Investigating orthosteric and allosteric modulation of G protein-coupled receptors using in silico approaches

G protein-coupled receptors (GPCRs) represent the largest family of proteins the human genome expresses. Stimuli recognized by GPCRs vary from photons to carbohydrates, lipids, peptides, and proteins, making them fundamental players in numerous physiological events and pathologies, including cancer, neurodegenerative and cardiovascular diseases. The relevance of these proteins to human health has made them major targets in drug development, with GPCR-targeting drugs accounting for more than 30% of all drugs currently on the market. Since the 1980s, molecular modeling has been largely applied to drug design. It allows the consideration of the three-dimensional properties of small- and macromolecules and permits correlating them to functionality. The introduction of artificial intelligence revolutionized the applicability of these methods to drug development, as in the case of AlphaFold, which led to an incredible enlargement of the available protein structural data. This thesis explores various molecular modeling techniques to investigate orthosteric and allosteric modulation of GPCRs and combines them with experimental evidence to prove the predictions. The features of orthosteric modulation and receptor-specificity are explored in chapters 3 & 4 for two class A GPCRs, the orexin receptor 2 (OX2R) and the histamine receptor 2 (H2R), respectively. Additionally, an in-depth analysis of allosteric modulation and its features is carried out in chapter 5 and applied in chapter 6. The orexin system is a major player in sleep/wake cycles, regulated through the activation of two class A GPCRs, orexin receptors 1 & 2, by two peptides, orexins A & B. An impaired production of these peptides or an alteration of the relative receptor’s signaling causes the onset of various pathologies, depending if the alteration causes overexpression of the system (insomnia), or its suppression (narcolepsy). The study reported in this thesis aimed at identifying novel OX2R-specific agonists for the treatment of narcolepsy. The binding of small molecules was investigated through docking calculations, and the e↵ect of agonist and antagonist binding on the receptor conformational landscape was studied through molecular dynamics simulations. Those compounds found promising from the in silico studies were then pharmacologically characterized in cell-based assays. Histamine receptors are widely known targets involved in many alterations of physiological functions (e.g., inflammation, allergy, cancer). However, very little is known about receptor subtype specificity due to the similarity between the two receptors that are addressed by current therapies, histamine receptors 1 & 2 (H1R & H2R). The orthosteric binding pocket of the H2R is therefore inspected to better understand the binding modes of available agonists for this receptor, and investigate, through docking and mutational studies, what determines the higher affinity of certain ligands for one of the two receptors. Thus the investigation shed light on the requirements for developing new subtype-specific active principles. A valuable approach for tackling receptor specificity is the development of allosteric modulators, which act on di↵erent and less conserved binding sites than the orthosteric pocket. They also allow regulation of receptor activation rather than inducing an on/o↵ e↵ect, as many orthosteric binders do. Known allosteric sites (KS) features are investigated in chapter 5, analyzing the distribution of allosteric pockets among di↵erent classes and which residues constitute hotspots in each site. Furthemore, the occupancy of these pockets by di↵erent molecular entities is inspected by studying the properties of compounds not directly acting on the receptors but showing a preference for a specific binding site, together with the function of allosteric modulators depending on the pocket they address. Attempts to develop new allosteric modulators for the class A GPCR, ETB the class C GPCR, GPRC5B are carried out, relying on the acquired knowledge. The studies reported in this thesis demonstrate the efficacy of integrating diverse computational and experimental techniques to elucidate structural and functional properties of an important drug target, such as the superfamily of G protein-coupled receptors.