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Ligand/protein binding (LPB) is a major topic in medicine, chemistry and biology. Since the advent of computers, many scientists have put efforts in developing theoretical models that could decode the alphabet of the LPB interaction. The success of this task passes by the resolution of the molecular mechanism of LPB. In the past century, major attention was dedicated to the thermodynamics of LPB, while more recent studies have revealed that ligand (un)binding kinetics is at least as important as ligand binding thermodynamics in determining the drug in vivo efficacy. In the present review, we introduce the most widely used computational methods to study LPB thermodynamics and kinetics. It is important to say that no method outperforms another, they all have pros and cons and the choice of the user should take carefully into account the system under investigation, the available structural and experimental data, and the goal of the research. A perspective on future directions of method development and research on LPB concludes the discussion. This article is categorized under: Molecular and Statistical Mechanics > Free Energy Methods Structure and Mechanism > Computational Biochemistry and Biophysics Molecular and Statistical Mechanics > Molecular Dynamics and Monte-Carlo Methods.

Ligand–protein binding kinetic rates are growing in importance as parameters to consider in drug discovery and lead optimization. In this study we analysed using surface plasmon resonance (SPR) the transition state (TS) properties of a set of six adenosine A2A receptor inhibitors, belonging to both the xanthine and the triazolo-triazine scaffolds. SPR highlighted interesting differences among the ligands in the enthalpic and entropic components of the TS energy barriers for the binding and unbinding events. To better understand at a molecular level these differences, we developed suMetaD, a novel molecular dynamics (MD)—based approach combining supervised MD and metadynamics. This method allows simulation of the ligand unbinding and binding events. It also provides the system conformation corresponding to the highest energy barrier the ligand is required to overcome to reach the final state. For the six ligands evaluated in this study their TS thermodynamic properties were linked in particular to the role of water molecules in solvating/desolvating the pocket and the small molecules. suMetaD identified kinetic bottleneck conformations near the bound state position or in the vestibule area. In the first case the barrier is mainly enthalpic, requiring the breaking of strong interactions with the protein. In the vestibule TS location the kinetic bottleneck is instead mainly of entropic nature, linked to the solvent behaviour.

Spectroscopic and functional properties of human cytochrome c and its Tyr67 residue mutants (i.e., Tyr67His and Tyr67Arg) have been investigated. In the case of the Tyr67His mutant, we have observed only a very limited structural alteration of the heme pocket and of the Ω-loop involving, among others, the residue Met80 and its bond with the heme iron. Conversely, in the Tyr67Arg mutant the Fe-Met80 bond is cleaved; consequently, a much more extensive structural alteration of the Ω-loop can be envisaged. The structural, and thus the functional modifications, of the Tyr67Arg mutant are present in both the ferric [Fe(III)] and the ferrous [Fe(II)] forms, indicating that the structural changes are independent of the heme iron oxidation state, depending instead on the type of substituting residue. Furthermore, a significant peroxidase activity is evident for the Tyr67Arg mutant, highlighting the role of Arg as a basic, positively charged residue at pH7.0, located in the heme distal pocket, which may act as an acid to cleave the O-O bond in H2O2. As a whole, our results indicate that a delicate equilibrium is associated with the spatial arrangement of the Ω-loop. Clearly, Arg, but not His, is able to stabilize and polarize the negative charge on the Fe(III)-OOH complex during the formation of Compound I, with important consequences on cytochrome peroxidation activity and its role in the apoptotic process, which is somewhat different in yeast and mammals.