Free-energy simulations applied to melatonin receptor ligands

Abstract

The present work reports molecular modelling investigation performed for melatonergic ligands, aimed at the design of new compounds, definition of structure-activity relationships and investigation of the mechanism of action. Some aspects of the binding mode of melatonergic ligands are studied for the first time exploiting three-dimensional structures of the two melatonin receptors, which were released just before the beginning of the Thesis. While providing a new starting point for structure-based drug design, experimental structures are representative of free-energy minima which are rarely escaped within unbiased molecular dynamics simulations. However, configurations far from these minima could hide important information for design of compounds of pharmaceutical interest. Therefore, free-energy differences must be assessed by either observing the probability of an alternative state to spontaneously occur in longer unbiased simulations or by introducing an external bias potential permitting the system to access new configurations in a shorter simulation time. A basic theoretical overview of such approaches, i.e., free-energy calculations, performed in the Thesis is provided in Chapter 3. While some events, such as most of the studies on ligand conformational space in Chapter 5, can spontaneously occur in unbiased molecular dynamics, most of the simulations require sampling of hidden degrees of freedom of the system, which are not immediately unveiled by crystal structures. Indeed, some states are inherently not observable in such experimental structures, remarking the importance of complex free-energy simulations in the context of drug discovery pipelines in lieu of routinary and simpler docking calculations, still useful to rationalize main structure-activity relationships. These techniques, while requiring more expensive calculations and an increasing complexity, are usually employed in projects of pharmaceutical interest beyond the hit discovery phase, when potent ligands and a richer occurrence of structure-activity relationships are available. In the Thesis, multiple aspects of melatonergic ligands activity have been investigated, including: i) the unbinding route from the ligand binding site, ii) the conformational space of the ligands in bound and unbound states, iii) molecular determinants leading to MT2 receptor subtype selectivity, iv) the impact of the ligand binding on the free-energy landscape of receptor activation. These aspects were analysed in the context of medicinal chemistry projects, in which molecular modelling simulations were performed to rationalize the structure-activity relationships of melatonergic ligands, additionally providing insights for the design of novel compounds. A general introduction to melatonin receptors, with a particular focus on the most important classes of melatonergic ligands, is provided in the Chapter 2, introducing the medicinal chemistry framework in which this work is inserted. In Chapter 4, simulations supporting the hypothesis that melatonergic ligands are recruited through a lipophilic pathway passing through TM helices IV and V are presented and discussed. The free-energy profile associated with the unbinding route of 2-iodomelatonin from the MT1 receptor was characterized and a molecular determinant rationalizing a faster dissociation of the ligand from the MT1 receptor is proposed. Additionally, simulations are commented in combination with experimental data provided by radioligand binding assays and mutagenesis experiments. The impact of conformational equilibria on the binding of melatonergic ligands is extensively analysed in Chapter 5. In particular, the abundance of the bioactive conformation of chiral ligands in the solvent was related to the measure of stereoselectivity for different melatonergic ligands. This approach is generalizable to different classes of ligands, comprising both simplified derivatives of the indole ring and analogues, such as tetrahydroquinolines, in which the alkylamide chain is partially constrained into a ring, dramatically increasing the binding affinity. In Chapter 6, different elements were considered regarding the ligand selectivity toward the MT2 receptor subtype, for the optimization of two important classes of melatonergic ligands: 2-aryl substituted indole derivatives and N-anilinoethylamides. Such properties comprise the study of the size and the orientation of 2-aryl substituents of the indole scaffold for the optimal occupation of an aromatic subpocket, as well as the introduction on both scaffolds of more hydrophilic groups, enhancing the water solubility, while preserving the high binding affinity and selectivity toward the MT2 receptor. Finally, in Chapter 7, molecular determinants driving receptor activation are studied through metadynamics simulations of the MT2 receptor in complex with a prototypical agonist or antagonist. While providing limited information, this study permitted an approximate dissection of structural motifs, scattered across the TM bundle, called microswitches, which are involved in structural changes related to receptor activation and inactivation.