Understanding protein structures can facilitate the development of therapeutic drugs. Traditionally, protein structures have been determined through experimental approaches such as X-ray crystallography, NMR spectroscopy, and cryo-electron microscopy. While these methods are effective and are considered the gold standard, they are very resource-intensive and time-consuming, ultimately limiting their scalability. However, with recent developments in computational biology and artificial intelligence (AI), the field of protein prediction has been revolutionized. Innovations like AlphaFold and RoseTTAFold enable protein structure predictions to be made directly from amino acid sequences with remarkable speed and accuracy. Despite the enormous enthusiasm associated with these newly developed AI-approaches, their true potential in structure-based drug discovery remains uncertain. In fact, although these algorithms generally predict overall protein structures well, essential details for computational ligand docking, such as the exact location of amino acid side chains within the binding pocket, are not predicted with the necessary accuracy. Additionally, docking methodologies are considered more as a hypothesis generator rather than a precise predictor of ligand–target interactions, and thus, usually identify many false-positive hits among only a few correctly predicted interactions. In this paper, we are reviewing the latest development in this cutting-edge field with emphasis on the GPCR target class to assess the potential role of AI approaches in structure-based drug discovery.

β-arrestins are multifunctional regulators of G protein-coupled receptor (GPCR) signaling, orchestrating diverse downstream signaling events and physiological responses across the vast GPCR superfamily. While GPCR pharmacology has advanced to target orthosteric and allosteric sites, as well as G proteins and GRKs, comparable chemical tools to study β-arrestins remain lacking. Here, we report the discovery of small-molecule inhibitors that selectively target β-arrestins and delineate their mechanism of action through integrated pharmacological, biochemical, biophysical, and structural analyses. These inhibitors disrupt β-arrestin-engagement with agonist-activated GPCRs, impairing desensitization, internalization, and β-arrestin-dependent functions while sparing G protein–receptor coupling. Cryo-EM, MD simulations, and structure-guided mutagenesis reveal that one modulator, Cmpd-5, engages a cryptic pocket formed by the middle, C-, and lariat loops of β-arrestin1—a critical receptor-binding interface—stabilizing a distinct conformation incompatible with GPCR engagement. Together, these findings provide a mechanistic framework for β-arrestin modulation, introducing transducer-targeted strategies to fine-tune GPCR signaling and guide the development of pathway-specific therapeutics.

β-Arrestins are multifunctional regulators of G-protein-coupled receptor (GPCR) signalling and orchestrate diverse downstream signalling events and physiological responses across the GPCR superfamily ^1–3 . Although GPCR pharmacology has advanced to target orthosteric and allosteric sites, as well as G proteins and GPCR kinases, direct chemical tools to modulate β-arrestin activities have remained conspicuously absent. Here we report the identification of small-molecule inhibitors that selectively target β-arrestins and delineate their mechanism of action through integrated pharmacological, biochemical, biophysical and structural analyses. These inhibitors disrupt β-arrestin engagement with agonist-activated GPCRs, impairing desensitization, internalization and β-arrestin-dependent physiological functions while sparing G protein–receptor coupling. Cryo-electron microscopy, molecular dynamics simulations and structure-guided mutagenesis reveal that one modulator, Cmpd-5, engages a pocket within the central crest of β-arrestin1 formed by the middle, C and lariat loops, a critical receptor-binding interface, stabilizing a distinct conformation that is incompatible with full β-arrestin–receptor engagement. Together, these findings establish a mechanistic framework for β-arrestin modulation, reveal a novel allosteric site for structure-based drug design, and open new avenues for transducer-targeted, pathway-specific GPCR therapeutic agents.

Chemokine receptors are GPCRs that regulate the chemotactic migration of a wide variety of cells including immune and cancer cells. Most chemokine receptors contain features associated with the ability to stimulate G protein signaling during β-arrestin-mediated receptor internalization into endosomes. As endosomal signaling of certain non-GPCR receptors plays a major role in cell migration, we chose to investigate the potential role of endosomal chemokine receptor signaling on mechanisms governing this function. Applying a combination of pharmacological and cell biological approaches, we demonstrate that the model chemokine receptor CCR7 recruits G protein and β-arrestin simultaneously upon chemokine stimulation, which enables internalized receptors to activate G protein from endosomes. Furthermore, spatiotemporal-resolved APEX2 proteome profiling shows that endosomal CCR7 uniquely enriches specific Rho GTPase regulators as compared to plasma membrane CCR7, which is directly associated with enhanced activity of the Rho GTPase Rac1 and chemotaxis of immune T cells. As Rac1 drives the formation of membrane protrusions during chemotaxis, our findings suggest an important integrated function of endosomal chemokine receptor signaling in cell migration.

G protein-coupled receptors (GPCRs) have traditionally been understood to signal through heterotrimeric G proteins exclusively from the cell surface followed by β-arrestin (βarr)-mediated desensitization and receptor internalization into endosomes. However, this view has evolved significantly with growing evidence showing that some GPCRs continue to signal from endosomes after their internalization as well as from other intracellular organelles. The vasopressin V2 receptor (V2R) exemplifies this paradigm shift as it promotes robust endosomal G protein before being sorted to lysosomes for degradation. Intriguingly, recent observations suggest that the lysosomal surface itself holds a substantial pool of heterotrimeric G proteins, raising the possibility that GPCRs such as the V2R may stimulate signaling from this subcellular region. To investigate this, we here employed a NanoBiT bystander approach to track intracellular V2R trafficking and transducer activation in real-time. Our results show that activated V2R is trafficked relatively fast to lysosomes where it retains the ability to couple to both G proteins and βarrs. Applying nanobody/intrabody biosensors, we further demonstrated that the V2R activates endogenous G proteins and βarrs at the lysosomal surface and that inhibition of V2R translocation to endolysosomal compartments blunts its ability to stimulate G protein signaling. Together, these findings suggest that the lysosomal surface serves as an unappreciated hub for signaling by some GPCRs before they eventually are engulfed into the lysosomal lumen for degradation. One-sentence summary: Upon activation, the vasopressin V2 receptor is internalized and sorted to the lysosomal membrane, where it activates G proteins and β-arrestins.