On the Implication of Water on Fragment‐to‐Ligand Growth in Kinase Binding Thermodynamics

Abstract

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.

Two Methods, One Goal: Structural Differences between Cocrystallization and Crystal Soaking to Discover Ligand Binding Poses

“…In the current PKA example, we are in the comfortable situation, that also NMR data in solution are available. Remarkably, we have matching results from the corresponding 2D‐NMR experiments; thus we believe our cocrystallization results suggest the more realistic picture of the binding geometry [23,24] …”

“…PKA, like most kinases, is a flexible protein, especially next to the active site. As we have observed high similarity of the cocrystallized structures presented here, and 2D‐NMR experiments, we can assume that our cocrystallized structures are the better approximation representing the relevant protein–ligand binding pose also in solution [23,24] . It is known that PKA performs conformational adaptations of the Gly‐rich loop upon ligand binding in solution.…”

“…Cocrystal structures of 1 and 3 – 7 have been documented in previous contributions [23,24,29] . The corresponding soaked structures were collected at the storage ring Bessy II Helmholtz‐Zentrum Berlin, Germany at Beamline 14.1 on a Pilatus 6 M pixel detector.…”

“…The cocrystallization protocols of 1 , 3 – 7 were discussed previously [23,24,29] . Thus, only differences to the soaking protocols will be reported here.…”

Thermodynamic profiling for fragment-based lead discovery and optimization

“…Ligand binding may be accompanied with protonation/ deprotonation processes whose thermodynamic consequences are pH and buffer dependent, and their dissection leads to intrinsic thermodynamic parameters most relevant in drug design [38,39]. These studies highlight the complexity of ligand binding thermodynamics and show that it is often difficult to pinpoint what the origins of the changes in entropy and enthalpy are [40,41]. Owing to their multifactorial character and non-additivity, thermodynamic quantities cannot be used as direct endpoints, but they are useful in analyzing and supporting the optimization of specific systems [42][43][44].…”