the selectivity signal below the resolution of the methods. From an enumerated series, EV043 showed the largest end-point free-energy preference for CK2α′ (ΔΔGb -7.74 kcal/mol), although this value remained sensitive to the starting pose in CK2α, while the non-selective inhibitor CX-4945 was reproduced correctly (+0.08 kcal/mol). Distinguishing CK2α′ from CK2α therefore requires alchemical free-energy methods supported by experimental validation.