Free energy and kinetics in protein-ligand binding: experimental measurements and computational estimates

Virtually all biochemical activities are mediated by the organization and recognition of biological macromolecules. An accurate characterization of the thermodynamics and kinetics governing the formation of supramolecular complexes is required to deeply understand the molecular principles driving all biological interactions. Thermodynamics provides the driving force of protein-ligand binding and is quantified by the binding free energies or the equilibrium dissociation constants. Since the interacting partners are out of equilibrium in vivo, the thermodynamic description of binding needs to be complemented by the knowledge of the kinetic rates. Nowadays, various biophysical experimental techniques can determine thermodynamic and kinetic properties, which are still difficult to be efficiently predicted by computational methods mainly because of the limited force field accuracy and the high computational cost. During my Ph.D., I applied molecular dynamics (MD)-based methods to characterize the thermodynamics and kinetics of inter-molecular interactions. First, I worked on a new enhanced MD-based protocol to simulate protein-ligand dissociation events. This approach provides a realistic description of the evolution of the system to an external perturbation accounting for the natural forces driving the dissociation mechanisms. By applying this computational approach to two pharmaceutically relevant kinases, I was able to rank two series of compounds on unbinding kinetics and to get qualitative mechanistic and path information on the underlying unbinding events, providing additional valuable information to be used in the optimization of lead compounds. Then, I developed an innovative computational method to estimate free energies applicable to systems of arbitrary complexity. Despite the number of challenges to be overcome, the method is very promising being able to provide accurate free energy estimates. Therefore, computer simulations emerged as a valuable tool to obtain information on both the thermodynamic and kinetic aspects governing the formation of supramolecular complexes, which might be used in the rational optimization of lead compounds.