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.

In lead optimization, protein crystallography is an indispensable tool to analyze drug binding. Binding modes and non‐covalent interaction inventories are essential to design follow‐up synthesis candidates. Two protocols are commonly applied to produce protein–ligand complexes: cocrystallization and soaking. Because of its time and cost effectiveness, soaking is the more popular method. Taking eight ligand hinge binders of protein kinase A, we demonstrate that cocrystallization is superior. Particularly for flexible proteins, such as kinases, and larger ligands cocrystallization captures more reliable the correct binding pose and induced protein adaptations. The geometrical discrepancies between soaking and cocrystallization appear smaller for fragment‐sized ligands. For larger flexible ligands that trigger conformational changes of the protein, soaking can be misleading and underestimates the number of possible polar interactions due to inadequate, highly impaired positions of protein amino‐acid side and main chain atoms. Thus, if applicable cocrystallization should be the gold standard to study protein–ligand complexes.

A ligand-binding study is presented focusing on thermodynamics of fragment expansion. The binding of four compounds with increasing molecular weight to protein kinase A (PKA) was analyzed. The ligands display affinities between low-micromolar to nanomolar potency despite their low molecular weight. Binding free energies were measured by isothermal titration calorimetry, revealing a trend toward more entropic and less enthalpic binding with increase in molecular weight. All protein-ligand complexes were analyzed by crystallography and solution NMR spectroscopy. Crystal structures and solution NMR data are highly consistent, and no major differences in complex dynamics across the series are observed that would explain the differences in the thermodynamic profiles. Instead, the thermodynamic trends result either from differences in the solvation patterns of the conformationally more flexible ligand in aqueous solution prior to protein binding as molecular dynamics simulations suggest, or from local shifts of the water structure in the ligand-bound state. Our data thus provide evidence that changes in the solvation pattern constitute an important parameter for the understanding of thermodynamic data in protein-ligand complex formation.

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