Beyond Affinity: Drug-Kinase Interaction Put under the Microscope

Over the last 20 years, the consideration of biophysical parameters such as kinetics and thermodynamics has been used to guide modern drug design. In contrast to the classical approach that mainly relies on affinity optimization, biophysical parameters allow a further discrimination of potential drug candidates with comparably high affinity. However, there is still a lack of systematic studies analyzing the impact of chemical ligand structure on, for example, thermodynamics. The studies presented in this thesis aim to bridge this gap. In this thesis a model protein, namely cAMP-dependent protein kinase (PKA) is used to gain particular insights into kinase behavior and thermodynamics upon ligand binding. Due to their implication in various diseases such as cancer, kinases are of utmost importance in drug design. The ligands used in this study were derived from the approved drug fasudil. They differ in their ligand degrees of freedom, molecular weight and decorations. Analyzing the impact of ligand degrees of freedom on the thermodynamic signatures, crystal structures were determined. The crystallographic analysis confirmed the flexible nature of the kinase. Particularly the position of the Gly-rich loop differs in the complex structures of ligands with varying ligand degrees of freedom. Thermodynamic signatures were determined using isothermal titration calorimetry. Remarkably, the ligand with the largest amount of internal degrees of freedom appeared to be the binder with the most beneficial entropic contribution. This counterintuitive observation is most likely the result of water displacement from the active site upon ligand binding and due to a higher ordered local water structure of the ligand in solution prior to protein binding. For the series of ligands with increasing molecular weight, differences in the ligand coordination with the protein could be observed. A clear trend toward a more entropic and less enthalpic binding upon increasing molecular weight could be observed. Again, this results from structural changes and probably from the state of the uncomplexed ligand in solution, an utterly underestimated factor. For ligand decoration, introduction of methyl groups is a simple but potentially powerful approach. For differentely methylated ligands not only position but also stereochemistry of the methyl group has an influence on binding potency as well as the thermodynamic signature of ligand binding. Strikingly, the combination of single methyl groups does not lead to additive effects, neither in the binding mode visible in the crystal structure nor in the thermodynamic profile. Further decorations and fragments of fasudil and adenosine triphosphate (ATP) were crystallographically analyzed focusing on their interaction with the hinge region of PKA. It is a key point of attack of ATP-competitive kinase inhibitors. Even minor changes in chemical ligand or fragment structure, result in severe changes of the hinge binding pose of the respective binders. A kinase screen testing 16 ligands against 39 different kinases was performed in order to evaluate if thermodynamic properties can be correlated to the selectivity profile of a potential drug. Especially for kinases, selectivity is challenging but of utmost importance. Remarkably, only ΔG correlated well with the determined selectivity profiles. Finally a methodology approach is presented comparing results from soaking and co-crystallization protocols. The results suggest that structural data from soaking experiments should ideally be verified by cocrystallization, since strong differences between the structures could be observed. There is still a lack of systematical studies correlating structural data, biophysical parameters and selectivity profiles of closely related ligand series. This is the only way to understand the interplay of these different factors, and only then biophysical parameters exceeding affinity information can reveal their full potential and predictive power for the selection of drug candidates

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 <i>prior</i> to protein binding, and by release of these waters, the favored entropic binding is observed

