Rational Design of Thermodynamic and Kinetic Binding Profiles by Optimizing Surface Water Networks Coating Protein-Bound Ligands

A previously studied congeneric series
of thermolysin inhibitors
addressing the solvent-accessible S2′ pocket with
different hydrophobic substituents showed modulations of the surface
water layers coating the protein-bound inhibitors. Increasing stabilization
of water molecules resulted in an enthalpically more favorable binding
signature, overall enhancing affinity. Based on this observation,
we optimized the series by designing tailored P2′
substituents to improve and further stabilize the surface water network.
MD simulations were applied to predict the putative water pattern
around the bound ligands. Subsequently, the inhibitors were synthesized
and characterized by high-resolution crystallography, microcalorimetry,
and surface plasmon resonance. One of the designed inhibitors established
the most pronounced water network of all inhibitors tested so far,
composed of several fused water polygons, and showed 50-fold affinity
enhancement with respect to the original methylated parent ligand.
Notably, the inhibitor forming the most perfect water network also
showed significantly prolonged residence time compared to the other
tested inhibitors.