Exploring the GPCR Surface

G protein-coupled receptors (GPCRs) are a large family of membrane proteins that play a central role in cellular signaling. Their conserved core architecture consists of seven transmembrane helices that span the cell membrane and enable the recognition of a wide range of extracellular ligands. Ligand binding induces conformational changes in the receptor that, in turn, promote intracellular signaling through the receptor's namesake G proteins as well as other intracellular effectors. Because GPCRs are involved in a broad spectrum of physiological processes, they represent one of the most important classes of drug targets, with approximately one-third of all marketed drugs acting on these receptors. However, drug design and discovery efforts are often hampered by ligandability and selectivity issues when compounds must compete with endogenous ligands at canonical binding sites. In many cases, either no suitable initial hits can be identified or the resulting hits show insufficient pharmacological activity and/or selectivity. This, in turn, makes it difficult to optimize them into potent and selective drug candidates with an acceptable side-effect profile. Within the field of GPCR drug discovery, allosteric modulation is widely regarded as a promising strategy to address these challenges. Allosteric ligands bind to sites distinct from the canonical binding site and thereby modulate receptor function in a more subtle and potentially more selective manner. However, structural and pharmacological data on allosteric binding sites and modulators remain limited, and the receptor surface beyond canonical binding sites is still insufficiently explored. The lack of a large-scale structural probing of the GPCR surface hampers hypothesis generation in allosteric drug discovery and limits the systematic identification of novel allosteric sites and modulators. This thesis investigates the structural landscape of G protein- coupled receptors, with a particular focus on allosteric modulation and the identification of novel binding sites for drug discovery. Although GPCRs are among the most important drug targets, drug design efforts often face challenges when targeting canonical binding sites. The results presented here therefore explore how structural and computational methods can be used to identify alternative receptor sites with therapeutic potential. The thesis is structured as a cumulative work and comprises two original publications and one submitted manuscript. The first part provides a systematic review of small-molecule allosteric modulation in GPCRs and summarizes structural and computational strategies for the discovery and design of allosteric modulators. It first introduces the core principles of allosteric modulation and discusses structural insights into the underlying modes of action. Although this part takes the form of a review, it also includes a dedicated analysis of structurally co-resolved allosteric modulators and their binding sites. Together with an extensive literature review of both structure-based and ligand-based approaches for the detection of allosteric sites and the design of allosteric modulators, this work provides a broad foundation for researchers interested in GPCR allosteric modulation. The second part presents the original GPCR pocketome, a structure-based atlas of predicted binding sites across 557 GPCR structures. Using density mapping based on probe docking and residue-contact network analysis, known binding sites were recapitulated and previously untargeted orphan sites were identified and functionally characterized. This work provided, for the first time, a comprehensive probing of the surface landscape of GPCRs and revealed a large number of potential binding sites, many of which appeared to be conserved across the structural receptorome. By combining structural mapping with functional interpretation, this study established a framework for addressing novel receptor sites with possible relevance for allosteric drug discovery. The third part expands this concept to 1,715 experimentally resolved GPCR structures and integrates algorithmically detected cavities with co-resolved ligands, ions, lipids, and cholesterol-like molecules. This analysis revealed conserved and class-specific cavity patterns, activation-state-dependent changes in cavity occurrence, and preferential hotspots for different types of chemical matter. Furthermore, the complete dataset generated in this work was made available through an interactive web service that allows researchers to explore the full set of detected cavities and known binding sites in conjunction with statistical analyses across receptor classes, activation states, and ligand types. Together, the studies presented in this thesis provide a structural framework for understanding GPCR ligandability beyond canonical binding sites and support the identification of novel allosteric ligands in future structure-guided drug discovery efforts. The findings presented here contribute to a more comprehensive view of the GPCR surface and help to advance the development of more fine-tuned and selective therapeutics targeting this important protein superfamily.