Beyond Affinity: Drug-Kinase Interaction Put under the Microscope

Over the last 20 years, the consideration of biophysical parameters such as kinetics and thermodynamics has been used to guide modern drug design. In contrast to the classical approach that mainly relies on affinity optimization, biophysical parameters allow a further discrimination of potential drug candidates with comparably high affinity. However, there is still a lack of systematic studies analyzing the impact of chemical ligand structure on, for example, thermodynamics. The studies presented in this thesis aim to bridge this gap. In this thesis a model protein, namely cAMP-dependent protein kinase (PKA) is used to gain particular insights into kinase behavior and thermodynamics upon ligand binding. Due to their implication in various diseases such as cancer, kinases are of utmost importance in drug design. The ligands used in this study were derived from the approved drug fasudil. They differ in their ligand degrees of freedom, molecular weight and decorations. Analyzing the impact of ligand degrees of freedom on the thermodynamic signatures, crystal structures were determined. The crystallographic analysis confirmed the flexible nature of the kinase. Particularly the position of the Gly-rich loop differs in the complex structures of ligands with varying ligand degrees of freedom. Thermodynamic signatures were determined using isothermal titration calorimetry. Remarkably, the ligand with the largest amount of internal degrees of freedom appeared to be the binder with the most beneficial entropic contribution. This counterintuitive observation is most likely the result of water displacement from the active site upon ligand binding and due to a higher ordered local water structure of the ligand in solution prior to protein binding. For the series of ligands with increasing molecular weight, differences in the ligand coordination with the protein could be observed. A clear trend toward a more entropic and less enthalpic binding upon increasing molecular weight could be observed. Again, this results from structural changes and probably from the state of the uncomplexed ligand in solution, an utterly underestimated factor. For ligand decoration, introduction of methyl groups is a simple but potentially powerful approach. For differentely methylated ligands not only position but also stereochemistry of the methyl group has an influence on binding potency as well as the thermodynamic signature of ligand binding. Strikingly, the combination of single methyl groups does not lead to additive effects, neither in the binding mode visible in the crystal structure nor in the thermodynamic profile. Further decorations and fragments of fasudil and adenosine triphosphate (ATP) were crystallographically analyzed focusing on their interaction with the hinge region of PKA. It is a key point of attack of ATP-competitive kinase inhibitors. Even minor changes in chemical ligand or fragment structure, result in severe changes of the hinge binding pose of the respective binders. A kinase screen testing 16 ligands against 39 different kinases was performed in order to evaluate if thermodynamic properties can be correlated to the selectivity profile of a potential drug. Especially for kinases, selectivity is challenging but of utmost importance. Remarkably, only ΔG correlated well with the determined selectivity profiles. Finally a methodology approach is presented comparing results from soaking and co-crystallization protocols. The results suggest that structural data from soaking experiments should ideally be verified by cocrystallization, since strong differences between the structures could be observed. There is still a lack of systematical studies correlating structural data, biophysical parameters and selectivity profiles of closely related ligand series. This is the only way to understand the interplay of these different factors, and only then biophysical parameters exceeding affinity information can reveal their full potential and predictive power for the selection of drug candidates

Surprising Non-Additivity of Methyl-Groups in Drug-Kinase Interaction

Drug optimization is guided by biophysical methods with increasing popularity. In the context of lead structure modifications, the introduction of methyl groups is a simple but potentially powerful approach. Hence, it is crucial to systematically investigate the influence of ligand methylation on biophysical characteristics such as thermodynamics. Here, we investigate the influence of ligand methylation in different positions and combinations on the drug–kinase interaction. Binding modes and complex structures were analyzed using protein crystallography. Thermodynamic signatures were measured via isothermal titration calorimetry (ITC). An extensive computational analysis supported the understanding of the underlying mechanisms. We found that not only position but also stereochemistry of the methyl group has an influence on binding potency as well as the thermodynamic signature of ligand binding to the protein. Strikingly, the combination of single methyl groups does not lead to additive effects. In our case, the merger of two methyl groups in one ligand leads to an entirely new alternative ligand binding mode in the protein ligand complex. Moreover, the combination of the two methyl groups also resulted in a nonadditive thermodynamic profile of ligand binding. Molecular dynamics (MD) simulations revealed distinguished characteristic motions of the ligands in solution explaining the pronounced thermodynamic changes. The unexpected drastic change in protein ligand interaction highlights the importance of crystallographic control even for minor modifications such as the introduction of a methyl group. For an in-depth understanding of ligand binding behavior, MD simulations have shown to be a powerful tool

Surprising Non-Additivity of Methyl-Groups in Drug-Kinase Interaction

Drug optimization is guided by biophysical methods with increasing popularity. In the context of lead structure modifications, the introduction of methyl groups is a simple but potentially powerful approach. Hence, it is crucial to systematically investigate the influence of ligand methylation on biophysical characteristics such as thermodynamics. Here, we investigate the influence of ligand methylation in different positions and combinations on the drug–kinase interaction. Binding modes and complex structures were analyzed using protein crystallography. Thermodynamic signatures were measured via isothermal titration calorimetry (ITC). An extensive computational analysis supported the understanding of the underlying mechanisms. We found that not only position but also stereochemistry of the methyl group has an influence on binding potency as well as the thermodynamic signature of ligand binding to the protein. Strikingly, the combination of single methyl groups does not lead to additive effects. In our case, the merger of two methyl groups in one ligand leads to an entirely new alternative ligand binding mode in the protein ligand complex. Moreover, the combination of the two methyl groups also resulted in a nonadditive thermodynamic profile of ligand binding. Molecular dynamics (MD) simulations revealed distinguished characteristic motions of the ligands in solution explaining the pronounced thermodynamic changes. The unexpected drastic change in protein ligand interaction highlights the importance of crystallographic control even for minor modifications such as the introduction of a methyl group. For an in-depth understanding of ligand binding behavior, MD simulations have shown to be a powerful tool