Paradoxically, Most Flexible Ligand Binds Most Entropy-Favored: Intriguing Impact of Ligand Flexibility and Solvation on Drug–Kinase Binding

Biophysical
parameters can accelerate drug development; e.g., rigid
ligands may reduce entropic penalty and improve binding affinity.
We studied systematically the impact of ligand rigidification on thermodynamics
using a series of fasudil derivatives inhibiting protein kinase A
by crystallography, isothermal titration calorimetry, nuclear magnetic
resonance, and molecular dynamics simulations. The ligands varied
in their internal degrees of freedom but conserve the number of heteroatoms.
Counterintuitively, the most flexible ligand displays the entropically
most favored binding. As experiment shows, this cannot be explained
by higher residual flexibility of ligand, protein, or formed complex
nor by a deviating or increased release of water molecules upon complex
formation. NMR and crystal structures show no differences in flexibility
and water release, although strong ligand-induced adaptations are
observed. Instead, the flexible ligand entraps more efficiently water
molecules in solution prior to protein binding, and
by release of these waters, the favored entropic binding is observed.